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Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes

Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes 2+ Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female 2+ hearts exhibit lower mito-[Ca ] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into 2+ supercomplexes os ff ets the deci fi t in mito-Ca accumulation, thereby reducing ROS production and stress-induced intracel- 2+ 2+ lular Ca mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca ] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed 2+ decreased mitochondria Ca uniporter expression and increased supercomplex assembly in rat and human female ventricu- lar tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced 2+ mito-ROS and spontaneous SR Ca release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in 2+ female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more effi- 2+ cient electron transport. Such organization coupled to lower levels of mito-[Ca ] limits mito-ROS under stress conditions 2+ 2+ and lowers propensity to pro-arrhythmic spontaneous SR Ca release. We conclude that sexual dimorphism in mito-Ca handling and ETC organization may contribute to cardioprotection in healthy premenopausal females. 2+ Keywords Cardiovascular diseases · Mitochondria · Sarcoplasmic reticulum Ca release · Sexual dimorphism · Oxidative stress · COX7RP Richard T. Clements and Radmila Terentyeva are co-first authors. * Dmitry Terentyev Department of Medicine, Cardiovascular Research Center, dmitry.terentyev@osumc.edu Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI, USA Department of Biomedical and Pharmaceutical Sciences, Department of Pathology, Anatomy and Cell Biology, University of Rhode Island College of Pharmacy, Kingston, MitoCare Center, Thomas Jefferson University, Philadelphia, RI, USA PA, USA Department of Medicine, Providence VAMC and Brown Division of Orthodontics, College of Dentistry, The Ohio University, Providence, RI, USA State University, Columbus, OH, USA Department of Physiology and Cell Biology, The Ohio State University, 460 Medical Center Dr, Columbus, OH 43210, USA Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Vol.:(0123456789) 1 3 15 Page 2 of 22 Basic Research in Cardiology (2023) 118:15 Abbreviations promote disturbances in sarcolemma membrane potential in BN-PAGE Blue native gel electrophoresis the form of delayed- and early-afterdepolarizations. These COX7RP Cytochrome c oxidase subunit 7A-related disturbances underlie pro-arrhythmic triggered activity at protein the whole heart level. The risk of pro-arrhythmic behav- CVD Cardiovascular disease ior profoundly escalates under stress conditions typically ETC Electron transport chain accompanied by β-adrenergic stimulation. Beta-adrenergic 2+ ISO Isoproterenol stimulation increases spontaneous SR Ca release and coin- 2+ MCU Mitochondrial Ca uniporter cidentally increases mito-ROS production [9]. Recently we + 2+ 2+ NCX1 Na Ca exchanger type 1 have shown that RyR2-mediated SR Ca release increases 2+ OVX Ovariectomy transfer of Ca to the mitochondrial matrix via mitochon- 2+ ROS Reactive oxygen speciesdrial Ca uniporter (MCU). This increase contributes to RyR2 Ryanodine receptor type 2 mito-ROS production in VCMs challenged with isoproter- 2+ SCW Spontaneous Ca wave enol (ISO), a selective β-adrenergic agonist [23]. Further- 2+ SERCA2a Sarcoplasmic endoplasmic reticulum Ca more, ROS produced by mitochondria lead to an increase ATPase type 2a in RyR2 oxidation and thereby channel activity, promoting SR Sarcoplasmic reticulum pro-arrhythmic spontaneous SCWs [15, 22, 23]. VCM Ventricular cardiomyocyte Cardiac contraction is a high-energy demanding process. Mitochondria are the major source of energy in VCMs and their ability to generate ATP must be tightly matched to Introduction rapidly changing metabolic demands [16, 21]. In addition to sensing ATP/ADP ratio, mitochondrial function can be 2+ The incidence of all causes of cardiovascular disease (CVD) tuned by changes in [Ca ] entering matrix via MCU. An 2+ is much lower in premenopausal females in comparison to increase in mitochondrial matrix [Ca ] activates several males of the same age [52]. However, later in life CVD dehydrogenases stimulating NADH and F AD production becomes as prevalent in females as males, often resulting that fuel electron transport chain (ETC). Interestingly, stud- in sudden cardiac death due to ventricular arrhythmias ies using mouse models with loss-of-MCU function dem- [40]. The major driver of cardiac dysfunction in old age has onstrated no change in basal cardiac function but showed been ascribed to defective mitochondrial function, leading significant loss of responsiveness to adrenergic stimula- 2+ to impaired metabolic flexibility and subsequent aberrant tion [38, 45]. This suggests that MCU-mediated mito-Ca stress responses [35]. Defective electron transport results uptake has a limited yet important function to boost energy in increased production of reactive oxygen species (ROS) production during stress as an integral part of the fight-or- by mitochondria, which can impair function of numerous flight response. Notably, it has been demonstrated that mito- 2+ enzymes and ion channels in cardiomyocytes [15, 19, 21]. [Ca ] is significantly lower in female VCMs than males [2 ]. Interestingly, in younger males, mitochondria are known to However, cardiac mitochondrial respiration capacity [29, 30] produce more ROS than mitochondria of younger females and cardiac output [25, 27] are comparable between males 2+ [14, 51], without differences in respiration capacity [29, 30]. and females, suggesting that SR Ca release-bioenergetics However, the exact mechanisms underlying this fundamental coupling is fundamentally different between sexes, and that difference between males and females, which may explain these differences may play a key role in lowering cardiovas- sexual dimorphism in rates of CVD, are yet to be defined. cular risk in premenopausal females. Cardiac contractility relies on tightly controlled release In order to provide similar metabolic output to males, it 2+ of Ca from the sarcoplasmic reticulum (SR) mediated by is reasonable to hypothesize that mitochondria from female cardiac ryanodine receptors type 2 (RyR2) [6]. The ampli- VCMs likely have more efficient electron transport to com- 2+ 2+ tude of SR Ca release in ventricular cardiomyocytes pensate for the reduction of matrix [Ca ] [2]. An additional (VCMs) during systole determines the strength of contrac- benefit of a more efficient ETC organization would include tile apparatus activation, while timely cessation of RyR2 reduced levels of ROS [14]. It was recently established that 2+ activity and effective clearance of Ca from the cytosol by elements of the ETC residing in mitochondrial cristae can 2+ SR Ca ATPase (SERCA2a) in diastole promotes relaxa- form large multimolecular supercomplexes, which substan- 2+ tion after each beat. Oxidative modifications of the SR Ca tially increase electron transport efficiency [12]. A group 2+ handling machinery profoundly impair intracellular Ca of proteins involved in facilitation of supercomplex assem- 2+ cycling by impeding SERCA2a-mediated Ca uptake and bly has recently been identified [3 ]. Of particular interest is facilitating untimely RyR2 channel opening [19]. This leads the supercomplex assembly factor, COX7RP (also known 2+ not only to the loss of SR Ca content, but also to gen- as Cox7a2l and SCAFI), given its expression is dependent 2+ eration of spontaneous diastolic Ca waves (SCWs) that on the sex hormone estrogen [33, 53]. However, whether 1 3 Basic Research in Cardiology (2023) 118:15 Page 3 of 22 15 potential differences in mitochondrial supercomplexes and Procurement Team, an entity not involved in the study, and COX7RP expression levels contribute to reduced mito-ROS were initially deemed potentially suitable for transplantation. production in female VCMs, thus exerting a stabilizing influ- The donor’s files do not contain specific entry why a specific 2+ ence on the SR Ca release, remains unknown. heart was not used. According to the coordinators, the most In the present study we investigated the role of sex and common reasons for not using a heart is that it was too small/ COX7RP-dependent changes in mitochondrial supercom- gender disparity, or the potential presence of a viral infec- 2+ plex assembly, mito-ROS production, and intracellular Ca tion. Demographic data of donors from whom tissue samples homeostasis in VCMs under conditions mimicking stress. were obtained are described in Table 1. Our results demonstrate a higher degree of ETC organiza- 2+ tion into supercomplexes and reduced mito-[Ca ] uptake Molecular biology in female VCMs vs males. This leads to reduced mito-ROS 2+ production and less pro-arrhythmic SCWs due to reduced The mitochondrial matrix Ca sensor mtRCamp1h was RyR2 oxidation under β-adrenergic stimulation. Our data constructed by fusing cytochrome C oxidase subunit IV at 2+ suggest that sexual dimorphism in mitochondrial Ca han- the N-terminus of RCamp1h coding region as described dling and ETC organization may contribute to cardioprotec- previously [1, 23]. Mito-GFP was constructed by fusing a tion in healthy premenopausal females. mitochondrial targeting sequence to GFP. The MLS-HyPer-7 Table 1 De-identified demographic data of human organ donors Methods Sample Sex Age (years) Race Study animals 1 Male 36 Caucasian 2 Male 22 African American All procedures involving animals were performed following 3 Male 44 African American the National Institutes of Health Guide for the Care and Use 4 Male 26 Caucasian of Laboratory Animals published by the US National Insti- 5 Male 60 Caucasian tutes of Health (NIH Publication No. 85-23, revised 2011) 6 Male 51 Unknown and were approved by the Institutional Animal Care and 7 Male 62 Unknown Use Committees of The Ohio State University, University 8 Female 43 Caucasian of Rhode Island and Rhode Island Hospital. Two-month- 9 Female 43 African American old male and female Sprague–Dawley rats (control) were 10 Female 68 Caucasian purchased from Charles River Laboratories (Wilmington, 11 Female 38 Caucasian MA, United States) and heart tissue samples and isolated 12 Female 52 Unknown ventricular myocytes were studied at 3–4  month of age. 13 Female 19 Caucasian In addition seven 6 month old Sprague–Dawley rats were 14 Female 24 African American bilaterally ovariectomized (OVX) and additional five rats 15 Female 26 Caucasian received a sham surgery (Sham) at Harlan Laboratories Inc. 16 Female 28 African American Indianapolis, IN, USA. These rats were used 3 months after 17 Female 29 Caucasian surgery to obtain heart tissue samples. To study potential 18 Female 30 Caucasian 2+ sex differences in intracellular Ca homeostasis, mito-ROS 19 Female 31 Caucasian 2+ and mito-Ca 22-month-old F344 rats of both sexes were 20 Female 32 Caucasian obtained from NIH aged rat colony. 21 Female 34 African American 22 Female 34 Caucasian Human samples 23 Female 60 Caucasian 24 Female 61 Caucasian Human left ventricular tissues were obtained from human 25 Female 62 Caucasian organ donors as previously described [20, 39]. These non- 26 Female 62 N/A failing hearts were from those organ donors who had no 27 Female 63 Caucasian history of AF and/or major cardiovascular diseases but not 28 Female 64 Caucasian used for heart transplantation. No one involved in the study 29 Female 65 Caucasian had any involvement in clinical decision making regarding 30 Female 67 Caucasian suitability of donor heart use. The studies were approved 31 Female 69 Caucasian by the Human Study Committee of The Ohio State Univer- 32 Female 72 Caucasian sity. The hearts were evaluated by Lifeline of Ohio Organ 1 3 15 Page 4 of 22 Basic Research in Cardiology (2023) 118:15 sensor was a gift from Vsevolod Belousov [42]. Adenovi- or propyleneoxide, embedded in Spurr’s or Epon (Embed ruses carrying biosensor constructs were generated using 812) resin. Longitudinal, ultrathin sections (65–80 nm) were the ViraPower Gateway expression system (Thermo Fisher cut from the resin-embedded blocks with a diamond knife Scientific, Waltham, MA, USA) [24]. COX7RP-FLAG and (Diatome-US, USA) using a Leica UCT ultramicrotome shRNA COX7RP adenoviruses were purchased from Vigene and caught on copper grid covered with formvar film or a Biosciences and Vector Biolabs, respectively. Gilder parallel bar grid (100 mesh). Images of longitudinal oriented cardiomyocytes were obtained via an FEI Tecnai Myocyte isolation and cell culture 12 TEM fitted with an AMT XR-111 10.5 Mpx or (later) AMT BioSprint 12 12 Mpx CCD camera at 3200–15,000 × To isolate ventricular cardiomyocytes (VCMs), rats were magnification (80 kV). Methods for analyzing mitochondria injected with pentobarbital (120 mg/kg) as a terminal pro- morphology were adapted from Khalifa et al. [30]. TEM cedure, and hearts removed via bilateral thoracotomy. Hearts images were uniformly adjusted for brightness/contrast and were placed in ice-cold Tyrode’s solution and mounted on mitochondria were manually selected by the freehand tool a Langendorff apparatus before retrograde perfusion with using ImageJ software (ImageJ, NIH). Individual mitochon- Tyrode’s solution (in mmol/L: 140 NaCl, 5.4 KCl, 1 MgCl , dria were analyzed for area, perimeter, aspect ratio (the ratio 10 HEPES, 5.6 glucose, pH 7.3) containing collagenase II between major and minor axes), and form factor. Form factor (Worthington Biochemical Corp., Lakewood, NJ, USA) at (F), a measure of mitochondrial length was determined for 37 °C for 15–20 min. Atria were removed and ventricles each image using the equation F = (perimeter /(4π × area). were minced and placed in a 37 °C water bath shaker in Mitochondria density was expressed as the percentage of collagenase solution. Isolated VCMs were plated in serum cell area occupied by mitochondrion. onto laminin-coated glass coverslips in 24-well plates 2+ (Medium 199 [Thermo Fisher Scientific] supplemented Measurements of intracellular Ca transients with 25 mmol/L N aHCO , 10 mmol/L HEPES, 5 mmol/L creatine, 5 mmol/L taurine, 10 μg/mL penicillin, 10 μg/mL Confocal imaging was performed using a Leica SP8 confo- streptomycin, and 10 μg/mL gentamycin; pH 7.3). Unat- cal microscope equipped with a 63 × 1.4 numerical aper- tached cells were removed after 1 h. Attached VCMs were ture oil objective. Cultured rat VCMs were loaded with infected with adenoviruses described below at multiplicity Fluo-3 AM (Invitrogen) at room temperature for 10 min 2+ of infection (MOI) of 10. Myocytes were cultured at 37 °C in Ca -free Tyrode’s solution, followed by a 10 min wash 2+ in 95% air, 5% CO for 36–44 h before analysis. in Tyrode’s solution containing 1 mmol/L Ca . Myocytes 2+ were perfused with Tyrode’s solution (1 mmol/L Ca ) dur- Transmission electron microscopy ing recordings, at room temperature. Myocytes were paced via field stimulation at 1 Hz with platinum electrodes. To 2+ For perfusion fixation, animals were heparinized, then euth- test for the propensity of spontaneous Ca waves (SCWs), anized by pentobarbital (120 mg/kg) injection. The hearts isoproterenol-treated (50 nmol/L) VCMs were paced for 20 s were cannulated through the aorta for retrograde perfusion and latency between the last pacing stimulus and the SCW on a Langendorff apparatus. First, the hearts were perfused was calculated. Caffeine (10 mmol/L) was applied at the 2+ 2+ by cold Ca -free Tyrode solution (in mmol/L: 140 NaCl, 5.4 end of recording to assess SR Ca content. Fluo-3AM was KCl, 1 MgCl , 10 HEPES, 5.6 glucose (pH 7.3) for ~ 5 min excited at 488 nm and fluorescence emission was collected at (until the heart stops beating). This is followed by perfusion 500–550 nm wavelengths in line scan mode at 200 Hz sam- + 2+ with 2.5% glutaraldehyde in 0.1 M Na -cacodylate buffer pling rate. Cytosolic Ca transient amplitude is presented (pH 7.4) for 5 min at room temperature. Then the heart was as ΔF/F , where F is basal fluorescence and ΔF = F–F . 0 0 0 immersed into the fixative for an additional 40 min before 2+ the apex was removed to cut the LV anterior wall open lon- Measurements of mitochondrial matrix Ca gitudinally along the anterior wall. After additional 30 min at room temperature, the heart in the excessive fixative was Myocytes were infected with mtRCamp1h adenovi- put on ice in individually labeled sealed vials, and shipped rus ± COX7RP or COX7RP-shRNA adenovirus and cultured to the MitoCare Center Electron Microscopy Facility at for 36–44 h [20]. mtRCamp1h was excited using 543 nm Thomas Jefferson University, for further processing. There, line of HeNe laser and fluorescence emission collected at small pieces from the LV wall were cut (~ 1  mm ), overnight 560–660  nm wavelengths, measured in the x–y mode at postfixed in 4 °C in 2% osmium tetroxide partially reduced 400 Hz sampling rate. After pacing, VCMs were washed + 2+ by 0.8% K Fe(CN) in 0.15  mol N a -cacodylate buffer. in Ca -free Tyrode’s solution before permeabilization 4 6 Samples were further contrasted en bloc with 1% aque- with saponin (0.001%). Tyrode’s solution was replaced ous uranylacetate, dehydrated in graded series of acetone with internal recording solution. The intracellular solution 1 3 Basic Research in Cardiology (2023) 118:15 Page 5 of 22 15 contained (mmol/L): 120  K aspartate, 20 KCl, 0.81 MgCl , USA; catalogue no. 524625) and protease inhibitor cock- 1 KH PO , 3 MgATP, 10 phosphocreatine, 20 HEPES (pH tails (Sigma; catalogue no. P8340) as described previously 2 4 7.2) and 5  U/mL creatine phosphokinase. Internal solu- [31]. Samples (20–30 μg of proteins) were resolved on a tion was supplemented with cytochalasin D (10 μmol/L) 4–20% gel via SDS‐PAGE, transferred onto nitrocellulose 2+ to inhibit contraction and Ca buffer EGTA (2 mmol/L) membranes, and probed with anti-COX7a2L (COX7RP), to obtain minimum mtRCamp1h fluorescence [20]. Maxi- anti-MCU, anti-Hsp60, anti-GAPDH antibodies and sub- 2+ mum fluorescence was achieved by application of Ca sequently probed with a goat anti‐mouse, goat anti‐rabbit (20  μmol/L) in EGTA-free solution. Using the equation secondary (Promega; Madison, WI, USA). Blots were devel- 2+ mito-[Ca ] = Kd × (F  −  Fmin)/(Fmax  −  F), where Kd of oped with ECL (Bio‐Rad Laboratories, Hercules, CA, USA; 2+ mtRCamp1h = 1.3 µmol/L for Ca , fluorescence was con- catalogue no. 1705061) and quantified and analyzed using 2+ verted to mito-[Ca ] for each VCM. Analysis parameters ImageJ (NIH, Bethesda, MD, USA;) and Origin, version 8 2+ included baseline mtRCamp1h mito-[Ca ] (μmol/L) in rest- (OriginLab Corp., Northampton, MA, USA). COX7RP and 2+ ing VCMs; peak mito-[Ca ] and the time to peak amplitude MCU signals were normalized to Hsp60 loading controls. (s) in VCMs undergoing periodic 1 Hz field stimulation [24]. List of antibodies used is present in Table 2. 