Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes

Figures

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  Fig. 1. Intracellular Ca2+ handling is divergent in healthy male and female ventricular myocytes. A Fluo-3 fluorescence (F/F0) profiles of isoproterenol treated (ISO, 50 nmol/L) rat ventricular cardiomyocytes (VCMs) undergoing 1 Hz pace-pause protocol. Arrows depict field stimulation-evoked Ca2+ transients, stars show proarrhythmic diastolic Ca2+ waves. B Mean ± SEM Ca2+ transient amplitude (ΔF/F0) and decay rate (s−1), n = 90 male (M) and n = 90 female (F) VCMs; and spontaneous Ca2+ wave (SCW) latency (s), n = 62 M and n = 59 F VCMs. N = 11–12 M and N = 11–13 F animals. C Representative traces of caffeine-induced Ca2+ transients (10 mmol/L). D Mean ± SEM caffeine transient amplitude (ΔF/F0) and decay rate (s−1), 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

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  Fig. 2. Matrix ROS biosensor MLS-HyPer7 reveals increased mito-ROS in male vs. female ventricular myocytes. A Mitochondrial MLS-Hyper-7 localization in live ventricular cardiomyocytes (VCMs) validated by Mitotracker. B Representative images of VCM infected with MLS-HyPer7, and treated with DTT (5 mmol/L) followed by DTDP (200 µmol/L) to achieve minimum and maximum fluorescence, demonstrating probe sensitivity. C Representative MLS-HyPer-7 recorded in male (M) and female (F) VCMs, respectively. Myocytes were treated with isoproterenol (ISO, 50 nmol/L) and paced at 1 Hz for 5 min. Fluorescence was normalized to minimum (DTT, 5 mmol/L) and maximum (DTDP, 200 µmol/L) fluorescence. D Mean ± SEM MLS-HyPer7 fluorescence, n = 37 M and n = 77 F VCMs, N = 11 M, N = 11 F animals. *p < 0.05, p values were calculated using two-level random intercept model

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  Fig. 3. Absence of sex‐related differences in mitochondrial counts and morphology in rat heart. A Mitochondrial density assessed by transmission electron microscopy (TEM) in the left ventricular cardiac tissue from male (M) and female (F) rats. Original magnification is × 3200 and the white bars represent 2 μm scale. Heart were fixed with 2.5% glutaraldehyde solution then processed for transmission electron microscopy. B–F Mitochondrial morphometric parameters, including mito area (B), perimeter (C), aspect ratio (D), form factor and density (% of the cell area, F), in male (M) and female (F) rat hearts. The analysis was performed randomly from 60 M (30 images), 60 F (21 images) cardiac mitochondria from three animals per group and were analyzed by three-level random intercept model

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  Fig. 4. Anti-oxidant defenses in male and female left ventricular cardiac tissues. A, B Representative western blots and pooled data for mitochondrial superoxide dismutase Mn-SOD and peroxiredoxin 5 (PRDX5) normalized optical density from male (M) and female (F) rat left ventricular (LV) tissue samples. GAPDH and Hsp60 was used as loading control. Mean ± SEM, N = 4 M and N = 4 F rat LV samples; *p < 0.05, Student’s t-test. C Total tissue GSH, reduced GSH, oxidized GSH (GSSG), and the ratio of GSH/GSSG was measured from male and female rat ventricles. Mean + SEM, N = 7 M and N = 6 F rat, *p < 0.05, Student’s t-test

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  Fig. 5. Mitochondrial ROS scavenging reduces spontaneous Ca2+ release is male ventricular myocytes. A Fluo-3 fluorescence (F/F0) profiles of isoproterenol treated (ISO, 50 nmol/L) rat ventricular cardiomyocytes (VCMs) undergoing 1 Hz pace-pause protocol. Arrows depict field stimulation-evoked Ca2+ transients, stars show proarrhythmic diastolic Ca2+ waves. Pretreatment with MitoTEMPO, a mitochondria-specific ROS scavenger (20 mmol/L, 8 min) reduces spontaneous Ca2+ waves. B Mean ± SEM Ca2+ transient amplitude (ΔF/F0) and decay rate (s−1), n = 26 male (M) and n = 37 MitoTEMPO treated male (M + MT) VCMs; and spontaneous Ca2+ wave (SCW) latency (s), n = 23 M and n = 25 M + MT VCMs. N = 4 M and N = 4 M + MT animals. C Representative traces of caffeine-induced Ca2+ transients (10 mmol/L). D Mean ± SEM caffeine transient amplitude (ΔF/F0) and decay rate (s−1), 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