2+ −1 The rate of mito-Ca decay (1/τ, s ) was derived from 2+ single exponential fit of VCM mito-[Ca ] decrease upon Western blot analysis of H9c2 cells cessation of pacing. H9c2 cells were plated in 6-well plates and infected with Measurements of mitochondrial matrix ROS COX7RP-FLAG and shRNA COX7RP adeno-viruses. At 48 h after infection, cells were lysed in lysis buffer from Cell The ratiometric MLS-HyPer7 probe was excited using Signaling (Cat # 9803S), supplemented with phosphatase 405 nm and 488 nm laser lines. Fluorescence emission was (Calbiochem, Cat#524,625) and protease inhibitor cocktails collected at 520–540 nm wavelengths, measured in the x–y (Sigma, Cat#P8340) as described previously [50]. Samples mode at 400 Hz sampling rate. Minimum fluorescence was were run on 4–20% TGX gels, transferred to nitrocellulose obtained by application of dithiothreitol (DTT, 5 mmol/L), membrane, and probed with anti-COX7a2L (COX7RP) anti- and maximum fluorescence was obtained by application of bodies from Sigma. Hsp60 was used as loading control (Cell 2,2′-dithiodipyridine (DTDP, 200 µmol/L). Data are pre- Signaling). sented as a percentage of ΔF/ΔFmax where ΔF = F − Fmin and ΔFmax = Fmax − Fmin [24]. RyR2 immunoprecipitation and immunoblotting Western blot analysis of cardiac mitochondrial For immunoprecipitation of RyR2, cultured rat VCMs proteins infected with Adv-COX7RP and Adv-shRNA COX7RP for 48 h were lysed using cell lysis buffer from Cell Signal- Tissue samples or cultured rat VCMs were lysed in lysis ing (catalogue no. 9803S), supplemented with phosphatase buffer from Cell Signaling (catalogue no. 9803S), supple- (Calbiochem; catalogue no. 524625) and protease inhibi- mented with phosphatase (Calbiochem, San Diego, CA, tor cocktails (Sigma; catalogue no. P8340). An overnight Table 2 Antibodies used in this Antibody/kit Species of used sample Source Identifier study Anti-COX7a2L H9C2,Rat and Human MilliporeSigma Cat# SAB1303595 Anti-RyR2 Rat and Human Alomone Cat# ARR-002 Anti-MCU Rat and Human MilliporeSigma Cat# HPA016480 Anti-COX IV Rat and Human Abcam Cat# ab16056 Anti-UQCRFS1 Rat and Human Abcam Cat# ab14746 Anti-NDUFA9 Rat and Human Abcam Cat# ab14713 Anti-Hsp60 H9C2, Rat and Human Cell Signaling Cat# 12165T Anti-GAPDH H9C2, Rat and Human Abcam Cat# ab8245 Anti-Rabbit IgG(H+L),HRP H9C2, Rat and Human Promega Cat# W4011 Anti-Mouse IgG(H+L),HRP H9C2, Rat and Human Promega Cat# W4021 Oxidized Protein Western blot kit Rat Abcam Cat# ab178020 Anti-MnSOD Rat Millipore Cat # 06-984 Anti-Peroxiredoxin 5 Rat Abcam Cat # ab180123 1 3 15 Page 6 of 22 Basic Research in Cardiology (2023) 118:15 immunoprecipitation of RyR2 was performed at 4 °C using III—anti-UQCRFS1; Complex IV—anti-COX IV; all from the Catch and Release v2.0 Kit (Millipore; catalogue no. Abcam. Hsp60 was used as loading control. Anti-Hsp60 17‐500) in accordance with the manufacturer’s instruc- antibodies were from Cell Signaling. The secondary anti- tions using anti‐RyR2 antibody (Alomone catalogue no. bodies used were goat anti‐mouse and goat anti‐rabbit ones ARR-002, 5 μg of antibody) and a negative control anti- (Promega, Madison, WI, USA). Blots were developed with body comprising normal rabbit IgG (Millipore; catalogue ECL (Bio-Rad Laboratories) and quantified using Image J no. 12-370, 5 μg of antibody). Samples were analyzed by (US National Institutes of Health) and Origin 8 software. immunoblotting. To determine oxidation status of RyR2, the Oxidized Oxygen consumption measurements Protein Western Blot Kit (Abcam catalogue no. ab178020) was used, whereby carbonyl groups of immunoprecipitated To assess mitochondrial function, oxygen consumption rates RyR2 were derivatized to 2,4 dinitrophenylhydrazone (DNP) (OCR) of H9c2 cells infected with Adv-COX7RP-FLAG by reaction with 2,4 dinitrophenylhydrazine [23]. For con- and Adv-shRNA COX7RP were measured by the Agilent trol, we used Derivatization Control Solution included in Seahorse XFe96 extracellular flux analyzer (Seahorse Bio- the kit. The DNP-RyR2 protein samples were separated on science, North Billerica, MA). Briefly, cells were seeded in 4–20% Mini-PROTEAN TGX gels (Bio-Rad Laboratories, XFe96 cell culture plates at 10,000 cells/well in standard Cat#456-1094) and DNP-associated signal was assessed DMEM + 10% FBS medium and placed in a 37 °C incubator by the kit-provided anti-DNP rabbit primary antibody and with 5% CO Adv-shRNA COX7RP was used for infection anti-RyR2, followed by HRP-conjugated anti-rabbit goat on day one and Adv-COX7RP on day 2. On day 3, cells lgG(H+L), secondary antibody. were washed twice with Seahorse assay medium (XF base Blots were developed with ECL (Bio-Rad Laboratories) medium supplemented with 10 mmol/L glucose, 2 mmol/L and quantified using Image J (US National Institutes of glutamine, and 1 mmol/L sodium pyruvate; pH 7.4) and Health, Bethesda, MD, USA) and Origin 8 software. incubated in a 37 °C incubator without CO for 1 h. Oxy- gen consumption rate (OCR) was measured according to Blue native PAGE manufacturer’s guidelines of the XF Cell Mito Stress Test kit (Agilent). Oligomycin A (1 μmol/L final concentration), For the assessment of native protein complexes using blue FCCP (1.5 μmol/L), and a combination of rotenone and anti- native polyacrylamide gel electrophoresis (BN‐PAGE) [20], mycin A (1 μmol/L each) were injected on cells as indicated. we used mitochondrial fractions isolated from LV heart tis- After the assay, cells were stained with Hoechst dye and sues suspended in buffer containing 225 mmol/L manni- nuclei counted on a Cytation 5 cell imaging plate reader tol, 70 mmol/L sucrose, 10 mmol/L HEPES, and 1 mmol/L (Agilent) to normalize results. Data are expressed as pmol EGTA (pH 7.4) [31]. The tissue was placed in a pre‐cooled O /min/100 cells. Plate location was randomized between 5 mL Wheaton™ Potter‐Elveheim Tissue Grinder (Fisher groups in each assay. Wells with no response to oligomycin, Scientific, Hampton, NH, USA; catalogue no. 22‐ 290067) FCCP, or rotenone/antimycin were removed from analysis and homogenized. Tissue homogenate was centrifuged at based on the assumption of premature injection/leak of 700g for 10 min. The pellet consisting of nuclei and cell assay drugs into the well from upper plate reservoir, which debris was discarded and the supernatant was then cen- confounds interpretation. There were no differences in the trifuged at 20,000g for 15 min. The pellet was considered proportion of these wells between groups. as mitochondrial enriched fraction. Samples were then solubilized using the mild detergent digitonin (1%) from Isolated heart oxygen consumption measurements NativePAGE™ Sample Prep Kit (Invitrogen; catalogue no. BN2008) in accordance with the manufacturer's instruc- Male and female rats of equal age and approximately tions. To demonstrate changes in association of supercom- 300–500 g were heparinized, anesthetized with 3% isoflu- plexes in VCMs infected with adenoviruses, cells were rane and heart and lungs removed en bloc following ster- lysed in digitonin (1%) for 30 min on ice, and centrifuged notomy. All procedures were approved by the Providence at 20,000g for 30 min. Processed samples were resolved VAMC IACUC committee. Hearts were rapidly placed in on NativePAGE™ 4–16% Bis‐Tris protein gels, 1.0 mm, ice-cold Krebs buffer, the aorta cannulated, and retrograde 15‐well (Invitrogen; catalogue no. BN2011B10) in Native- flow initiated with oxygenated Krebs buffer (in mmol/L: Page cathode and anode buffers electrophoresed at 150 V 118.00 NaCl, 4.70 KCl, 1.40 CaCl , 1.70 MgSO , 24.88 2 4 for 1.5. followed by 250 V for 2 h. Samples were trans- NaHCO , 6.00 glucose, 1.20 KH PO , and 2.00 Na pyru- 3 2 4 ferred onto nitrocellulose membranes before being probed vate) to wash blood and supply oxygen to the heart during with antibodies, as described above. The primary antibodies working heart preparation. Pulmonary veins were ligated, used were as follows: Complex I—anti-NDUFA9; Complex the lungs removed under a microscope and the heart rapidly 1 3 Basic Research in Cardiology (2023) 118:15 Page 7 of 22 15 moved to working heart apparatus (IHS5, Hugo Sachs Elek- as mean ± SEM. P values are provided with two significant tronik, March, Germany). Hearts were then slowly increased figures, where < 0.050 were considered significant (*). For to a constant perfusion pressure of 70 mmHg over 5 min. experiments involving multiple VCMs (data points) isolated A cannula was inserted into the left atria and a pressure from one individual rat heart, a hierarchical level random catheter inserted into the LV through the base of the heart intercept model was used [47]. The model tests for data with care to avoid large coronary vessels. A small catheter clustering for each cell isolation and adjusts for any cluster- attached to an in-line optical O sensor (PreSens Precision ing with significance testing. Posthoc pairwise comparisons Testing, Regensburg, Germany) and withdrawal syringe were carried out for hierarchical testing. No experiment- pump was placed in the pulmonary artery to continuously wide/across-test multiple test correction was applied; only monitor O tension and temperature of coronary effluent. within-test corrections were made. Statistical tests used for An additional in-line O sensor monitored O and tempera- each dataset are presented in Table 3. 2 2 ture of perfusate. Hearts were switched to working mode via opening gravity flow of oxygenated Krebs buffer to the left atria (preload set to 10 mmHg). Langendorff perfusion was reduced and hearts allowed to equilibrate for 5 min before Results O consumption measurements. Coronary flow was assessed 2+ via eluent collection over 1 min. O consumption was nor- Increased spontaneous SR Ca release in rat male malized to heart weight and coronary flow and calculated VCMs vs females under β‑adrenergic stimulation according to Neely et al. [41]. Beta-adrenergic stimulation increases global pro-arrhyth- 2+ Glutathione GSH/GSSG measurement mic spontaneous Ca release events, SCWs [9]. In order to 2+ assess potential differences in intracellular Ca homeosta- 2+ Tissue total GSH and GSSG in rat left ventricles were meas- sis, male and female VCMs were loaded with Ca indicator ured using assay kit from Sigma-Aldrich (Cat MAK440) Fluo-3AM and treated with 50 nmol/L β-adrenergic agonist [34]. Briefly, stored tissue chunks were weighed and isoproterenol (ISO). After 5 min incubation in ISO, VCMs homogenates were prepared using Bullet Blender Strom 24 were subjected to field stimulation at 1 Hz for 5 min at room 2+ (Next Advance Inc, NY) in PBS, pH 7.4, 1 mmol/L EDTA temperature. Ca transients and SCWs were recorded using with or without scavenger (provided with the kit) for the a Leica SP8 confocal microscope in line scan mode. Rep- measurement of GSSG and total GSH respectively. All the resentative cell-averaged fluorescence profile traces (male 2+ samples were deproteinated with 5% meta-phosphoric acid. black, female red) are presented in Fig. 1A. Stimulated Ca The clarified and diluted samples were mixed with work - transients are indicated with arrows, while spontaneous 2+ ing reagent and spectrophotometric reading was obtained at global SR Ca releases are denoted with asterisks. Analysis 2+ 412 nm at 0 and 10 min using SpectraMax 190 microplate of Ca transient amplitudes expressed as ΔF/F , where F is 0 0 reader (Molecular Devices, CA). Total GSH, reduced GSH, basal fluorescence and ΔF = F −  F , revealed no difference 2+ and GSSG were calculated following assay protocol. between sexes (Fig. 1B left panel). Ca transient decay in male VCMs showed small but significant slowing in com- Data presentation and statistics parison to females, indicative of reduced SERCA2a activity (Fig. 1B center panel). Furthermore, male VCMs exhibited Group sample sizes were determined by power analysis shorter latency of SCW initiation vs females (Fig. 1B right based on our previous experience [20] performing two-tailed panel). Latency, a well-established readout of RyR2-medi- 2+ Student’s t-test to provide adequate power to detect 20% dif- ated SR Ca release refractoriness [4, 11], was measured as 2+ ference, assuming power of 80% (β = 0.80) and an α of 0.05. time interval from the start of the last stimulated Ca tran- No animals or samples were excluded from the study. sient in the train to the initiation point of the first SCW. Total 2+ Animals were grouped with no blinding, but randomized in SR Ca content was measured by rapid 10 mmol/L caffeine cellular experiments. Data acquisition and analysis were not application at the end of the experiment (Fig. 1C). As shown 2+ fully blinded as the same person carried out these processes. in Fig. 1D, SR Ca content was significantly higher in ISO- Representative traces for cellular imaging experiments treated female rat VCMs in comparison to males. The decay and representative western blot images were chosen to rates of the caffeine transient were not different between + 2+ closely match the mean for the parameters assessed. Sta- male and female VCMs, indicative of unchanged Na /Ca tistical analyses were performed using Origin 2020Pro (NCX1) exchanger activity. Taken together, these data sug- (OriginLab) and R software [20, 47]. For each experiment, gest that SERCA2a activity is reduced, while the activity of 2+ the number of animals (uppercase n [N]) and the number the SR Ca release channel RyR2 is significantly higher in of VCMs (lowercase n) used is indicated. Data is expressed male VCMs than in females under β-adrenergic stimulation. 1 3 15 Page 8 of 22 Basic Research in Cardiology (2023) 118:15 Table 3 Statistical tests used in this study Figure Type of sample Statistical test applied 1B One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 1D One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 2D One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 3B-F One rat per N, multiple cells (n) used per image per rat Three-level random intercept model with Tukey’s posthoc 4B LV tissue, one rat heart per N Two-sample Student’s t test 4C Ventricular tissue, one rat heart per N Two-sample Student’s t test 5B One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 5D One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 6C One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 6E LV tissue, one heart (rat/human) per N Two-sample Student’s t test 7B One rat per N, multiple cells (n) used per rat Transient amplitude/decay/latency—two-level random intercept model with Tukey’s posthoc 2+ Ca waves—Fisher’s exact test 7E One rat per N, multiple cells (n) used per rat One Way ANOVA with Tukey’s posthoc 8C One rat heart per N Two-sample Student’s t test 8D One rat heart per N Two-sample Student’s t test 8E One rat heart per N Two-sample Student’s t test 9A LV tissue, one rat heart per N Two-sample Student’s t test 9B LV tissue, one human heart per N Two-sample Student’s t test 9C LV tissue, one rat heart per N Two-sample Student’s t test 9D LV tissue, one human heart per N Two-sample Student’s t test 10B LV tissue, one human heart per N Two-sample Student’s t test 10D LV tissue, one rat heart per N Two-sample Student’s t test 10F LV tissue, one rat heart per N Two-sample Student’s t test 11C H9c2 cells, one independent experiment per N One way ANOVA with Tukey’s postdoc 12B Isolated VMs from one rat heart per N Two-sample Student’s t test 12D Isolated VMs from one rat heart per N Two-sample Student’s t test 13A Isolated VMs from one rat heart per N Two-sample Student’s t test 13B Isolated VMs from one rat heart per N Two-sample Student’s t test 13C One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 13D One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 14B One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 14E Isolated VMs from one rat heart per N Two-sample Student’s t test MLS-HyPer7 fluorescence ratio (male black, female red). Decreased mito‑ROS levels in female VCMs vs males After 5 min incubation in 50 nmol/L ISO, VCMs were field- under β‑adrenergic stimulation stimulated for 5 min before application of DTT and DTDP. As shown in Fig.  2D, male ISO-treated VCMs exhibit a Increased activity of RyR2 and decreased SERCA2a activity higher MLS-HyPer7 signal than female myocytes both may be explained by mito-ROS-mediated oxidation of these before and during field stimulation. Of note, it was previ- proteins [19]. To test this possibility, we expressed the dual ously shown using TEM that mitochondria content and size excitation wavelength ROS sensor MLS-HyPer7 targeted differ in VCMs from male and female mouse hearts [30]. to the mitochondrial matrix in VCMs [24]. Correct MLS- However, our TEM experiments in rat hearts showed no sig- HyPer7 localization was confirmed by co-staining VCMs nificant differences between sexes (Fig.  3). Therefore, sex with mitochondria-specific dye Mitotracker Red (Fig.  2A). differences in MLS-HyPer7 fluorescence cannot be ascribed The MLS-HyPer7 signal was normalized to minimal u fl ores - to differences in mitochondria content in rat VCMs. In addi- cence obtained by application of 5 mmol/L ROS scavenger tion, as the probe is ratiometric as well as normalized to DTT, and maximum fluorescence obtained by treatment of maxima and minima, there is minimal concern for sex- cells with 200 μmol/L DTDP, an oxidizing agent (Fig. 2B). dependent expression differences. There is a possibility that Figure 2C demonstrates representative normalized traces of 1 3 Basic Research in Cardiology (2023) 118:15 Page 9 of 22 15 2+ Fig. 1 Intracellular Ca handling is divergent in healthy male and female ventricular myocytes. A Fluo-3 fluores- cence (F/F ) profiles of isopro- terenol treated (ISO, 50 nmol/L) rat ventricular cardiomyocytes (VCMs) undergoing 1 Hz pace-pause protocol. Arrows depict field stimulation-evoked 2+ Ca transients, stars show 2+ proarrhythmic diastolic Ca 2+ waves. B Mean ± SEM Ca transient amplitude (ΔF/F ) −1 and decay rate (s ), n = 90 male (M) and n = 90 female (F) VCMs; and spontaneous 2+ Ca wave (SCW) latency (s), n = 62 M and n = 59 F VCMs. N = 11–12 M and N = 11–13 F animals. C Representative 2+ traces of caffeine-induced Ca transients (10 mmol/L). D Mean ± SEM caffeine transient amplitude (ΔF/F ) and decay −1 rate (s ), n = 69 M, n = 75 F VCMs, N = 11 M, N = 13 F animals. *p < 0.05, p values were calculated using two-level random intercept model heart antioxidant defenses differ between sexes as shown in the major role of differential mito-ROS production in sex 2+ other tissues [13]. To test this we performed western blot dimorphism in intracellular Ca homeostasis in VCMs analysis of mitochondrial superoxide dismutase (MnSOD) under β-adrenergic stimulation. and Peroxiredoxin-5 (PRXD5) and found no differences in 2+ samples from male and female rat cardiac tissues (Fig. 4A, Decreased mito‑Ca levels in female VCMs vs males B). Furthermore, we did not find statistically significant dif- under β‑adrenergic stimulation ference in GSH/GSSG ratio (Fig. 4C), indicating that there are no sex-dependent differences in antioxidant capacity. We next tested potential differences between sexes in 2+ 2+ Taken together, these data are in line with previous reports mito-[Ca ], given that changes in matrix [Ca ] are often that male VCMs exhibit significantly higher mito-ROS pro- associated with changes in mito-ROS production [24, 36, 2+ duction than female VCMs [14, 51]. 48]. Using the matrix-targeted Ca probe mtRCamp1h 2+ To test if increased mito-ROS plays a significant role in we determined that mitochondrial [Ca ] is significantly 2+ SR Ca release destabilization we performed experiments lower in field-stimulated female VCMs pretreated with treating male rat VCMs with MitoTEMPO, a mitochondria 50 nmol/L ISO for 5 min (Fig. 6A–C). Correct mitochon- specific ROS scavenger (20  μmol/L, 20  min preincuba- drial localization of mtRCamp1h was confirmed in VCMs tion [24]). As depicted in Fig. 5, MitoTEMPO significantly co-expressing mito-targeted GFP. As seen in Fig. 6A, red 2+ attenuated SCWs, accelerated Ca transient decay, and mtRCamp1h fluorescent signal overlaps with green signal 2+ 2+ increased SR Ca load in periodically paced male VCMs of mito-GFP. Figure 6B shows representative mito-Ca treated with ISO (50 nmol/L, 5 min). These results highlight 1 3 15 Page 10 of 22 Basic Research in Cardiology (2023) 118:15 Fig. 2 Matrix ROS biosensor MLS-HyPer7 reveals increased mito- in male (M) and female (F) VCMs, respectively. Myocytes were ROS in male vs. female ventricular myocytes. A Mitochondrial MLS- treated with isoproterenol (ISO, 50  nmol/L) and paced at 1  Hz for Hyper-7 localization in live ventricular cardiomyocytes (VCMs) vali- 5  min. Fluorescence was normalized to minimum (DTT, 5  mmol/L) dated by Mitotracker. B Representative images of VCM infected with and maximum (DTDP, 200  µmol/L) fluorescence. D Mean ± SEM MLS-HyPer7, and treated with DTT (5 mmol/L) followed by DTDP MLS-HyPer7 fluorescence, n = 37 M and n = 77 F VCMs, N = 11 M, (200 µmol/L) to achieve minimum and maximum fluorescence, dem- N = 11 F animals. *p < 0.05, p values were calculated using two-level onstrating probe sensitivity. C Representative MLS-HyPer-7 recorded random intercept model 2+ traces from male (black) and female (red) VCMs derived Aging abolishes sex differences in intracellular Ca from mRCamp1h signal. To convert mtRCamp1h fluores- homeostasis and mito‑ROS production 2+ cence into [Ca ], cells were permeabilized with saponin 2+ at the end of recording and exposed to 2 mmol/L Ca Using young and old VCMs from female rabbit hearts we 2+ buffer EGTA to obtain Fmin followed by application previously showed that disturbances in Ca handling are 2+ 20 μmol/L [Ca ] to obtain Fmax. Although no significant governed by increased mito-ROS production [15]. Here we 2+ 2+ changes in mito-[Ca ] were detected at rest, field stimu- tested potential sex differences in Ca cycling in VCMs from lation of VCMs at 1 Hz revealed that in female VCMs it 22-month-old F344 rats obtained from NIH aged rat colony. 2+ 2+ takes significantly longer to reach maximum mito-[Ca ], As demonstrated in Fig. 7, we find no differences in Ca tran- 2+ and that this maximum concentration is significantly lower sient amplitude and decay rate, as well as SR Ca content than in male VCMs (Fig. 6C). Importantly, western blot and propensity to generate pro-arrhythmic SCWs (Fig. 7A–D). analysis revealed significantly lower expression levels of This was accompanied by similar rates of mito-ROS produc- MCU in rat female VCMs vs males, which could poten- tion in old male and female VCMs (Fig.  7E), which is in 2+ tially explain why matrix Ca is lower (Fig. 6D). Nota- striking contrast to young cells (Fig. 6B). Of note, the sex 2+ bly, lower MCU levels were detected in female human differences in mito-Ca uptake and removal were preserved LV donor heart (≤ 52 year old) samples vs males as well with aging (Fig. 7E), suggesting that the main source of age- 2+ (Fig. 6E). Given that mito-[Ca ], in addition to uptake, related mito-ROS increase in females is not related to mito- + 2+ 2+ is determined by removal via mitochondrial Na /Ca Ca mishandling. exchanger [17], we assessed the rate of removal by meas- 2+ uring mito-Ca decay upon cessation of pacing. Of note, 2+ the rate of Ca removal from mitochondrial matrix was significantly faster in females. 1 3 Basic Research in Cardiology (2023) 118:15 Page 11 of 22 15 Fig. 3 Absence of sex‐related differences in mitochondrial counts and microscopy. B–F Mitochondrial morphometric parameters, including morphology in rat heart. A Mitochondrial density assessed by trans- mito area (B), perimeter (C), aspect ratio (D), form factor and density mission electron microscopy (TEM) in the left ventricular cardiac (% of the cell area, F), in male (M) and female (F) rat hearts. The tissue from male (M) and female (F) rats. Original magnification is analysis was performed randomly from 60 M (30 images), 60 F (21 × 3200 and the white bars represent 2 μm scale. Heart were fixed with images) cardiac mitochondria from three animals per group and were 2.5% glutaraldehyde solution then processed for transmission electron analyzed by three-level random intercept model LV tissues. Importantly, experiments using LV tissues from Female mitochondria exhibit higher levels of ETC healthy human donor hearts exhibited a similar pattern supercomplexes and COX7RP (Fig. 9B). Notably, the risk of CVD and cardiac arrhythmia dramatically increases in postmenopausal females match- Differences in cardiac respiratory capacity between sexes ing or even outpacing males [52], suggesting that hormonal has not been previously described [29, 30]. In line with this, changes may play a role in these processes. Of note, expres- our measurements of oxygen consumption in Langendorff- sion of COX7RP, a protein critically involved in supercom- perfused working heart preparations showed no difference plex formation, is estrogen dependent [33, 53]. We tested in myocardial O consumption between males and females potential sex differences in COX7RP expression levels and (Fig. 8). Therefore, we surmised that a compensatory mecha- 2+ found that they are significantly higher in females than males nism must exist to offset the effects of lower matrix [Ca ] both in rat and human hearts from heathy donors (Fig. 9C, in females vs males. One plausible mechanism to explain D). Furthermore, using samples from young and old female this discrepancy could include enhanced incorporation of human hearts we confirmed that COX7RP levels decrease individual ETC complexes into ETC supercomplexes to with aging in contrast to MCU (Fig. 10A, B). Experiments increase electron transport efficiency [3 , 12]. Indeed, as using sham and ovariectomized (OVX) female rats showed depicted in Fig. 9A, Blue Native Gel Electrophoresis (BN- an identical expression pattern (Fig. 10C, D). In addition to PAGE) of isolated mitochondrial proteins showed increased a decrease in COX7RP levels, we found that supercomplexes high molecular weight assemblies consisting of comigrating are significantly reduced in OVX LV tissue samples as well subunits for Complex I, III and IV in rat female vs male 1 3 15 Page 12 of 22 Basic Research in Cardiology (2023) 118:15 Fig. 4 Anti-oxidant defenses in male and female left ventricular car- as loading control. Mean ± SEM, N = 4 M and N = 4 F rat LV sam- diac tissues. A, B Representative western blots and pooled data for ples; *p < 0.05, Student’s t-test. C Total tissue GSH, reduced GSH, mitochondrial superoxide dismutase Mn-SOD and peroxiredoxin 5 oxidized GSH (GSSG), and the ratio of GSH/GSSG was measured (PRDX5) normalized optical density from male (M) and female (F) from male and female rat ventricles. Mean + SEM, N = 7 M and N = 6 rat left ventricular (LV) tissue samples. GAPDH and Hsp60 was used F rat, *p < 0.05, Student’s t-test (Fig. 10E, F). These data support the key role of estrogen COX7RP were subjected to BN-PAGE analysis to test dependent COX7RP expression in supercomplex assembly. potential COX7RP influence on ETC supercomplex assem- bly (Fig. 12C, D). Importantly, COX7RP KD significantly COX7RP regulates mitochondria oxygen reduced supercomplexes in female VCMs, while COX7RP consumption and supercomplexes overexpression increased them in VCMs from males. These data further corroborates the key role of COX7RP in mito- In order to modify COX7RP expression levels, we utilized chondrial ETC supercomplex assembly in cardiomyocytes. adenoviral vectors carrying human COX7RP-FLAG and Importantly, altering COX7RP levels did not change MCU 2+ 2+ shRNA COX7RP sequences. Efficiency of adenovirus- expression levels, mito-Ca uptake and peak [Ca ] in male mediated COX7RP overexpression and shRNA-COX7RP and female rat VCMs (Fig. 13). knockdown (KD) was confirmed in H9c2 cells 48 h after 2+ transduction with western blot (Fig. 11A). As seen in repre- COX7RP regulates SR Ca release, mito‑ROS sentative western blot, FLAG-tagged COX7RP runs slower production and RyR2 oxidation in VCMs on the gel than wild type COX7RP. Mitochondrial protein Hsp60 was used as loading control. Next, we performed Next, we tested the effects of adenoviral-mediated measurement of oxygen consumption in H9c2 cells with a COX7RP-FLAG overexpression in male VCMs and Seahorse Analyzer in cells expressing shRNA-COX7RP or shRNA-mediated KD in females treated with ISO COX7RP. Averaged traces are presented in Fig.  11B. As (50 nmol/L, 5 min) on RyR2-mediated spontaneous SR 2+ seen in Fig. 11C, COX7RP overexpression increases basal, Ca release. Representative line scan images and cor- maximum and ATP-linked respiration in H9c2 cells, while responding Fluo-3 fluorescence time profiles are depicted COX7RP KD reduces it. Next, viral constructs were tested in Fig. 14A. The latency of SCWs appearing after cessa- in cultured rat VCMs. COX7RP levels were increased ~ 50% tion of 1 Hz field-stimulation was significantly lengthened in male VCMs 48 h after transduction (Fig. 12A, B). Expres- in male VCMs overexpressing COX7RP, while COX7RP sion of shRNA COX7RP (shC) led to ~ twofold reduction of KD significantly shortened it (Fig.  14A, B). Importantly, the protein in female VCMs 48 h after infection (Fig. 12A, parallel experiments in VCMs coexpressing mito-ROS B). Next, samples from cultured male VCMs expressing sensor MLS-HyPer7 subjected to the same experimental COX7RP-FLAG and female VCMs expressing shRNA protocol showed significant mito-ROS reduction in male 1 3 Basic Research in Cardiology (2023) 118:15 Page 13 of 22 15 Fig. 5 Mitochondrial ROS scavenging reduces spontaneous 2+ Ca release is male ventricular myocytes. A Fluo-3 fluores- cence (F/F ) profiles of isopro- terenol treated (ISO, 50 nmol/L) rat ventricular cardiomyocytes (VCMs) undergoing 1 Hz pace- pause protocol. Arrows depict 2+ field stimulation-evoked Ca transients, stars show proar- 2+ rhythmic diastolic Ca waves. Pretreatment with MitoTEMPO, a mitochondria-specific ROS scavenger (20 mmol/L, 8 min) 2+ reduces spontaneous Ca 2+ waves. B Mean ± SEM Ca transient amplitude (ΔF/F ) and −1 decay rate (s ), n = 26 male (M) and n = 37 MitoTEMPO treated male (M + MT) VCMs; 2+ and spontaneous Ca wave (SCW) latency (s), n = 23 M and n = 25 M + MT VCMs. N = 4 M and N = 4 M + MT ani- mals. C Representative traces of 2+ caffeine-induced Ca transients (10 mmol/L). D Mean ± SEM caffeine transient amplitude −1 (ΔF/F ) and decay rate (s ), n = 11 M, n = 15 M + MT VCMs, N = 4 M, N = 4 M + MT animals. *p < 0.05, p values were calculated using two-level random intercept model COX7RP-overexpressing VCMs, while mito-ROS in Discussion female shRNA-COX7RP-expressing VCMs was signifi- cantly increased (Fig.  14C). We then assessed possible In the present study we investigated the mechanisms underly- COX7RP expression-dependent changes in RyR2 oxida- ing reduced mito-ROS production in female VCMs. We have tion state. Control and COX7RP-overexpressing male and shown that male rat VCMs exhibit higher propensity to pro- 2+ shRNA COX7RP-expressing female VCMs were exposed arrhythmic diastolic SR Ca release governed by hyperactive to 50 nmol/L ISO pretreatment and then paced at 1 Hz for RyR2s due to oxidation by mito-ROS. We found that lower 5 min in the presence of ISO. Then cells were immediately mito-ROS production in females is accompanied by lower 2+ collected for RyR2 immunoprecipitation. The relative mito-[Ca ] and heightened ETC supercomplex assembly. changes in immunoprecipitated RyR2s oxidized cysteine We found the more efficient electron transport in females is content were assessed using anti-DNP antibodies [23]. As likely related to increased expression levels of the estrogen- shown in Fig. 14D, COX7RP overexpression significantly dependent mitochondrial assembly protein COX7RP. reduces RyR2 oxidation in male VCMs, while COX7RP KD in female VCMs increases it. To summarize, COX7RP KD increased RyR2 oxidation, mito-ROS, and profoundly 2+ disturbed SR Ca release in female VCMs promoting gen- eration of pro-arrhythmic SCWs, while COX7RP overex- pression led to opposite results in males. 1 3 15 Page 14 of 22 Basic Research in Cardiology (2023) 118:15 2+ 2+ 2+ 2+ Fig. 6 Female ventricular myocytes have reduced Ca levels in matrix [Ca ], and peak matrix [Ca ], time to peak matrix [Ca ], mitochondria matrix vs. males. A Representative confocal images and decay rate during pacing, n = 13  M and n = 15 F VMs. N = 8  M of a ventricular myocyte (VCM) infected with matrix-targeted mtR- and 8 F animals. *p < 0.05, p values were calculated using two-level Camp1h (Kd ~ 1.3 mmol/L) and mito-GFP, with a merged figure dem- random intercept model. D, E Representative western blots and onstrating correct probe localization. B Representative time course pooled data for COX7RP normalized optical density in rat and human of mtRCamp1h fluorescence recorded in male (M) and female (F) left ventricular (LV) tissue samples. Mean ± SEM, N = 8 M and N = 8 VCMs. Myocytes were treated with isoproterenol (ISO, 50  nmol/L) F rat; and N = 4 M and N = 4 F human samples. *p < 0.05, Student’s for 3  min before pacing at 1  Hz for 5  min. C Mean ± SEM baseline t-test 2+ −1 Fig. 7 Intracellular Ca handling is similar in male and female ven- feine transient amplitude (ΔF/F ) and decay rate (s ), n = 7 M and tricular myocytes from aged (22  month old) hearts. A Fluo-4 fluo- F VMs, N = 4 M and F animals. p values were calculated using two- rescence (F/F ) profiles of isoproterenol treated (ISO, 50 nmol/L) rat level random intercept model except where indicated. E Aged male 2+ ventricular myocytes (VCMs) paced at 1  Hz. B Mean ± SEM Ca (M) and female (F) VCMs exhibit similar mito-ROS levels, while −1 2+ transient amplitude (ΔF/F ) and decay rate (s ), n = 23 male (M) and sexual dimorphism in matrix [Ca ] preserved with aging. VCMs 2+ n = 25 female (F) VCMs; proportion of cells displaying Ca waves were passed at 1  Hz for 5  min in the presence of 50  nmol/L ISO, 2+ following 2 Hz pacing and distribution of latency to Ca waves (s), Mean ± SEM, N = 2–4 M, 2–4 F, n = 6–20, *p < 0.05, p values were n = 33 for M and n = 33 for F, N = 4 M and F animals. C Representa- calculated using one way ANOVA 2+ tive traces of caffeine-induced Ca transients. D Mean ± SEM caf- Recent studies using mouse models with loss of MCU Increased mito‑ROS and unstable intracellular 2+ 2+ RyR2‑mediated Ca cycling in male vs female VCMs function revealed that SR Ca release is directly coupled to mitochondrial metabolic output in stress conditions [38, during β‑adrenergic stimulation 1 3 Basic Research in Cardiology (2023) 118:15 Page 15 of 22 15 Fig. 8 Oxygen consumption is similar in male and female work- Quantitation of male and female isolated rat heart myocardial O con- ing heart preparations. A Picture of rat working heart preparation. B sumption N = 3 males and N = 3 females. p values were calculated Representative left ventricular (LVP), aortic and atrial pressure traces using Student’s t-test (mmHg) and LVP dP/dt of isolated rat heart in working mode. C–E Fig. 9 Female left ventricular tissues demonstrate increased mito- black bars. Mean ± SEM, N = 8 M and N = 8 F rat LV samples; and chondrial supercomplex formation and expression of COX7RP N = 6 M and N = 4 F human LV samples, *p < 0.05, Student’s t-test. compared to males. A, B Representative BN-PAGE images of ETC C, D Representative western blots and pooled data for COX7RP nor- complexes from male (M) and female (F) rat (A) and human (B) left malized optical density in rat (C) and human (D) LV tissue samples. ventricular (LV) tissue samples. Antibodies used: Complex I—anti- Hsp60 was used as loading control (inset, Anti-Hsp60 from Cell NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti-COX Signaling). Mean ± SEM, N = 8 M and N = 8 F rat samples; and N = 4 IV all from Abcam. Hsp60 was used as loading control (inset, Anti- M and N = 4 F human samples, *p < 0.05, Student’s t-test Hsp60 from Cell Signaling). Supercomplexes (SCs) are indicated by 1 3 15 Page 16 of 22 Basic Research in Cardiology (2023) 118:15 Fig. 10 Reduced COX7RP levels in LV heart tissues from old female Sham and ovariectomised (OVX) rats. Hsp60 was used as loading donors and ovariectomized female rats. A, B Representative west- control (Cell Signaling). E Representative BN-PAGE images of ETC ern blots and pooled data for COX7RP and MCU normalized opti- complexes from rat LV tissue samples. Antibodies used: Complex I— cal density in left ventricular (LV) tissue samples from young (less anti-NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti- than 35  year old, N = 10) and old (more than 60  years old, N = 10) COX IV; all from Abcam. Hsp60 was used as loading control (inset, female donor hearts. Mean ± SEM, *p < 0.05, Student’s t-test. C, D Anti-Hsp60 from Cell Signaling). Supercomplexes (SCs) are indi- Representative western blots and pooled data for COX7RP and MCU cated by black bar. F Pooled normalized optical density data for (E). normalized optical density in left ventricular (LV) tissue samples Mean ± SEM, N = 5 Sham and N = 7 OVX, *p < 0.05, Student’s t-test Fig. 11 COX7RP modulates oxygen consumption in H9C2 myo- ings of oxygen consumption in H9C2 cells in control cells and cells blasts. A Representative western blot demonstrating efficiency of expressing COX7RP or COX7RP shRNA. C Pooled respirometry COX7RP overexpression and COX7RP shRNA constructs in H9C2 data, Mean ± SEM, n ≥ 18, N = 3 independent experiments, *p < 0.05, cells infected with adenoviruses for 48  h. Hsp60 and GAPDH were one way ANOVA used as loading controls. B Representative Seahorse Analyzer record- 2+ 2+ 45]. β-adrenergic stimulation increases SR Ca release SR Ca content in male VCMs. Furthermore, male VCMs 2+ amplitude to levels sufficient for MCU to transmit Ca into exhibited shorter latency of SCW initiation. Together, these 2+ the mitochondrial matrix [23], activating Ca -dependent data are a strong indication that RyR2 activity is higher in dehydrogenases in the Krebs Cycle and promoting ATP male than in female VCMs under β-adrenergic stimulation. 2+ production [21]. Thus, our main focus was to characterize Previous reports directly linked impaired SR Ca handling 2+ potential differences in Ca homeostasis in VCMs from in VCMs incubated with β-adrenergic agonists to increased female and male rat hearts in the presence of ISO (Fig. 1). RyR2 oxidation by mito-ROS resulting in increased channel Notably, although we did not find statistically significant dif- activity and high propensity to pro-arrhythmic SCWs [9, 15, 2+ ferences in Ca transient amplitudes in periodically stimu- 23]. In line with these findings, our measurements using the lated male and female VCMs, we saw a significantly lower mitochondria-targeted ROS biosensor MLS-HyPer-7 suggest 1 3 Basic Research in Cardiology (2023) 118:15 Page 17 of 22 15 Fig. 12 COX7RP expression levels regulate formation of mitochon- tion of multimolecular ETC supercomplexes in Ms, while COX7RP drial supercomplexes in rat ventricular myocytes. A, B Transfection shRNA decreases it in Fs. Antibodies used: Complex I—anti- of rat ventricular cardiomyocytes with Ad-COX7RP-FLAG (male NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti-COX (M)) and with shRNA COX7RP (female (F)) alters expression levels IV; anti-GAPDH all from Abcam. Hsp60 was used as loading control of COX7RP protein 48  h after infection (10 MOI). Hsp60 was used (inset, Anti-Hsp60 from Cell Signaling). Supercomplexes (SCs) are as loading control. B Pooled data for (A), Mean ± SEM, N = 4  M and indicated by black bars. D Pooled normalized optical density data for N = 5 F, *p < 0.05, Student’s t-test. C Representative BN-page experi- (C) mean ± SEM, N = 4  M and N = 5 F animals, *p < 0.05, Student’s ments demonstrating that COX7RP overexpression increases forma- t-test 2+ that sex differences in RyR2-mediated spontaneous SR Ca treatment of male VCMs with MitoTEMPO significantly 2+ 2+ release are indeed associated with different levels of mito- attenuated spontaneous SR Ca release, increased SR Ca 2+ ROS (Fig. 2). Importantly, RyR2 hyperoxidation has been load, and accelerated Ca transient decay (Fig. 5). Taken associated with reduced cardiac function and/or increased together, our data suggest that high mito-ROS in ISO-treated 2+ arrhythmic potential in various cardiac diseases including male VCMs markedly disturbs intracellular Ca homeo- heart failure [49], infarct [5, 10], diabetic cardiomyopathy stasis consistent with increased RyR2 activity and lower [7, 28, 37], age-related cardiac dysfunction [15, 18], and SERCA2a function. even inherited cardiac arrhythmia syndromes [23]. Increased 2+ mito-ROS production in VCMs from male hearts is expected Sexual dimorphism in mitochondria [Ca ] uptake to carry additional risk and may explain heightened protec- and ETC supercomplex assembly in the heart tion in healthy premenopausal females. 2+ Interestingly, the Ca transient decay in ISO-treated Mitochondria are increasingly acknowledged as a key tar- female VCMs was ~ 10% faster than males (Fig.  1A, B). get for sex differences in pathologies [51]. However, under - 2+ Changes in Ca transient decay rate could be primarily standing of exact mechanisms underlying sex differences ascribed to differences in SERCA2a expression and activity in mito-ROS production in health remains limited. Given [6]. It is not likely that there are differences in SERCA2a that ETC is the major source of ROS in mitochondria, expression levels between sexes [43]. However, SERCA2a increased ETC activity under conditions of higher workload activity can be modulated, i.e. lowered, by redox modifi - or stress is expected to accelerate ROS production [21, 26]. 2+ cation at cysteine 674 [44]. Slow C a transient decay in Indeed, we recently reported that MCU-mediated influx of 2+ male VCMs treated with ISO is consistent with reduced Ca into the mitochondrial matrix, which increases ETC SERCA2a activity under higher ROS levels. Importantly, activity by stimulating production of NADH and FAD , 1 3 15 Page 18 of 22 Basic Research in Cardiology (2023) 118:15 Fig. 13 Altering COX7RP expression levels does not affect MCU D Adenovirus-mediated changes in COX7RP expression levels do 2+ 2+ expression and mito-Ca uptake in male and female rat ventricu- not affect mitochondrial Ca uptake measured using mtRCamp1h 2+ lar myocytes. A, B Transfection of rat ventricular cardiomyocytes Ca biosensor in M and F VCMs. Mean ± SEM, n = 9 M and (VCMs) with Ad-COX7RP-FLAG (male (M)) and with shRNA n = 20 M + COX, N = 4 M; n = 27 F and n = 21 F + shC and N = 3 F, COX7RP (female (F)) does not change expression levels of MCU *p < 0.05, p values were calculated using two-level random intercept 48  h after infection (10 MOI). Hsp60 was used as loading control. model Mean ± SEM, N = 4 M and N = 3 F, *p < 0.05, Student’s t-test. C, 2+ is essential for mito-ROS production in VCMs challenged offset the effects of lower matrix [Ca ] in females vs males. 2+ 2+ with β-adrenergic agonist [23]. Importantly, mito-[Ca ] in We confirmed that despite lower mitochondrial [Ca ] in VCMs rapidly changes in response to changes in frequency female VCMs, female working hearts have the same myo- 2+ and amplitude of cytosolic Ca transients, directly link-cardial O consumption as male hearts when operating at ing contractile activity and mitochondria ATP production to similar work levels (Fig. 8). Mitochondrial supercomplex closely match varying metabolic demand with output. Our assembly is one plausible mechanism of respiratory com- 2+ experiments in VCMs expressing mito-[Ca ] biosensor pensation in females [14]. Furthermore, more compact ETC 2+ mRCamp1h (Fig. 6A) showed that mito-[Ca ] is drastically organization is expected to reduce emission of ROS due to lower in field-stimulated ISO-treated VCMs from female rat more efficient electron transport. Indeed, our BN-PAGE 2+ hearts vs males (Fig. 6B, C), despite similar Ca transient experiments demonstrate higher abundance of supercom- 2+ amplitudes (Fig. 1A, B). Previously it was reported that Ca plexes in LV tissues from female hearts in both rats and uptake is lower in isolated female rat cardiac mitochondria humans (Fig. 9A, B). The importance of supercomplexes than in males [2]. The authors suggested that this decrease in the heart is underscored by the fact that in certain heart could be ascribed to lower MCU activity. In support of this failure models, a reduction in supercomplexes was shown to notion, our western blot analysis clearly shows lower expres- be sufficient to reduce respiration capacity without detect- sion levels of MCU in mitochondria from female rat and able changes in individual complexes’ activities [46]. The human LV tissues vs males (Fig. 6D, E). Furthermore, our assembly of supercomplexes is governed by several proteins, 2+ data suggests that Ca removal from the matrix is more effi- including COX7RP that brings together Complex I, III, and cient in female mitochondria. Taken together, these results IV [3, 12]. The transcription of COX7RP is regulated in part suggest that female cardiac mitochondria are less reliant on by estrogen [33, 53], which prompted us to compare expres- 2+ [Ca ] accumulation to satisfy increased metabolic demand sion levels between sexes. Indeed, COX7RP was more abun- during high workload or stress. dant in rat and human female LV heart samples (Fig. 9C, Given that cardiac mitochondrial respiratory capacity is D). Notably, COX7RP decreased in aged vs young female not known to be grossly different between sexes [29, 30] and human LV heart tissues (Fig. 10A, B). Furthermore, ovariec- cardiac output is similar even at higher workloads [25, 27], tomy significantly reduced COX7RP and supercomplexes we surmised that a compensatory mechanism must exist to in female LV tissue samples (Fig. 10C, D). Of note, it was 1 3 Basic Research in Cardiology (2023) 118:15 Page 19 of 22 15 Fig. 14 COX7RP overexpression reduces spontaneous SR data for normalized fluorescence of mitochondria matrix ROS biosen- 2+ Ca release and mito-ROS in male ventricular myocytes under sor MLS-HyPer-7. Mean ± SEM, n = 29–42 VCMs, N = 6–9 animals, β-adrenergic stimulation. Conversely, COX7RP knock-down *p < 0.05, p values were calculated using two-level random intercept 2+ increases Ca waves and mito-ROS in females. A, B Representa- model. D Adenovirus–mediated expression of COX7RP in males 2+ 2+ tive Ca traces (A) and pooled data (B) for spontaneous Ca and shRNA COX7RP in females alter RyR2 oxidation levels in rat waves (SCW) latency in male (M), female (F), male overexpress- VMs exposed to isoproterenol (ISO, 50  nmol/L) paced at 1  Hz for ing COX7RP (M + COX), and female expressing shRNA COX7RP 5  min cultured for 48  h after infection (10 MOI). Immunoprecipi- (F + shC) ventricular cardiomyocytes (VCMs) exposed to 50  nmol/L tated RyR2s from M and F cultured VCM samples were probed with isoproterenol (ISO, 5 min) after cessation of field stimulation (1 Hz). Anti-DNP antibody to detect amount of oxidized cysteines. E Pooled Mean ± SEM, n = 58–64, N = 6–9 preparations, *p < 0.05, p values DNP optical density normalized to corresponding RyR2 signal. were calculated using two-level random intercept model. C Pooled Mean ± SEM, N = 3 M, N = 4 F. *p < 0.05, Student’s t-test previously reported that ovariectomy does not change dimin- VCMs led to entirely opposite results producing adverse 2+ 2+ ished [Ca ] uptake in female cardiac mitochondria [2]. In effects on cytosolic Ca cycling and ROS. Our results line with this report, our data shows no change in MCU point to the possibility that downregulation of COX7RP levels in hearts from OVX rats vs shams and human tissue [33, 53] and concomitant acceleration of mito-ROS pro- 2+ samples from old vs young females. Moreover, mito-Ca duction in the aging heart [15, 18] might contribute to measurements in VCMs from old rats showed that sex dif- abrupt increase in CVD risk in postmenopausal females. 2+ ferences in mito-Ca uptake and removal were still present This would add COX7RP in females to the growing list of 2+ despite loss of differences in intracellular Ca handling and proteins involved in cardioprotection that show age-related levels of mito-ROS (Fig. 7). Together, these data implicate changes in expression/activity, explaining loss of cardio- loss of COX7RP as a key driver for increased mito-ROS protection with age [8]. Interestingly, a recent study [32] production in low estrogen conditions. ruled out any sex-related differences in infarct size and Our in vitro gain- and loss-of-function experiments in protection by ischemic pre-conditioning in minipigs, ques- H9c2 cells demonstrated a strong capacity of COX7RP tioning the cardioprotection paradigm in premenopausal to regulate respiration in these cells (Fig. 11). In VCMs females. Our data may explain sexual dimorphism in some from male rat hearts, COX7RP overexpression not only aspects of CVD risk, especially pathophysiology associ- 2+ increased supercomplexes (Fig.  12), but also reduced ated with arrhythmia and Ca handling disturbances. mito-ROS and RyR2 oxidation, which led to increased However, these differences may not offer improved car - 2+ SR Ca load and reduced propensity for pro-arrhythmic dioprotection in response to severe irreversible ischemic SCWs (Fig.  14). Importantly, COX7RP KD in female injury resulting in necrosis and infarct. 1 3 15 Page 20 of 22 Basic Research in Cardiology (2023) 118:15 Conclusions References 1. Akerboom J, Carreras Calderón N, Tian L, Wabnig S, Prigge To summarize, our data highlight the fundamental differ - M, Tolö J, Gordus A, Orger MB, Severi KE, Macklin JJ, Patel ences in bidirectional SR-mitochondria communication in R, Pulver SR, Wardill TJ, Fischer E, Schüler C, Chen TW, Sark- VCMs from heathy adult male and female hearts. 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Azuma K, Ikeda K, Inoue S (2020) Functional mechanisms of mitochondrial respiratory chain supercomplex assembly factors Acknowledgements We thank The OSU Medical Center Biobank for and their involvement in muscle quality. Int J Mol Sci 21:3182. providing human donor cardiac tissues. https:// doi. org/ 10. 3390/ ijms2 10931 82 4. Belevych AE, Terentyev D, Terentyeva R, Ho HT, Gyorke I, Funding This work was supported by The Ohio State University Presi- Bonilla IM, Carnes CA, Billman GE, Györke S (2012) Shortened dent’s Postdoctoral Scholars Award (S. Hamilton), National Institutes 2+ Ca signaling refractoriness underlies cellular arrhythmogen- of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI) esis in a postinfarction model of sudden cardiac death. Circ Res K99HL155492 and K99HL155492-02S1 (S. Hamilton), NIH NHLBI 110:569–577. https://doi. or g/10. 1161/ CIR CRES AHA.111. 260455 R01HL135236 (R.T. Clements), NIH NHLBI R01HL063043, NIH 5. Belevych AE, Terentyev D, Viatchenko-Karpinski S, Terentyeva NHLBI R01HL074045 (S. Gyorke), NIH National Institute of Aging R, Sridhar A, Nishijima Y, Wilson LD, Cardounel AJ, Laurita KR, K01AG056848 (M. Stratton), NIH R01 HL136951 and R01 HL154001 Carnes CA, Billman GE, Gyorke S (2009) Redox modification of (F. Accornero), NIH NHLBI R01HL138579 (S. Gyorke and J.P. Davis), ryanodine receptors underlies calcium alternans in a canine model NIH NHLBI R01HL142588 (G. Csordas and D. Terentyev), and NIH of sudden cardiac death. Cardiovasc Res 84:387–395. https://doi. NHLBI HL121796 (D. Terentyev). org/ 10. 1093/ cvr/ cvp246 6. Bers DM (2002) Cardiac excitation-contraction coupling. Nature Data availability All available data are incorporated into the article, 415:198–205. https:// doi. org/ 10. 1038/ 41519 8a raw data can be made avaliable upon reasonable request. 7. Bidasee KR, Nallani K, Besch HR Jr, Dincer UD (2003) Strep- tozotocin-induced diabetes increases disulfide bond formation Declarations on cardiac ryanodine receptor (RyR2). J Pharmacol Exp Ther 305:989–998. https:// doi. org/ 10. 1124/ jpet. 102. 046201 Conflict of interest The authors declare that they have no conflict of 8. Boengler K, Schulz R, Heusch G (2009) Loss of cardioprotection interest. with ageing. Cardiovasc Res 83(2):247–261. https:// doi. org/ 10. 1093/ cvr/ cvp033 Ethical approval All procedures performed in studies involving ani- 9. Bovo E, Lipsius SL, Zima AV (2012) Reactive oxygen species 2+ mals were in accordance with the ethical standards of the institution contribute to the development of arrhythmogenic Ca waves dur- or practice at which the studies were conducted (The Ohio State Uni- ing β-adrenergic receptor stimulation in rabbit cardiomyocytes. versity Institutional Animal Care and Use Committee (IACUC), per- J Physiol 590:3291–3304. https:// doi. org/ 10. 1113/ jphys iol. 2012. mit number 2019A00000041 and 2010A00000117-R3). The studies 230748 were approved by the Human Study Committee of The Ohio State 10. Bovo E, Mazurek SR, Zima AV (2018) Oxidation of ryanodine 2+ University. receptor after ischemia-reperfusion increases propensity of Ca waves during β-adrenergic receptor stimulation. Am J Physiol Heart Circ Physiol 315:H1032–H1040. https:// doi. org/ 10. 1152/ Open Access This article is licensed under a Creative Commons Attri- ajphe art. 00334. 2018 bution 4.0 International License, which permits use, sharing, adapta- 11. Brunello L, Slabaugh JL, Radwanski PB, Ho HT, Belevych AE, tion, distribution and reproduction in any medium or format, as long Lou Q, Chen H, Napolitano C, Lodola F, Priori SG, Fedorov VV, as you give appropriate credit to the original author(s) and the source, Volpe P, Fill M, Janssen PM, Györke S (2013) Decreased RyR2 provide a link to the Creative Commons licence, and indicate if changes refractoriness determines myocardial synchronization of aberrant were made. The images or other third party material in this article are 2+ Ca release in a genetic model of arrhythmia. 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Springer Journals
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Copyright © The Author(s) 2023
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0300-8428
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1435-1803
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10.1007/s00395-023-00988-1
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Abstract

2+ Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female 2+ hearts exhibit lower mito-[Ca ] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into 2+ supercomplexes os ff ets the deci fi t in mito-Ca accumulation, thereby reducing ROS production and stress-induced intracel- 2+ 2+ lular Ca mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca ] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed 2+ decreased mitochondria Ca uniporter expression and increased supercomplex assembly in rat and human female ventricu- lar tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced 2+ mito-ROS and spontaneous SR Ca release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in 2+ female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more effi- 2+ cient electron transport. Such organization coupled to lower levels of mito-[Ca ] limits mito-ROS under stress conditions 2+ 2+ and lowers propensity to pro-arrhythmic spontaneous SR Ca release. We conclude that sexual dimorphism in mito-Ca handling and ETC organization may contribute to cardioprotection in healthy premenopausal females. 2+ Keywords Cardiovascular diseases · Mitochondria · Sarcoplasmic reticulum Ca release · Sexual dimorphism · Oxidative stress · COX7RP Richard T. Clements and Radmila Terentyeva are co-first authors. * Dmitry Terentyev Department of Medicine, Cardiovascular Research Center, dmitry.terentyev@osumc.edu Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI, USA Department of Biomedical and Pharmaceutical Sciences, Department of Pathology, Anatomy and Cell Biology, University of Rhode Island College of Pharmacy, Kingston, MitoCare Center, Thomas Jefferson University, Philadelphia, RI, USA PA, USA Department of Medicine, Providence VAMC and Brown Division of Orthodontics, College of Dentistry, The Ohio University, Providence, RI, USA State University, Columbus, OH, USA Department of Physiology and Cell Biology, The Ohio State University, 460 Medical Center Dr, Columbus, OH 43210, USA Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Vol.:(0123456789) 1 3 15 Page 2 of 22 Basic Research in Cardiology (2023) 118:15 Abbreviations promote disturbances in sarcolemma membrane potential in BN-PAGE Blue native gel electrophoresis the form of delayed- and early-afterdepolarizations. These COX7RP Cytochrome c oxidase subunit 7A-related disturbances underlie pro-arrhythmic triggered activity at protein the whole heart level. The risk of pro-arrhythmic behav- CVD Cardiovascular disease ior profoundly escalates under stress conditions typically ETC Electron transport chain accompanied by β-adrenergic stimulation. Beta-adrenergic 2+ ISO Isoproterenol stimulation increases spontaneous SR Ca release and coin- 2+ MCU Mitochondrial Ca uniporter cidentally increases mito-ROS production [9]. Recently we + 2+ 2+ NCX1 Na Ca exchanger type 1 have shown that RyR2-mediated SR Ca release increases 2+ OVX Ovariectomy transfer of Ca to the mitochondrial matrix via mitochon- 2+ ROS Reactive oxygen speciesdrial Ca uniporter (MCU). This increase contributes to RyR2 Ryanodine receptor type 2 mito-ROS production in VCMs challenged with isoproter- 2+ SCW Spontaneous Ca wave enol (ISO), a selective β-adrenergic agonist [23]. Further- 2+ SERCA2a Sarcoplasmic endoplasmic reticulum Ca more, ROS produced by mitochondria lead to an increase ATPase type 2a in RyR2 oxidation and thereby channel activity, promoting SR Sarcoplasmic reticulum pro-arrhythmic spontaneous SCWs [15, 22, 23]. VCM Ventricular cardiomyocyte Cardiac contraction is a high-energy demanding process. Mitochondria are the major source of energy in VCMs and their ability to generate ATP must be tightly matched to Introduction rapidly changing metabolic demands [16, 21]. In addition to sensing ATP/ADP ratio, mitochondrial function can be 2+ The incidence of all causes of cardiovascular disease (CVD) tuned by changes in [Ca ] entering matrix via MCU. An 2+ is much lower in premenopausal females in comparison to increase in mitochondrial matrix [Ca ] activates several males of the same age [52]. However, later in life CVD dehydrogenases stimulating NADH and F AD production becomes as prevalent in females as males, often resulting that fuel electron transport chain (ETC). Interestingly, stud- in sudden cardiac death due to ventricular arrhythmias ies using mouse models with loss-of-MCU function dem- [40]. The major driver of cardiac dysfunction in old age has onstrated no change in basal cardiac function but showed been ascribed to defective mitochondrial function, leading significant loss of responsiveness to adrenergic stimula- 2+ to impaired metabolic flexibility and subsequent aberrant tion [38, 45]. This suggests that MCU-mediated mito-Ca stress responses [35]. Defective electron transport results uptake has a limited yet important function to boost energy in increased production of reactive oxygen species (ROS) production during stress as an integral part of the fight-or- by mitochondria, which can impair function of numerous flight response. Notably, it has been demonstrated that mito- 2+ enzymes and ion channels in cardiomyocytes [15, 19, 21]. [Ca ] is significantly lower in female VCMs than males [2 ]. Interestingly, in younger males, mitochondria are known to However, cardiac mitochondrial respiration capacity [29, 30] produce more ROS than mitochondria of younger females and cardiac output [25, 27] are comparable between males 2+ [14, 51], without differences in respiration capacity [29, 30]. and females, suggesting that SR Ca release-bioenergetics However, the exact mechanisms underlying this fundamental coupling is fundamentally different between sexes, and that difference between males and females, which may explain these differences may play a key role in lowering cardiovas- sexual dimorphism in rates of CVD, are yet to be defined. cular risk in premenopausal females. Cardiac contractility relies on tightly controlled release In order to provide similar metabolic output to males, it 2+ of Ca from the sarcoplasmic reticulum (SR) mediated by is reasonable to hypothesize that mitochondria from female cardiac ryanodine receptors type 2 (RyR2) [6]. The ampli- VCMs likely have more efficient electron transport to com- 2+ 2+ tude of SR Ca release in ventricular cardiomyocytes pensate for the reduction of matrix [Ca ] [2]. An additional (VCMs) during systole determines the strength of contrac- benefit of a more efficient ETC organization would include tile apparatus activation, while timely cessation of RyR2 reduced levels of ROS [14]. It was recently established that 2+ activity and effective clearance of Ca from the cytosol by elements of the ETC residing in mitochondrial cristae can 2+ SR Ca ATPase (SERCA2a) in diastole promotes relaxa- form large multimolecular supercomplexes, which substan- 2+ tion after each beat. Oxidative modifications of the SR Ca tially increase electron transport efficiency [12]. A group 2+ handling machinery profoundly impair intracellular Ca of proteins involved in facilitation of supercomplex assem- 2+ cycling by impeding SERCA2a-mediated Ca uptake and bly has recently been identified [3 ]. Of particular interest is facilitating untimely RyR2 channel opening [19]. This leads the supercomplex assembly factor, COX7RP (also known 2+ not only to the loss of SR Ca content, but also to gen- as Cox7a2l and SCAFI), given its expression is dependent 2+ eration of spontaneous diastolic Ca waves (SCWs) that on the sex hormone estrogen [33, 53]. However, whether 1 3 Basic Research in Cardiology (2023) 118:15 Page 3 of 22 15 potential differences in mitochondrial supercomplexes and Procurement Team, an entity not involved in the study, and COX7RP expression levels contribute to reduced mito-ROS were initially deemed potentially suitable for transplantation. production in female VCMs, thus exerting a stabilizing influ- The donor’s files do not contain specific entry why a specific 2+ ence on the SR Ca release, remains unknown. heart was not used. According to the coordinators, the most In the present study we investigated the role of sex and common reasons for not using a heart is that it was too small/ COX7RP-dependent changes in mitochondrial supercom- gender disparity, or the potential presence of a viral infec- 2+ plex assembly, mito-ROS production, and intracellular Ca tion. Demographic data of donors from whom tissue samples homeostasis in VCMs under conditions mimicking stress. were obtained are described in Table 1. Our results demonstrate a higher degree of ETC organiza- 2+ tion into supercomplexes and reduced mito-[Ca ] uptake Molecular biology in female VCMs vs males. This leads to reduced mito-ROS 2+ production and less pro-arrhythmic SCWs due to reduced The mitochondrial matrix Ca sensor mtRCamp1h was RyR2 oxidation under β-adrenergic stimulation. Our data constructed by fusing cytochrome C oxidase subunit IV at 2+ suggest that sexual dimorphism in mitochondrial Ca han- the N-terminus of RCamp1h coding region as described dling and ETC organization may contribute to cardioprotec- previously [1, 23]. Mito-GFP was constructed by fusing a tion in healthy premenopausal females. mitochondrial targeting sequence to GFP. The MLS-HyPer-7 Table 1 De-identified demographic data of human organ donors Methods Sample Sex Age (years) Race Study animals 1 Male 36 Caucasian 2 Male 22 African American All procedures involving animals were performed following 3 Male 44 African American the National Institutes of Health Guide for the Care and Use 4 Male 26 Caucasian of Laboratory Animals published by the US National Insti- 5 Male 60 Caucasian tutes of Health (NIH Publication No. 85-23, revised 2011) 6 Male 51 Unknown and were approved by the Institutional Animal Care and 7 Male 62 Unknown Use Committees of The Ohio State University, University 8 Female 43 Caucasian of Rhode Island and Rhode Island Hospital. Two-month- 9 Female 43 African American old male and female Sprague–Dawley rats (control) were 10 Female 68 Caucasian purchased from Charles River Laboratories (Wilmington, 11 Female 38 Caucasian MA, United States) and heart tissue samples and isolated 12 Female 52 Unknown ventricular myocytes were studied at 3–4  month of age. 13 Female 19 Caucasian In addition seven 6 month old Sprague–Dawley rats were 14 Female 24 African American bilaterally ovariectomized (OVX) and additional five rats 15 Female 26 Caucasian received a sham surgery (Sham) at Harlan Laboratories Inc. 16 Female 28 African American Indianapolis, IN, USA. These rats were used 3 months after 17 Female 29 Caucasian surgery to obtain heart tissue samples. To study potential 18 Female 30 Caucasian 2+ sex differences in intracellular Ca homeostasis, mito-ROS 19 Female 31 Caucasian 2+ and mito-Ca 22-month-old F344 rats of both sexes were 20 Female 32 Caucasian obtained from NIH aged rat colony. 21 Female 34 African American 22 Female 34 Caucasian Human samples 23 Female 60 Caucasian 24 Female 61 Caucasian Human left ventricular tissues were obtained from human 25 Female 62 Caucasian organ donors as previously described [20, 39]. These non- 26 Female 62 N/A failing hearts were from those organ donors who had no 27 Female 63 Caucasian history of AF and/or major cardiovascular diseases but not 28 Female 64 Caucasian used for heart transplantation. No one involved in the study 29 Female 65 Caucasian had any involvement in clinical decision making regarding 30 Female 67 Caucasian suitability of donor heart use. The studies were approved 31 Female 69 Caucasian by the Human Study Committee of The Ohio State Univer- 32 Female 72 Caucasian sity. The hearts were evaluated by Lifeline of Ohio Organ 1 3 15 Page 4 of 22 Basic Research in Cardiology (2023) 118:15 sensor was a gift from Vsevolod Belousov [42]. Adenovi- or propyleneoxide, embedded in Spurr’s or Epon (Embed ruses carrying biosensor constructs were generated using 812) resin. Longitudinal, ultrathin sections (65–80 nm) were the ViraPower Gateway expression system (Thermo Fisher cut from the resin-embedded blocks with a diamond knife Scientific, Waltham, MA, USA) [24]. COX7RP-FLAG and (Diatome-US, USA) using a Leica UCT ultramicrotome shRNA COX7RP adenoviruses were purchased from Vigene and caught on copper grid covered with formvar film or a Biosciences and Vector Biolabs, respectively. Gilder parallel bar grid (100 mesh). Images of longitudinal oriented cardiomyocytes were obtained via an FEI Tecnai Myocyte isolation and cell culture 12 TEM fitted with an AMT XR-111 10.5 Mpx or (later) AMT BioSprint 12 12 Mpx CCD camera at 3200–15,000 × To isolate ventricular cardiomyocytes (VCMs), rats were magnification (80 kV). Methods for analyzing mitochondria injected with pentobarbital (120 mg/kg) as a terminal pro- morphology were adapted from Khalifa et al. [30]. TEM cedure, and hearts removed via bilateral thoracotomy. Hearts images were uniformly adjusted for brightness/contrast and were placed in ice-cold Tyrode’s solution and mounted on mitochondria were manually selected by the freehand tool a Langendorff apparatus before retrograde perfusion with using ImageJ software (ImageJ, NIH). Individual mitochon- Tyrode’s solution (in mmol/L: 140 NaCl, 5.4 KCl, 1 MgCl , dria were analyzed for area, perimeter, aspect ratio (the ratio 10 HEPES, 5.6 glucose, pH 7.3) containing collagenase II between major and minor axes), and form factor. Form factor (Worthington Biochemical Corp., Lakewood, NJ, USA) at (F), a measure of mitochondrial length was determined for 37 °C for 15–20 min. Atria were removed and ventricles each image using the equation F = (perimeter /(4π × area). were minced and placed in a 37 °C water bath shaker in Mitochondria density was expressed as the percentage of collagenase solution. Isolated VCMs were plated in serum cell area occupied by mitochondrion. onto laminin-coated glass coverslips in 24-well plates 2+ (Medium 199 [Thermo Fisher Scientific] supplemented Measurements of intracellular Ca transients with 25 mmol/L N aHCO , 10 mmol/L HEPES, 5 mmol/L creatine, 5 mmol/L taurine, 10 μg/mL penicillin, 10 μg/mL Confocal imaging was performed using a Leica SP8 confo- streptomycin, and 10 μg/mL gentamycin; pH 7.3). Unat- cal microscope equipped with a 63 × 1.4 numerical aper- tached cells were removed after 1 h. Attached VCMs were ture oil objective. Cultured rat VCMs were loaded with infected with adenoviruses described below at multiplicity Fluo-3 AM (Invitrogen) at room temperature for 10 min 2+ of infection (MOI) of 10. Myocytes were cultured at 37 °C in Ca -free Tyrode’s solution, followed by a 10 min wash 2+ in 95% air, 5% CO for 36–44 h before analysis. in Tyrode’s solution containing 1 mmol/L Ca . Myocytes 2+ were perfused with Tyrode’s solution (1 mmol/L Ca ) dur- Transmission electron microscopy ing recordings, at room temperature. Myocytes were paced via field stimulation at 1 Hz with platinum electrodes. To 2+ For perfusion fixation, animals were heparinized, then euth- test for the propensity of spontaneous Ca waves (SCWs), anized by pentobarbital (120 mg/kg) injection. The hearts isoproterenol-treated (50 nmol/L) VCMs were paced for 20 s were cannulated through the aorta for retrograde perfusion and latency between the last pacing stimulus and the SCW on a Langendorff apparatus. First, the hearts were perfused was calculated. Caffeine (10 mmol/L) was applied at the 2+ 2+ by cold Ca -free Tyrode solution (in mmol/L: 140 NaCl, 5.4 end of recording to assess SR Ca content. Fluo-3AM was KCl, 1 MgCl , 10 HEPES, 5.6 glucose (pH 7.3) for ~ 5 min excited at 488 nm and fluorescence emission was collected at (until the heart stops beating). This is followed by perfusion 500–550 nm wavelengths in line scan mode at 200 Hz sam- + 2+ with 2.5% glutaraldehyde in 0.1 M Na -cacodylate buffer pling rate. Cytosolic Ca transient amplitude is presented (pH 7.4) for 5 min at room temperature. Then the heart was as ΔF/F , where F is basal fluorescence and ΔF = F–F . 0 0 0 immersed into the fixative for an additional 40 min before 2+ the apex was removed to cut the LV anterior wall open lon- Measurements of mitochondrial matrix Ca gitudinally along the anterior wall. After additional 30 min at room temperature, the heart in the excessive fixative was Myocytes were infected with mtRCamp1h adenovi- put on ice in individually labeled sealed vials, and shipped rus ± COX7RP or COX7RP-shRNA adenovirus and cultured to the MitoCare Center Electron Microscopy Facility at for 36–44 h [20]. mtRCamp1h was excited using 543 nm Thomas Jefferson University, for further processing. There, line of HeNe laser and fluorescence emission collected at small pieces from the LV wall were cut (~ 1  mm ), overnight 560–660  nm wavelengths, measured in the x–y mode at postfixed in 4 °C in 2% osmium tetroxide partially reduced 400 Hz sampling rate. After pacing, VCMs were washed + 2+ by 0.8% K Fe(CN) in 0.15  mol N a -cacodylate buffer. in Ca -free Tyrode’s solution before permeabilization 4 6 Samples were further contrasted en bloc with 1% aque- with saponin (0.001%). Tyrode’s solution was replaced ous uranylacetate, dehydrated in graded series of acetone with internal recording solution. The intracellular solution 1 3 Basic Research in Cardiology (2023) 118:15 Page 5 of 22 15 contained (mmol/L): 120  K aspartate, 20 KCl, 0.81 MgCl , USA; catalogue no. 524625) and protease inhibitor cock- 1 KH PO , 3 MgATP, 10 phosphocreatine, 20 HEPES (pH tails (Sigma; catalogue no. P8340) as described previously 2 4 7.2) and 5  U/mL creatine phosphokinase. Internal solu- [31]. Samples (20–30 μg of proteins) were resolved on a tion was supplemented with cytochalasin D (10 μmol/L) 4–20% gel via SDS‐PAGE, transferred onto nitrocellulose 2+ to inhibit contraction and Ca buffer EGTA (2 mmol/L) membranes, and probed with anti-COX7a2L (COX7RP), to obtain minimum mtRCamp1h fluorescence [20]. Maxi- anti-MCU, anti-Hsp60, anti-GAPDH antibodies and sub- 2+ mum fluorescence was achieved by application of Ca sequently probed with a goat anti‐mouse, goat anti‐rabbit (20  μmol/L) in EGTA-free solution. Using the equation secondary (Promega; Madison, WI, USA). Blots were devel- 2+ mito-[Ca ] = Kd × (F  −  Fmin)/(Fmax  −  F), where Kd of oped with ECL (Bio‐Rad Laboratories, Hercules, CA, USA; 2+ mtRCamp1h = 1.3 µmol/L for Ca , fluorescence was con- catalogue no. 1705061) and quantified and analyzed using 2+ verted to mito-[Ca ] for each VCM. Analysis parameters ImageJ (NIH, Bethesda, MD, USA;) and Origin, version 8 2+ included baseline mtRCamp1h mito-[Ca ] (μmol/L) in rest- (OriginLab Corp., Northampton, MA, USA). COX7RP and 2+ ing VCMs; peak mito-[Ca ] and the time to peak amplitude MCU signals were normalized to Hsp60 loading controls. (s) in VCMs undergoing periodic 1 Hz field stimulation [24]. List of antibodies used is present in Table 2. 2+ −1 The rate of mito-Ca decay (1/τ, s ) was derived from 2+ single exponential fit of VCM mito-[Ca ] decrease upon Western blot analysis of H9c2 cells cessation of pacing. H9c2 cells were plated in 6-well plates and infected with Measurements of mitochondrial matrix ROS COX7RP-FLAG and shRNA COX7RP adeno-viruses. At 48 h after infection, cells were lysed in lysis buffer from Cell The ratiometric MLS-HyPer7 probe was excited using Signaling (Cat # 9803S), supplemented with phosphatase 405 nm and 488 nm laser lines. Fluorescence emission was (Calbiochem, Cat#524,625) and protease inhibitor cocktails collected at 520–540 nm wavelengths, measured in the x–y (Sigma, Cat#P8340) as described previously [50]. Samples mode at 400 Hz sampling rate. Minimum fluorescence was were run on 4–20% TGX gels, transferred to nitrocellulose obtained by application of dithiothreitol (DTT, 5 mmol/L), membrane, and probed with anti-COX7a2L (COX7RP) anti- and maximum fluorescence was obtained by application of bodies from Sigma. Hsp60 was used as loading control (Cell 2,2′-dithiodipyridine (DTDP, 200 µmol/L). Data are pre- Signaling). sented as a percentage of ΔF/ΔFmax where ΔF = F − Fmin and ΔFmax = Fmax − Fmin [24]. RyR2 immunoprecipitation and immunoblotting Western blot analysis of cardiac mitochondrial For immunoprecipitation of RyR2, cultured rat VCMs proteins infected with Adv-COX7RP and Adv-shRNA COX7RP for 48 h were lysed using cell lysis buffer from Cell Signal- Tissue samples or cultured rat VCMs were lysed in lysis ing (catalogue no. 9803S), supplemented with phosphatase buffer from Cell Signaling (catalogue no. 9803S), supple- (Calbiochem; catalogue no. 524625) and protease inhibi- mented with phosphatase (Calbiochem, San Diego, CA, tor cocktails (Sigma; catalogue no. P8340). An overnight Table 2 Antibodies used in this Antibody/kit Species of used sample Source Identifier study Anti-COX7a2L H9C2,Rat and Human MilliporeSigma Cat# SAB1303595 Anti-RyR2 Rat and Human Alomone Cat# ARR-002 Anti-MCU Rat and Human MilliporeSigma Cat# HPA016480 Anti-COX IV Rat and Human Abcam Cat# ab16056 Anti-UQCRFS1 Rat and Human Abcam Cat# ab14746 Anti-NDUFA9 Rat and Human Abcam Cat# ab14713 Anti-Hsp60 H9C2, Rat and Human Cell Signaling Cat# 12165T Anti-GAPDH H9C2, Rat and Human Abcam Cat# ab8245 Anti-Rabbit IgG(H+L),HRP H9C2, Rat and Human Promega Cat# W4011 Anti-Mouse IgG(H+L),HRP H9C2, Rat and Human Promega Cat# W4021 Oxidized Protein Western blot kit Rat Abcam Cat# ab178020 Anti-MnSOD Rat Millipore Cat # 06-984 Anti-Peroxiredoxin 5 Rat Abcam Cat # ab180123 1 3 15 Page 6 of 22 Basic Research in Cardiology (2023) 118:15 immunoprecipitation of RyR2 was performed at 4 °C using III—anti-UQCRFS1; Complex IV—anti-COX IV; all from the Catch and Release v2.0 Kit (Millipore; catalogue no. Abcam. Hsp60 was used as loading control. Anti-Hsp60 17‐500) in accordance with the manufacturer’s instruc- antibodies were from Cell Signaling. The secondary anti- tions using anti‐RyR2 antibody (Alomone catalogue no. bodies used were goat anti‐mouse and goat anti‐rabbit ones ARR-002, 5 μg of antibody) and a negative control anti- (Promega, Madison, WI, USA). Blots were developed with body comprising normal rabbit IgG (Millipore; catalogue ECL (Bio-Rad Laboratories) and quantified using Image J no. 12-370, 5 μg of antibody). Samples were analyzed by (US National Institutes of Health) and Origin 8 software. immunoblotting. To determine oxidation status of RyR2, the Oxidized Oxygen consumption measurements Protein Western Blot Kit (Abcam catalogue no. ab178020) was used, whereby carbonyl groups of immunoprecipitated To assess mitochondrial function, oxygen consumption rates RyR2 were derivatized to 2,4 dinitrophenylhydrazone (DNP) (OCR) of H9c2 cells infected with Adv-COX7RP-FLAG by reaction with 2,4 dinitrophenylhydrazine [23]. For con- and Adv-shRNA COX7RP were measured by the Agilent trol, we used Derivatization Control Solution included in Seahorse XFe96 extracellular flux analyzer (Seahorse Bio- the kit. The DNP-RyR2 protein samples were separated on science, North Billerica, MA). Briefly, cells were seeded in 4–20% Mini-PROTEAN TGX gels (Bio-Rad Laboratories, XFe96 cell culture plates at 10,000 cells/well in standard Cat#456-1094) and DNP-associated signal was assessed DMEM + 10% FBS medium and placed in a 37 °C incubator by the kit-provided anti-DNP rabbit primary antibody and with 5% CO Adv-shRNA COX7RP was used for infection anti-RyR2, followed by HRP-conjugated anti-rabbit goat on day one and Adv-COX7RP on day 2. On day 3, cells lgG(H+L), secondary antibody. were washed twice with Seahorse assay medium (XF base Blots were developed with ECL (Bio-Rad Laboratories) medium supplemented with 10 mmol/L glucose, 2 mmol/L and quantified using Image J (US National Institutes of glutamine, and 1 mmol/L sodium pyruvate; pH 7.4) and Health, Bethesda, MD, USA) and Origin 8 software. incubated in a 37 °C incubator without CO for 1 h. Oxy- gen consumption rate (OCR) was measured according to Blue native PAGE manufacturer’s guidelines of the XF Cell Mito Stress Test kit (Agilent). Oligomycin A (1 μmol/L final concentration), For the assessment of native protein complexes using blue FCCP (1.5 μmol/L), and a combination of rotenone and anti- native polyacrylamide gel electrophoresis (BN‐PAGE) [20], mycin A (1 μmol/L each) were injected on cells as indicated. we used mitochondrial fractions isolated from LV heart tis- After the assay, cells were stained with Hoechst dye and sues suspended in buffer containing 225 mmol/L manni- nuclei counted on a Cytation 5 cell imaging plate reader tol, 70 mmol/L sucrose, 10 mmol/L HEPES, and 1 mmol/L (Agilent) to normalize results. Data are expressed as pmol EGTA (pH 7.4) [31]. The tissue was placed in a pre‐cooled O /min/100 cells. Plate location was randomized between 5 mL Wheaton™ Potter‐Elveheim Tissue Grinder (Fisher groups in each assay. Wells with no response to oligomycin, Scientific, Hampton, NH, USA; catalogue no. 22‐ 290067) FCCP, or rotenone/antimycin were removed from analysis and homogenized. Tissue homogenate was centrifuged at based on the assumption of premature injection/leak of 700g for 10 min. The pellet consisting of nuclei and cell assay drugs into the well from upper plate reservoir, which debris was discarded and the supernatant was then cen- confounds interpretation. There were no differences in the trifuged at 20,000g for 15 min. The pellet was considered proportion of these wells between groups. as mitochondrial enriched fraction. Samples were then solubilized using the mild detergent digitonin (1%) from Isolated heart oxygen consumption measurements NativePAGE™ Sample Prep Kit (Invitrogen; catalogue no. BN2008) in accordance with the manufacturer's instruc- Male and female rats of equal age and approximately tions. To demonstrate changes in association of supercom- 300–500 g were heparinized, anesthetized with 3% isoflu- plexes in VCMs infected with adenoviruses, cells were rane and heart and lungs removed en bloc following ster- lysed in digitonin (1%) for 30 min on ice, and centrifuged notomy. All procedures were approved by the Providence at 20,000g for 30 min. Processed samples were resolved VAMC IACUC committee. Hearts were rapidly placed in on NativePAGE™ 4–16% Bis‐Tris protein gels, 1.0 mm, ice-cold Krebs buffer, the aorta cannulated, and retrograde 15‐well (Invitrogen; catalogue no. BN2011B10) in Native- flow initiated with oxygenated Krebs buffer (in mmol/L: Page cathode and anode buffers electrophoresed at 150 V 118.00 NaCl, 4.70 KCl, 1.40 CaCl , 1.70 MgSO , 24.88 2 4 for 1.5. followed by 250 V for 2 h. Samples were trans- NaHCO , 6.00 glucose, 1.20 KH PO , and 2.00 Na pyru- 3 2 4 ferred onto nitrocellulose membranes before being probed vate) to wash blood and supply oxygen to the heart during with antibodies, as described above. The primary antibodies working heart preparation. Pulmonary veins were ligated, used were as follows: Complex I—anti-NDUFA9; Complex the lungs removed under a microscope and the heart rapidly 1 3 Basic Research in Cardiology (2023) 118:15 Page 7 of 22 15 moved to working heart apparatus (IHS5, Hugo Sachs Elek- as mean ± SEM. P values are provided with two significant tronik, March, Germany). Hearts were then slowly increased figures, where < 0.050 were considered significant (*). For to a constant perfusion pressure of 70 mmHg over 5 min. experiments involving multiple VCMs (data points) isolated A cannula was inserted into the left atria and a pressure from one individual rat heart, a hierarchical level random catheter inserted into the LV through the base of the heart intercept model was used [47]. The model tests for data with care to avoid large coronary vessels. A small catheter clustering for each cell isolation and adjusts for any cluster- attached to an in-line optical O sensor (PreSens Precision ing with significance testing. Posthoc pairwise comparisons Testing, Regensburg, Germany) and withdrawal syringe were carried out for hierarchical testing. No experiment- pump was placed in the pulmonary artery to continuously wide/across-test multiple test correction was applied; only monitor O tension and temperature of coronary effluent. within-test corrections were made. Statistical tests used for An additional in-line O sensor monitored O and tempera- each dataset are presented in Table 3. 2 2 ture of perfusate. Hearts were switched to working mode via opening gravity flow of oxygenated Krebs buffer to the left atria (preload set to 10 mmHg). Langendorff perfusion was reduced and hearts allowed to equilibrate for 5 min before Results O consumption measurements. Coronary flow was assessed 2+ via eluent collection over 1 min. O consumption was nor- Increased spontaneous SR Ca release in rat male malized to heart weight and coronary flow and calculated VCMs vs females under β‑adrenergic stimulation according to Neely et al. [41]. Beta-adrenergic stimulation increases global pro-arrhyth- 2+ Glutathione GSH/GSSG measurement mic spontaneous Ca release events, SCWs [9]. In order to 2+ assess potential differences in intracellular Ca homeosta- 2+ Tissue total GSH and GSSG in rat left ventricles were meas- sis, male and female VCMs were loaded with Ca indicator ured using assay kit from Sigma-Aldrich (Cat MAK440) Fluo-3AM and treated with 50 nmol/L β-adrenergic agonist [34]. Briefly, stored tissue chunks were weighed and isoproterenol (ISO). After 5 min incubation in ISO, VCMs homogenates were prepared using Bullet Blender Strom 24 were subjected to field stimulation at 1 Hz for 5 min at room 2+ (Next Advance Inc, NY) in PBS, pH 7.4, 1 mmol/L EDTA temperature. Ca transients and SCWs were recorded using with or without scavenger (provided with the kit) for the a Leica SP8 confocal microscope in line scan mode. Rep- measurement of GSSG and total GSH respectively. All the resentative cell-averaged fluorescence profile traces (male 2+ samples were deproteinated with 5% meta-phosphoric acid. black, female red) are presented in Fig. 1A. Stimulated Ca The clarified and diluted samples were mixed with work - transients are indicated with arrows, while spontaneous 2+ ing reagent and spectrophotometric reading was obtained at global SR Ca releases are denoted with asterisks. Analysis 2+ 412 nm at 0 and 10 min using SpectraMax 190 microplate of Ca transient amplitudes expressed as ΔF/F , where F is 0 0 reader (Molecular Devices, CA). Total GSH, reduced GSH, basal fluorescence and ΔF = F −  F , revealed no difference 2+ and GSSG were calculated following assay protocol. between sexes (Fig. 1B left panel). Ca transient decay in male VCMs showed small but significant slowing in com- Data presentation and statistics parison to females, indicative of reduced SERCA2a activity (Fig. 1B center panel). Furthermore, male VCMs exhibited Group sample sizes were determined by power analysis shorter latency of SCW initiation vs females (Fig. 1B right based on our previous experience [20] performing two-tailed panel). Latency, a well-established readout of RyR2-medi- 2+ Student’s t-test to provide adequate power to detect 20% dif- ated SR Ca release refractoriness [4, 11], was measured as 2+ ference, assuming power of 80% (β = 0.80) and an α of 0.05. time interval from the start of the last stimulated Ca tran- No animals or samples were excluded from the study. sient in the train to the initiation point of the first SCW. Total 2+ Animals were grouped with no blinding, but randomized in SR Ca content was measured by rapid 10 mmol/L caffeine cellular experiments. Data acquisition and analysis were not application at the end of the experiment (Fig. 1C). As shown 2+ fully blinded as the same person carried out these processes. in Fig. 1D, SR Ca content was significantly higher in ISO- Representative traces for cellular imaging experiments treated female rat VCMs in comparison to males. The decay and representative western blot images were chosen to rates of the caffeine transient were not different between + 2+ closely match the mean for the parameters assessed. Sta- male and female VCMs, indicative of unchanged Na /Ca tistical analyses were performed using Origin 2020Pro (NCX1) exchanger activity. Taken together, these data sug- (OriginLab) and R software [20, 47]. For each experiment, gest that SERCA2a activity is reduced, while the activity of 2+ the number of animals (uppercase n [N]) and the number the SR Ca release channel RyR2 is significantly higher in of VCMs (lowercase n) used is indicated. Data is expressed male VCMs than in females under β-adrenergic stimulation. 1 3 15 Page 8 of 22 Basic Research in Cardiology (2023) 118:15 Table 3 Statistical tests used in this study Figure Type of sample Statistical test applied 1B One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 1D One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 2D One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 3B-F One rat per N, multiple cells (n) used per image per rat Three-level random intercept model with Tukey’s posthoc 4B LV tissue, one rat heart per N Two-sample Student’s t test 4C Ventricular tissue, one rat heart per N Two-sample Student’s t test 5B One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 5D One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 6C One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 6E LV tissue, one heart (rat/human) per N Two-sample Student’s t test 7B One rat per N, multiple cells (n) used per rat Transient amplitude/decay/latency—two-level random intercept model with Tukey’s posthoc 2+ Ca waves—Fisher’s exact test 7E One rat per N, multiple cells (n) used per rat One Way ANOVA with Tukey’s posthoc 8C One rat heart per N Two-sample Student’s t test 8D One rat heart per N Two-sample Student’s t test 8E One rat heart per N Two-sample Student’s t test 9A LV tissue, one rat heart per N Two-sample Student’s t test 9B LV tissue, one human heart per N Two-sample Student’s t test 9C LV tissue, one rat heart per N Two-sample Student’s t test 9D LV tissue, one human heart per N Two-sample Student’s t test 10B LV tissue, one human heart per N Two-sample Student’s t test 10D LV tissue, one rat heart per N Two-sample Student’s t test 10F LV tissue, one rat heart per N Two-sample Student’s t test 11C H9c2 cells, one independent experiment per N One way ANOVA with Tukey’s postdoc 12B Isolated VMs from one rat heart per N Two-sample Student’s t test 12D Isolated VMs from one rat heart per N Two-sample Student’s t test 13A Isolated VMs from one rat heart per N Two-sample Student’s t test 13B Isolated VMs from one rat heart per N Two-sample Student’s t test 13C One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 13D One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 14B One rat per N, multiple cells (n) used per rat Two-level random intercept model with Tukey’s posthoc 14E Isolated VMs from one rat heart per N Two-sample Student’s t test MLS-HyPer7 fluorescence ratio (male black, female red). Decreased mito‑ROS levels in female VCMs vs males After 5 min incubation in 50 nmol/L ISO, VCMs were field- under β‑adrenergic stimulation stimulated for 5 min before application of DTT and DTDP. As shown in Fig.  2D, male ISO-treated VCMs exhibit a Increased activity of RyR2 and decreased SERCA2a activity higher MLS-HyPer7 signal than female myocytes both may be explained by mito-ROS-mediated oxidation of these before and during field stimulation. Of note, it was previ- proteins [19]. To test this possibility, we expressed the dual ously shown using TEM that mitochondria content and size excitation wavelength ROS sensor MLS-HyPer7 targeted differ in VCMs from male and female mouse hearts [30]. to the mitochondrial matrix in VCMs [24]. Correct MLS- However, our TEM experiments in rat hearts showed no sig- HyPer7 localization was confirmed by co-staining VCMs nificant differences between sexes (Fig.  3). Therefore, sex with mitochondria-specific dye Mitotracker Red (Fig.  2A). differences in MLS-HyPer7 fluorescence cannot be ascribed The MLS-HyPer7 signal was normalized to minimal u fl ores - to differences in mitochondria content in rat VCMs. In addi- cence obtained by application of 5 mmol/L ROS scavenger tion, as the probe is ratiometric as well as normalized to DTT, and maximum fluorescence obtained by treatment of maxima and minima, there is minimal concern for sex- cells with 200 μmol/L DTDP, an oxidizing agent (Fig. 2B). dependent expression differences. There is a possibility that Figure 2C demonstrates representative normalized traces of 1 3 Basic Research in Cardiology (2023) 118:15 Page 9 of 22 15 2+ Fig. 1 Intracellular Ca handling is divergent in healthy male and female ventricular myocytes. A Fluo-3 fluores- cence (F/F ) profiles of isopro- terenol treated (ISO, 50 nmol/L) rat ventricular cardiomyocytes (VCMs) undergoing 1 Hz pace-pause protocol. Arrows depict field stimulation-evoked 2+ Ca transients, stars show 2+ proarrhythmic diastolic Ca 2+ waves. B Mean ± SEM Ca transient amplitude (ΔF/F ) −1 and decay rate (s ), n = 90 male (M) and n = 90 female (F) VCMs; and spontaneous 2+ Ca wave (SCW) latency (s), n = 62 M and n = 59 F VCMs. N = 11–12 M and N = 11–13 F animals. C Representative 2+ traces of caffeine-induced Ca transients (10 mmol/L). D Mean ± SEM caffeine transient amplitude (ΔF/F ) and decay −1 rate (s ), n = 69 M, n = 75 F VCMs, N = 11 M, N = 13 F animals. *p < 0.05, p values were calculated using two-level random intercept model heart antioxidant defenses differ between sexes as shown in the major role of differential mito-ROS production in sex 2+ other tissues [13]. To test this we performed western blot dimorphism in intracellular Ca homeostasis in VCMs analysis of mitochondrial superoxide dismutase (MnSOD) under β-adrenergic stimulation. and Peroxiredoxin-5 (PRXD5) and found no differences in 2+ samples from male and female rat cardiac tissues (Fig. 4A, Decreased mito‑Ca levels in female VCMs vs males B). Furthermore, we did not find statistically significant dif- under β‑adrenergic stimulation ference in GSH/GSSG ratio (Fig. 4C), indicating that there are no sex-dependent differences in antioxidant capacity. We next tested potential differences between sexes in 2+ 2+ Taken together, these data are in line with previous reports mito-[Ca ], given that changes in matrix [Ca ] are often that male VCMs exhibit significantly higher mito-ROS pro- associated with changes in mito-ROS production [24, 36, 2+ duction than female VCMs [14, 51]. 48]. Using the matrix-targeted Ca probe mtRCamp1h 2+ To test if increased mito-ROS plays a significant role in we determined that mitochondrial [Ca ] is significantly 2+ SR Ca release destabilization we performed experiments lower in field-stimulated female VCMs pretreated with treating male rat VCMs with MitoTEMPO, a mitochondria 50 nmol/L ISO for 5 min (Fig. 6A–C). Correct mitochon- specific ROS scavenger (20  μmol/L, 20  min preincuba- drial localization of mtRCamp1h was confirmed in VCMs tion [24]). As depicted in Fig. 5, MitoTEMPO significantly co-expressing mito-targeted GFP. As seen in Fig. 6A, red 2+ attenuated SCWs, accelerated Ca transient decay, and mtRCamp1h fluorescent signal overlaps with green signal 2+ 2+ increased SR Ca load in periodically paced male VCMs of mito-GFP. Figure 6B shows representative mito-Ca treated with ISO (50 nmol/L, 5 min). These results highlight 1 3 15 Page 10 of 22 Basic Research in Cardiology (2023) 118:15 Fig. 2 Matrix ROS biosensor MLS-HyPer7 reveals increased mito- in male (M) and female (F) VCMs, respectively. Myocytes were ROS in male vs. female ventricular myocytes. A Mitochondrial MLS- treated with isoproterenol (ISO, 50  nmol/L) and paced at 1  Hz for Hyper-7 localization in live ventricular cardiomyocytes (VCMs) vali- 5  min. Fluorescence was normalized to minimum (DTT, 5  mmol/L) dated by Mitotracker. B Representative images of VCM infected with and maximum (DTDP, 200  µmol/L) fluorescence. D Mean ± SEM MLS-HyPer7, and treated with DTT (5 mmol/L) followed by DTDP MLS-HyPer7 fluorescence, n = 37 M and n = 77 F VCMs, N = 11 M, (200 µmol/L) to achieve minimum and maximum fluorescence, dem- N = 11 F animals. *p < 0.05, p values were calculated using two-level onstrating probe sensitivity. C Representative MLS-HyPer-7 recorded random intercept model 2+ traces from male (black) and female (red) VCMs derived Aging abolishes sex differences in intracellular Ca from mRCamp1h signal. To convert mtRCamp1h fluores- homeostasis and mito‑ROS production 2+ cence into [Ca ], cells were permeabilized with saponin 2+ at the end of recording and exposed to 2 mmol/L Ca Using young and old VCMs from female rabbit hearts we 2+ buffer EGTA to obtain Fmin followed by application previously showed that disturbances in Ca handling are 2+ 20 μmol/L [Ca ] to obtain Fmax. Although no significant governed by increased mito-ROS production [15]. Here we 2+ 2+ changes in mito-[Ca ] were detected at rest, field stimu- tested potential sex differences in Ca cycling in VCMs from lation of VCMs at 1 Hz revealed that in female VCMs it 22-month-old F344 rats obtained from NIH aged rat colony. 2+ 2+ takes significantly longer to reach maximum mito-[Ca ], As demonstrated in Fig. 7, we find no differences in Ca tran- 2+ and that this maximum concentration is significantly lower sient amplitude and decay rate, as well as SR Ca content than in male VCMs (Fig. 6C). Importantly, western blot and propensity to generate pro-arrhythmic SCWs (Fig. 7A–D). analysis revealed significantly lower expression levels of This was accompanied by similar rates of mito-ROS produc- MCU in rat female VCMs vs males, which could poten- tion in old male and female VCMs (Fig.  7E), which is in 2+ tially explain why matrix Ca is lower (Fig. 6D). Nota- striking contrast to young cells (Fig. 6B). Of note, the sex 2+ bly, lower MCU levels were detected in female human differences in mito-Ca uptake and removal were preserved LV donor heart (≤ 52 year old) samples vs males as well with aging (Fig. 7E), suggesting that the main source of age- 2+ (Fig. 6E). Given that mito-[Ca ], in addition to uptake, related mito-ROS increase in females is not related to mito- + 2+ 2+ is determined by removal via mitochondrial Na /Ca Ca mishandling. exchanger [17], we assessed the rate of removal by meas- 2+ uring mito-Ca decay upon cessation of pacing. Of note, 2+ the rate of Ca removal from mitochondrial matrix was significantly faster in females. 1 3 Basic Research in Cardiology (2023) 118:15 Page 11 of 22 15 Fig. 3 Absence of sex‐related differences in mitochondrial counts and microscopy. B–F Mitochondrial morphometric parameters, including morphology in rat heart. A Mitochondrial density assessed by trans- mito area (B), perimeter (C), aspect ratio (D), form factor and density mission electron microscopy (TEM) in the left ventricular cardiac (% of the cell area, F), in male (M) and female (F) rat hearts. The tissue from male (M) and female (F) rats. Original magnification is analysis was performed randomly from 60 M (30 images), 60 F (21 × 3200 and the white bars represent 2 μm scale. Heart were fixed with images) cardiac mitochondria from three animals per group and were 2.5% glutaraldehyde solution then processed for transmission electron analyzed by three-level random intercept model LV tissues. Importantly, experiments using LV tissues from Female mitochondria exhibit higher levels of ETC healthy human donor hearts exhibited a similar pattern supercomplexes and COX7RP (Fig. 9B). Notably, the risk of CVD and cardiac arrhythmia dramatically increases in postmenopausal females match- Differences in cardiac respiratory capacity between sexes ing or even outpacing males [52], suggesting that hormonal has not been previously described [29, 30]. In line with this, changes may play a role in these processes. Of note, expres- our measurements of oxygen consumption in Langendorff- sion of COX7RP, a protein critically involved in supercom- perfused working heart preparations showed no difference plex formation, is estrogen dependent [33, 53]. We tested in myocardial O consumption between males and females potential sex differences in COX7RP expression levels and (Fig. 8). Therefore, we surmised that a compensatory mecha- 2+ found that they are significantly higher in females than males nism must exist to offset the effects of lower matrix [Ca ] both in rat and human hearts from heathy donors (Fig. 9C, in females vs males. One plausible mechanism to explain D). Furthermore, using samples from young and old female this discrepancy could include enhanced incorporation of human hearts we confirmed that COX7RP levels decrease individual ETC complexes into ETC supercomplexes to with aging in contrast to MCU (Fig. 10A, B). Experiments increase electron transport efficiency [3 , 12]. Indeed, as using sham and ovariectomized (OVX) female rats showed depicted in Fig. 9A, Blue Native Gel Electrophoresis (BN- an identical expression pattern (Fig. 10C, D). In addition to PAGE) of isolated mitochondrial proteins showed increased a decrease in COX7RP levels, we found that supercomplexes high molecular weight assemblies consisting of comigrating are significantly reduced in OVX LV tissue samples as well subunits for Complex I, III and IV in rat female vs male 1 3 15 Page 12 of 22 Basic Research in Cardiology (2023) 118:15 Fig. 4 Anti-oxidant defenses in male and female left ventricular car- as loading control. Mean ± SEM, N = 4 M and N = 4 F rat LV sam- diac tissues. A, B Representative western blots and pooled data for ples; *p < 0.05, Student’s t-test. C Total tissue GSH, reduced GSH, mitochondrial superoxide dismutase Mn-SOD and peroxiredoxin 5 oxidized GSH (GSSG), and the ratio of GSH/GSSG was measured (PRDX5) normalized optical density from male (M) and female (F) from male and female rat ventricles. Mean + SEM, N = 7 M and N = 6 rat left ventricular (LV) tissue samples. GAPDH and Hsp60 was used F rat, *p < 0.05, Student’s t-test (Fig. 10E, F). These data support the key role of estrogen COX7RP were subjected to BN-PAGE analysis to test dependent COX7RP expression in supercomplex assembly. potential COX7RP influence on ETC supercomplex assem- bly (Fig. 12C, D). Importantly, COX7RP KD significantly COX7RP regulates mitochondria oxygen reduced supercomplexes in female VCMs, while COX7RP consumption and supercomplexes overexpression increased them in VCMs from males. These data further corroborates the key role of COX7RP in mito- In order to modify COX7RP expression levels, we utilized chondrial ETC supercomplex assembly in cardiomyocytes. adenoviral vectors carrying human COX7RP-FLAG and Importantly, altering COX7RP levels did not change MCU 2+ 2+ shRNA COX7RP sequences. Efficiency of adenovirus- expression levels, mito-Ca uptake and peak [Ca ] in male mediated COX7RP overexpression and shRNA-COX7RP and female rat VCMs (Fig. 13). knockdown (KD) was confirmed in H9c2 cells 48 h after 2+ transduction with western blot (Fig. 11A). As seen in repre- COX7RP regulates SR Ca release, mito‑ROS sentative western blot, FLAG-tagged COX7RP runs slower production and RyR2 oxidation in VCMs on the gel than wild type COX7RP. Mitochondrial protein Hsp60 was used as loading control. Next, we performed Next, we tested the effects of adenoviral-mediated measurement of oxygen consumption in H9c2 cells with a COX7RP-FLAG overexpression in male VCMs and Seahorse Analyzer in cells expressing shRNA-COX7RP or shRNA-mediated KD in females treated with ISO COX7RP. Averaged traces are presented in Fig.  11B. As (50 nmol/L, 5 min) on RyR2-mediated spontaneous SR 2+ seen in Fig. 11C, COX7RP overexpression increases basal, Ca release. Representative line scan images and cor- maximum and ATP-linked respiration in H9c2 cells, while responding Fluo-3 fluorescence time profiles are depicted COX7RP KD reduces it. Next, viral constructs were tested in Fig. 14A. The latency of SCWs appearing after cessa- in cultured rat VCMs. COX7RP levels were increased ~ 50% tion of 1 Hz field-stimulation was significantly lengthened in male VCMs 48 h after transduction (Fig. 12A, B). Expres- in male VCMs overexpressing COX7RP, while COX7RP sion of shRNA COX7RP (shC) led to ~ twofold reduction of KD significantly shortened it (Fig.  14A, B). Importantly, the protein in female VCMs 48 h after infection (Fig. 12A, parallel experiments in VCMs coexpressing mito-ROS B). Next, samples from cultured male VCMs expressing sensor MLS-HyPer7 subjected to the same experimental COX7RP-FLAG and female VCMs expressing shRNA protocol showed significant mito-ROS reduction in male 1 3 Basic Research in Cardiology (2023) 118:15 Page 13 of 22 15 Fig. 5 Mitochondrial ROS scavenging reduces spontaneous 2+ Ca release is male ventricular myocytes. A Fluo-3 fluores- cence (F/F ) profiles of isopro- terenol treated (ISO, 50 nmol/L) rat ventricular cardiomyocytes (VCMs) undergoing 1 Hz pace- pause protocol. Arrows depict 2+ field stimulation-evoked Ca transients, stars show proar- 2+ rhythmic diastolic Ca waves. Pretreatment with MitoTEMPO, a mitochondria-specific ROS scavenger (20 mmol/L, 8 min) 2+ reduces spontaneous Ca 2+ waves. B Mean ± SEM Ca transient amplitude (ΔF/F ) and −1 decay rate (s ), n = 26 male (M) and n = 37 MitoTEMPO treated male (M + MT) VCMs; 2+ and spontaneous Ca wave (SCW) latency (s), n = 23 M and n = 25 M + MT VCMs. N = 4 M and N = 4 M + MT ani- mals. C Representative traces of 2+ caffeine-induced Ca transients (10 mmol/L). D Mean ± SEM caffeine transient amplitude −1 (ΔF/F ) and decay rate (s ), n = 11 M, n = 15 M + MT VCMs, N = 4 M, N = 4 M + MT animals. *p < 0.05, p values were calculated using two-level random intercept model COX7RP-overexpressing VCMs, while mito-ROS in Discussion female shRNA-COX7RP-expressing VCMs was signifi- cantly increased (Fig.  14C). We then assessed possible In the present study we investigated the mechanisms underly- COX7RP expression-dependent changes in RyR2 oxida- ing reduced mito-ROS production in female VCMs. We have tion state. Control and COX7RP-overexpressing male and shown that male rat VCMs exhibit higher propensity to pro- 2+ shRNA COX7RP-expressing female VCMs were exposed arrhythmic diastolic SR Ca release governed by hyperactive to 50 nmol/L ISO pretreatment and then paced at 1 Hz for RyR2s due to oxidation by mito-ROS. We found that lower 5 min in the presence of ISO. Then cells were immediately mito-ROS production in females is accompanied by lower 2+ collected for RyR2 immunoprecipitation. The relative mito-[Ca ] and heightened ETC supercomplex assembly. changes in immunoprecipitated RyR2s oxidized cysteine We found the more efficient electron transport in females is content were assessed using anti-DNP antibodies [23]. As likely related to increased expression levels of the estrogen- shown in Fig. 14D, COX7RP overexpression significantly dependent mitochondrial assembly protein COX7RP. reduces RyR2 oxidation in male VCMs, while COX7RP KD in female VCMs increases it. To summarize, COX7RP KD increased RyR2 oxidation, mito-ROS, and profoundly 2+ disturbed SR Ca release in female VCMs promoting gen- eration of pro-arrhythmic SCWs, while COX7RP overex- pression led to opposite results in males. 1 3 15 Page 14 of 22 Basic Research in Cardiology (2023) 118:15 2+ 2+ 2+ 2+ Fig. 6 Female ventricular myocytes have reduced Ca levels in matrix [Ca ], and peak matrix [Ca ], time to peak matrix [Ca ], mitochondria matrix vs. males. A Representative confocal images and decay rate during pacing, n = 13  M and n = 15 F VMs. N = 8  M of a ventricular myocyte (VCM) infected with matrix-targeted mtR- and 8 F animals. *p < 0.05, p values were calculated using two-level Camp1h (Kd ~ 1.3 mmol/L) and mito-GFP, with a merged figure dem- random intercept model. D, E Representative western blots and onstrating correct probe localization. B Representative time course pooled data for COX7RP normalized optical density in rat and human of mtRCamp1h fluorescence recorded in male (M) and female (F) left ventricular (LV) tissue samples. Mean ± SEM, N = 8 M and N = 8 VCMs. Myocytes were treated with isoproterenol (ISO, 50  nmol/L) F rat; and N = 4 M and N = 4 F human samples. *p < 0.05, Student’s for 3  min before pacing at 1  Hz for 5  min. C Mean ± SEM baseline t-test 2+ −1 Fig. 7 Intracellular Ca handling is similar in male and female ven- feine transient amplitude (ΔF/F ) and decay rate (s ), n = 7 M and tricular myocytes from aged (22  month old) hearts. A Fluo-4 fluo- F VMs, N = 4 M and F animals. p values were calculated using two- rescence (F/F ) profiles of isoproterenol treated (ISO, 50 nmol/L) rat level random intercept model except where indicated. E Aged male 2+ ventricular myocytes (VCMs) paced at 1  Hz. B Mean ± SEM Ca (M) and female (F) VCMs exhibit similar mito-ROS levels, while −1 2+ transient amplitude (ΔF/F ) and decay rate (s ), n = 23 male (M) and sexual dimorphism in matrix [Ca ] preserved with aging. VCMs 2+ n = 25 female (F) VCMs; proportion of cells displaying Ca waves were passed at 1  Hz for 5  min in the presence of 50  nmol/L ISO, 2+ following 2 Hz pacing and distribution of latency to Ca waves (s), Mean ± SEM, N = 2–4 M, 2–4 F, n = 6–20, *p < 0.05, p values were n = 33 for M and n = 33 for F, N = 4 M and F animals. C Representa- calculated using one way ANOVA 2+ tive traces of caffeine-induced Ca transients. D Mean ± SEM caf- Recent studies using mouse models with loss of MCU Increased mito‑ROS and unstable intracellular 2+ 2+ RyR2‑mediated Ca cycling in male vs female VCMs function revealed that SR Ca release is directly coupled to mitochondrial metabolic output in stress conditions [38, during β‑adrenergic stimulation 1 3 Basic Research in Cardiology (2023) 118:15 Page 15 of 22 15 Fig. 8 Oxygen consumption is similar in male and female work- Quantitation of male and female isolated rat heart myocardial O con- ing heart preparations. A Picture of rat working heart preparation. B sumption N = 3 males and N = 3 females. p values were calculated Representative left ventricular (LVP), aortic and atrial pressure traces using Student’s t-test (mmHg) and LVP dP/dt of isolated rat heart in working mode. C–E Fig. 9 Female left ventricular tissues demonstrate increased mito- black bars. Mean ± SEM, N = 8 M and N = 8 F rat LV samples; and chondrial supercomplex formation and expression of COX7RP N = 6 M and N = 4 F human LV samples, *p < 0.05, Student’s t-test. compared to males. A, B Representative BN-PAGE images of ETC C, D Representative western blots and pooled data for COX7RP nor- complexes from male (M) and female (F) rat (A) and human (B) left malized optical density in rat (C) and human (D) LV tissue samples. ventricular (LV) tissue samples. Antibodies used: Complex I—anti- Hsp60 was used as loading control (inset, Anti-Hsp60 from Cell NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti-COX Signaling). Mean ± SEM, N = 8 M and N = 8 F rat samples; and N = 4 IV all from Abcam. Hsp60 was used as loading control (inset, Anti- M and N = 4 F human samples, *p < 0.05, Student’s t-test Hsp60 from Cell Signaling). Supercomplexes (SCs) are indicated by 1 3 15 Page 16 of 22 Basic Research in Cardiology (2023) 118:15 Fig. 10 Reduced COX7RP levels in LV heart tissues from old female Sham and ovariectomised (OVX) rats. Hsp60 was used as loading donors and ovariectomized female rats. A, B Representative west- control (Cell Signaling). E Representative BN-PAGE images of ETC ern blots and pooled data for COX7RP and MCU normalized opti- complexes from rat LV tissue samples. Antibodies used: Complex I— cal density in left ventricular (LV) tissue samples from young (less anti-NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti- than 35  year old, N = 10) and old (more than 60  years old, N = 10) COX IV; all from Abcam. Hsp60 was used as loading control (inset, female donor hearts. Mean ± SEM, *p < 0.05, Student’s t-test. C, D Anti-Hsp60 from Cell Signaling). Supercomplexes (SCs) are indi- Representative western blots and pooled data for COX7RP and MCU cated by black bar. F Pooled normalized optical density data for (E). normalized optical density in left ventricular (LV) tissue samples Mean ± SEM, N = 5 Sham and N = 7 OVX, *p < 0.05, Student’s t-test Fig. 11 COX7RP modulates oxygen consumption in H9C2 myo- ings of oxygen consumption in H9C2 cells in control cells and cells blasts. A Representative western blot demonstrating efficiency of expressing COX7RP or COX7RP shRNA. C Pooled respirometry COX7RP overexpression and COX7RP shRNA constructs in H9C2 data, Mean ± SEM, n ≥ 18, N = 3 independent experiments, *p < 0.05, cells infected with adenoviruses for 48  h. Hsp60 and GAPDH were one way ANOVA used as loading controls. B Representative Seahorse Analyzer record- 2+ 2+ 45]. β-adrenergic stimulation increases SR Ca release SR Ca content in male VCMs. Furthermore, male VCMs 2+ amplitude to levels sufficient for MCU to transmit Ca into exhibited shorter latency of SCW initiation. Together, these 2+ the mitochondrial matrix [23], activating Ca -dependent data are a strong indication that RyR2 activity is higher in dehydrogenases in the Krebs Cycle and promoting ATP male than in female VCMs under β-adrenergic stimulation. 2+ production [21]. Thus, our main focus was to characterize Previous reports directly linked impaired SR Ca handling 2+ potential differences in Ca homeostasis in VCMs from in VCMs incubated with β-adrenergic agonists to increased female and male rat hearts in the presence of ISO (Fig. 1). RyR2 oxidation by mito-ROS resulting in increased channel Notably, although we did not find statistically significant dif- activity and high propensity to pro-arrhythmic SCWs [9, 15, 2+ ferences in Ca transient amplitudes in periodically stimu- 23]. In line with these findings, our measurements using the lated male and female VCMs, we saw a significantly lower mitochondria-targeted ROS biosensor MLS-HyPer-7 suggest 1 3 Basic Research in Cardiology (2023) 118:15 Page 17 of 22 15 Fig. 12 COX7RP expression levels regulate formation of mitochon- tion of multimolecular ETC supercomplexes in Ms, while COX7RP drial supercomplexes in rat ventricular myocytes. A, B Transfection shRNA decreases it in Fs. Antibodies used: Complex I—anti- of rat ventricular cardiomyocytes with Ad-COX7RP-FLAG (male NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti-COX (M)) and with shRNA COX7RP (female (F)) alters expression levels IV; anti-GAPDH all from Abcam. Hsp60 was used as loading control of COX7RP protein 48  h after infection (10 MOI). Hsp60 was used (inset, Anti-Hsp60 from Cell Signaling). Supercomplexes (SCs) are as loading control. B Pooled data for (A), Mean ± SEM, N = 4  M and indicated by black bars. D Pooled normalized optical density data for N = 5 F, *p < 0.05, Student’s t-test. C Representative BN-page experi- (C) mean ± SEM, N = 4  M and N = 5 F animals, *p < 0.05, Student’s ments demonstrating that COX7RP overexpression increases forma- t-test 2+ that sex differences in RyR2-mediated spontaneous SR Ca treatment of male VCMs with MitoTEMPO significantly 2+ 2+ release are indeed associated with different levels of mito- attenuated spontaneous SR Ca release, increased SR Ca 2+ ROS (Fig. 2). Importantly, RyR2 hyperoxidation has been load, and accelerated Ca transient decay (Fig. 5). Taken associated with reduced cardiac function and/or increased together, our data suggest that high mito-ROS in ISO-treated 2+ arrhythmic potential in various cardiac diseases including male VCMs markedly disturbs intracellular Ca homeo- heart failure [49], infarct [5, 10], diabetic cardiomyopathy stasis consistent with increased RyR2 activity and lower [7, 28, 37], age-related cardiac dysfunction [15, 18], and SERCA2a function. even inherited cardiac arrhythmia syndromes [23]. Increased 2+ mito-ROS production in VCMs from male hearts is expected Sexual dimorphism in mitochondria [Ca ] uptake to carry additional risk and may explain heightened protec- and ETC supercomplex assembly in the heart tion in healthy premenopausal females. 2+ Interestingly, the Ca transient decay in ISO-treated Mitochondria are increasingly acknowledged as a key tar- female VCMs was ~ 10% faster than males (Fig.  1A, B). get for sex differences in pathologies [51]. However, under - 2+ Changes in Ca transient decay rate could be primarily standing of exact mechanisms underlying sex differences ascribed to differences in SERCA2a expression and activity in mito-ROS production in health remains limited. Given [6]. It is not likely that there are differences in SERCA2a that ETC is the major source of ROS in mitochondria, expression levels between sexes [43]. However, SERCA2a increased ETC activity under conditions of higher workload activity can be modulated, i.e. lowered, by redox modifi - or stress is expected to accelerate ROS production [21, 26]. 2+ cation at cysteine 674 [44]. Slow C a transient decay in Indeed, we recently reported that MCU-mediated influx of 2+ male VCMs treated with ISO is consistent with reduced Ca into the mitochondrial matrix, which increases ETC SERCA2a activity under higher ROS levels. Importantly, activity by stimulating production of NADH and FAD , 1 3 15 Page 18 of 22 Basic Research in Cardiology (2023) 118:15 Fig. 13 Altering COX7RP expression levels does not affect MCU D Adenovirus-mediated changes in COX7RP expression levels do 2+ 2+ expression and mito-Ca uptake in male and female rat ventricu- not affect mitochondrial Ca uptake measured using mtRCamp1h 2+ lar myocytes. A, B Transfection of rat ventricular cardiomyocytes Ca biosensor in M and F VCMs. Mean ± SEM, n = 9 M and (VCMs) with Ad-COX7RP-FLAG (male (M)) and with shRNA n = 20 M + COX, N = 4 M; n = 27 F and n = 21 F + shC and N = 3 F, COX7RP (female (F)) does not change expression levels of MCU *p < 0.05, p values were calculated using two-level random intercept 48  h after infection (10 MOI). Hsp60 was used as loading control. model Mean ± SEM, N = 4 M and N = 3 F, *p < 0.05, Student’s t-test. C, 2+ is essential for mito-ROS production in VCMs challenged offset the effects of lower matrix [Ca ] in females vs males. 2+ 2+ with β-adrenergic agonist [23]. Importantly, mito-[Ca ] in We confirmed that despite lower mitochondrial [Ca ] in VCMs rapidly changes in response to changes in frequency female VCMs, female working hearts have the same myo- 2+ and amplitude of cytosolic Ca transients, directly link-cardial O consumption as male hearts when operating at ing contractile activity and mitochondria ATP production to similar work levels (Fig. 8). Mitochondrial supercomplex closely match varying metabolic demand with output. Our assembly is one plausible mechanism of respiratory com- 2+ experiments in VCMs expressing mito-[Ca ] biosensor pensation in females [14]. Furthermore, more compact ETC 2+ mRCamp1h (Fig. 6A) showed that mito-[Ca ] is drastically organization is expected to reduce emission of ROS due to lower in field-stimulated ISO-treated VCMs from female rat more efficient electron transport. Indeed, our BN-PAGE 2+ hearts vs males (Fig. 6B, C), despite similar Ca transient experiments demonstrate higher abundance of supercom- 2+ amplitudes (Fig. 1A, B). Previously it was reported that Ca plexes in LV tissues from female hearts in both rats and uptake is lower in isolated female rat cardiac mitochondria humans (Fig. 9A, B). The importance of supercomplexes than in males [2]. The authors suggested that this decrease in the heart is underscored by the fact that in certain heart could be ascribed to lower MCU activity. In support of this failure models, a reduction in supercomplexes was shown to notion, our western blot analysis clearly shows lower expres- be sufficient to reduce respiration capacity without detect- sion levels of MCU in mitochondria from female rat and able changes in individual complexes’ activities [46]. The human LV tissues vs males (Fig. 6D, E). Furthermore, our assembly of supercomplexes is governed by several proteins, 2+ data suggests that Ca removal from the matrix is more effi- including COX7RP that brings together Complex I, III, and cient in female mitochondria. Taken together, these results IV [3, 12]. The transcription of COX7RP is regulated in part suggest that female cardiac mitochondria are less reliant on by estrogen [33, 53], which prompted us to compare expres- 2+ [Ca ] accumulation to satisfy increased metabolic demand sion levels between sexes. Indeed, COX7RP was more abun- during high workload or stress. dant in rat and human female LV heart samples (Fig. 9C, Given that cardiac mitochondrial respiratory capacity is D). Notably, COX7RP decreased in aged vs young female not known to be grossly different between sexes [29, 30] and human LV heart tissues (Fig. 10A, B). Furthermore, ovariec- cardiac output is similar even at higher workloads [25, 27], tomy significantly reduced COX7RP and supercomplexes we surmised that a compensatory mechanism must exist to in female LV tissue samples (Fig. 10C, D). Of note, it was 1 3 Basic Research in Cardiology (2023) 118:15 Page 19 of 22 15 Fig. 14 COX7RP overexpression reduces spontaneous SR data for normalized fluorescence of mitochondria matrix ROS biosen- 2+ Ca release and mito-ROS in male ventricular myocytes under sor MLS-HyPer-7. Mean ± SEM, n = 29–42 VCMs, N = 6–9 animals, β-adrenergic stimulation. Conversely, COX7RP knock-down *p < 0.05, p values were calculated using two-level random intercept 2+ increases Ca waves and mito-ROS in females. A, B Representa- model. D Adenovirus–mediated expression of COX7RP in males 2+ 2+ tive Ca traces (A) and pooled data (B) for spontaneous Ca and shRNA COX7RP in females alter RyR2 oxidation levels in rat waves (SCW) latency in male (M), female (F), male overexpress- VMs exposed to isoproterenol (ISO, 50  nmol/L) paced at 1  Hz for ing COX7RP (M + COX), and female expressing shRNA COX7RP 5  min cultured for 48  h after infection (10 MOI). Immunoprecipi- (F + shC) ventricular cardiomyocytes (VCMs) exposed to 50  nmol/L tated RyR2s from M and F cultured VCM samples were probed with isoproterenol (ISO, 5 min) after cessation of field stimulation (1 Hz). Anti-DNP antibody to detect amount of oxidized cysteines. E Pooled Mean ± SEM, n = 58–64, N = 6–9 preparations, *p < 0.05, p values DNP optical density normalized to corresponding RyR2 signal. were calculated using two-level random intercept model. C Pooled Mean ± SEM, N = 3 M, N = 4 F. *p < 0.05, Student’s t-test previously reported that ovariectomy does not change dimin- VCMs led to entirely opposite results producing adverse 2+ 2+ ished [Ca ] uptake in female cardiac mitochondria [2]. In effects on cytosolic Ca cycling and ROS. Our results line with this report, our data shows no change in MCU point to the possibility that downregulation of COX7RP levels in hearts from OVX rats vs shams and human tissue [33, 53] and concomitant acceleration of mito-ROS pro- 2+ samples from old vs young females. Moreover, mito-Ca duction in the aging heart [15, 18] might contribute to measurements in VCMs from old rats showed that sex dif- abrupt increase in CVD risk in postmenopausal females. 2+ ferences in mito-Ca uptake and removal were still present This would add COX7RP in females to the growing list of 2+ despite loss of differences in intracellular Ca handling and proteins involved in cardioprotection that show age-related levels of mito-ROS (Fig. 7). Together, these data implicate changes in expression/activity, explaining loss of cardio- loss of COX7RP as a key driver for increased mito-ROS protection with age [8]. Interestingly, a recent study [32] production in low estrogen conditions. ruled out any sex-related differences in infarct size and Our in vitro gain- and loss-of-function experiments in protection by ischemic pre-conditioning in minipigs, ques- H9c2 cells demonstrated a strong capacity of COX7RP tioning the cardioprotection paradigm in premenopausal to regulate respiration in these cells (Fig. 11). In VCMs females. Our data may explain sexual dimorphism in some from male rat hearts, COX7RP overexpression not only aspects of CVD risk, especially pathophysiology associ- 2+ increased supercomplexes (Fig.  12), but also reduced ated with arrhythmia and Ca handling disturbances. mito-ROS and RyR2 oxidation, which led to increased However, these differences may not offer improved car - 2+ SR Ca load and reduced propensity for pro-arrhythmic dioprotection in response to severe irreversible ischemic SCWs (Fig.  14). 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Azuma K, Ikeda K, Inoue S (2020) Functional mechanisms of mitochondrial respiratory chain supercomplex assembly factors Acknowledgements We thank The OSU Medical Center Biobank for and their involvement in muscle quality. Int J Mol Sci 21:3182. providing human donor cardiac tissues. https:// doi. org/ 10. 3390/ ijms2 10931 82 4. Belevych AE, Terentyev D, Terentyeva R, Ho HT, Gyorke I, Funding This work was supported by The Ohio State University Presi- Bonilla IM, Carnes CA, Billman GE, Györke S (2012) Shortened dent’s Postdoctoral Scholars Award (S. Hamilton), National Institutes 2+ Ca signaling refractoriness underlies cellular arrhythmogen- of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI) esis in a postinfarction model of sudden cardiac death. Circ Res K99HL155492 and K99HL155492-02S1 (S. Hamilton), NIH NHLBI 110:569–577. https://doi. or g/10. 1161/ CIR CRES AHA.111. 260455 R01HL135236 (R.T. Clements), NIH NHLBI R01HL063043, NIH 5. Belevych AE, Terentyev D, Viatchenko-Karpinski S, Terentyeva NHLBI R01HL074045 (S. Gyorke), NIH National Institute of Aging R, Sridhar A, Nishijima Y, Wilson LD, Cardounel AJ, Laurita KR, K01AG056848 (M. Stratton), NIH R01 HL136951 and R01 HL154001 Carnes CA, Billman GE, Gyorke S (2009) Redox modification of (F. Accornero), NIH NHLBI R01HL138579 (S. Gyorke and J.P. Davis), ryanodine receptors underlies calcium alternans in a canine model NIH NHLBI R01HL142588 (G. Csordas and D. Terentyev), and NIH of sudden cardiac death. Cardiovasc Res 84:387–395. https://doi. NHLBI HL121796 (D. Terentyev). org/ 10. 1093/ cvr/ cvp246 6. Bers DM (2002) Cardiac excitation-contraction coupling. Nature Data availability All available data are incorporated into the article, 415:198–205. https:// doi. org/ 10. 1038/ 41519 8a raw data can be made avaliable upon reasonable request. 7. Bidasee KR, Nallani K, Besch HR Jr, Dincer UD (2003) Strep- tozotocin-induced diabetes increases disulfide bond formation Declarations on cardiac ryanodine receptor (RyR2). J Pharmacol Exp Ther 305:989–998. https:// doi. org/ 10. 1124/ jpet. 102. 046201 Conflict of interest The authors declare that they have no conflict of 8. Boengler K, Schulz R, Heusch G (2009) Loss of cardioprotection interest. with ageing. Cardiovasc Res 83(2):247–261. https:// doi. org/ 10. 1093/ cvr/ cvp033 Ethical approval All procedures performed in studies involving ani- 9. Bovo E, Lipsius SL, Zima AV (2012) Reactive oxygen species 2+ mals were in accordance with the ethical standards of the institution contribute to the development of arrhythmogenic Ca waves dur- or practice at which the studies were conducted (The Ohio State Uni- ing β-adrenergic receptor stimulation in rabbit cardiomyocytes. versity Institutional Animal Care and Use Committee (IACUC), per- J Physiol 590:3291–3304. https:// doi. org/ 10. 1113/ jphys iol. 2012. mit number 2019A00000041 and 2010A00000117-R3). The studies 230748 were approved by the Human Study Committee of The Ohio State 10. Bovo E, Mazurek SR, Zima AV (2018) Oxidation of ryanodine 2+ University. receptor after ischemia-reperfusion increases propensity of Ca waves during β-adrenergic receptor stimulation. Am J Physiol Heart Circ Physiol 315:H1032–H1040. https:// doi. org/ 10. 1152/ Open Access This article is licensed under a Creative Commons Attri- ajphe art. 00334. 2018 bution 4.0 International License, which permits use, sharing, adapta- 11. Brunello L, Slabaugh JL, Radwanski PB, Ho HT, Belevych AE, tion, distribution and reproduction in any medium or format, as long Lou Q, Chen H, Napolitano C, Lodola F, Priori SG, Fedorov VV, as you give appropriate credit to the original author(s) and the source, Volpe P, Fill M, Janssen PM, Györke S (2013) Decreased RyR2 provide a link to the Creative Commons licence, and indicate if changes refractoriness determines myocardial synchronization of aberrant were made. The images or other third party material in this article are 2+ Ca release in a genetic model of arrhythmia. 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Journal

Basic Research in CardiologySpringer Journals

Published: May 3, 2023

Keywords: Cardiovascular diseases; Mitochondria; Sarcoplasmic reticulum Ca2+ release; Sexual dimorphism; Oxidative stress; COX7RP

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