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  Fig. 6. Female ventricular myocytes have reduced Ca2+ levels in mitochondria matrix vs. males. A Representative confocal images of a ventricular myocyte (VCM) infected with matrix-targeted mtRCamp1h (Kd ~ 1.3 mmol/L) and mito-GFP, with a merged figure demonstrating correct probe localization. B Representative time course of mtRCamp1h fluorescence recorded in male (M) and female (F) VCMs. Myocytes were treated with isoproterenol (ISO, 50 nmol/L) for 3 min before pacing at 1 Hz for 5 min. C Mean ± SEM baseline matrix [Ca2+], and peak matrix [Ca2+], time to peak matrix [Ca2+], and decay rate during pacing, n = 13 M and n = 15 F VMs. N = 8 M and 8 F animals. *p < 0.05, p values were calculated using two-level random intercept model. D, E Representative western blots and pooled data for COX7RP normalized optical density in rat and human left ventricular (LV) tissue samples. Mean ± SEM, N = 8 M and N = 8 F rat; and N = 4 M and N = 4 F human samples. *p < 0.05, Student’s t-test

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  Fig. 7. Intracellular Ca2+ handling is similar in male and female ventricular myocytes from aged (22 month old) hearts. A Fluo-4 fluorescence (F/F0) profiles of isoproterenol treated (ISO, 50 nmol/L) rat ventricular myocytes (VCMs) paced at 1 Hz. B Mean ± SEM Ca2+ transient amplitude (ΔF/F0) and decay rate (s−1), n = 23 male (M) and n = 25 female (F) VCMs; proportion of cells displaying Ca2+ waves following 2 Hz pacing and distribution of latency to Ca2+ waves (s), n = 33 for M and n = 33 for F, N = 4 M and F animals. C Representative traces of caffeine-induced Ca2+ transients. D Mean ± SEM caffeine transient amplitude (ΔF/F0) and decay rate (s−1), n = 7 M and F VMs, N = 4 M and F animals. p values were calculated using two-level random intercept model except where indicated. E Aged male (M) and female (F) VCMs exhibit similar mito-ROS levels, while sexual dimorphism in matrix [Ca2+] preserved with aging. VCMs were passed at 1 Hz for 5 min in the presence of 50 nmol/L ISO, Mean ± SEM, N = 2–4 M, 2–4 F, n = 6–20, *p < 0.05, p values were calculated using one way ANOVA

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  Fig. 8. Oxygen consumption is similar in male and female working heart preparations. A Picture of rat working heart preparation. B Representative left ventricular (LVP), aortic and atrial pressure traces (mmHg) and LVP dP/dt of isolated rat heart in working mode. C–E Quantitation of male and female isolated rat heart myocardial O2 consumption N = 3 males and N = 3 females. p values were calculated using Student’s t-test

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  Fig. 9. Female left ventricular tissues demonstrate increased mitochondrial supercomplex formation and expression of COX7RP compared to males. A, B Representative BN-PAGE images of ETC complexes from male (M) and female (F) rat (A) and human (B) left ventricular (LV) tissue samples. Antibodies used: Complex I—anti-NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti-COX IV all from Abcam. Hsp60 was used as loading control (inset, Anti-Hsp60 from Cell Signaling). Supercomplexes (SCs) are indicated by black bars. Mean ± SEM, N = 8 M and N = 8 F rat LV samples; and N = 6 M and N = 4 F human LV samples, *p < 0.05, Student’s t-test. C, D Representative western blots and pooled data for COX7RP normalized optical density in rat (C) and human (D) LV tissue samples. Hsp60 was used as loading control (inset, Anti-Hsp60 from Cell Signaling). Mean ± SEM, N = 8 M and N = 8 F rat samples; and N = 4 M and N = 4 F human samples, *p < 0.05, Student’s t-test

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  Fig. 10. Reduced COX7RP levels in LV heart tissues from old female donors and ovariectomized female rats. A, B Representative western blots and pooled data for COX7RP and MCU normalized optical density in left ventricular (LV) tissue samples from young (less than 35 year old, N = 10) and old (more than 60 years old, N = 10) female donor hearts. Mean ± SEM, *p < 0.05, Student’s t-test. C, D Representative western blots and pooled data for COX7RP and MCU normalized optical density in left ventricular (LV) tissue samples Sham and ovariectomised (OVX) rats. Hsp60 was used as loading control (Cell Signaling). E Representative BN-PAGE images of ETC complexes from rat LV tissue samples. Antibodies used: Complex I—anti-NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti-COX IV; all from Abcam. Hsp60 was used as loading control (inset, Anti-Hsp60 from Cell Signaling). Supercomplexes (SCs) are indicated by black bar. F Pooled normalized optical density data for (E). Mean ± SEM, N = 5 Sham and N = 7 OVX, *p < 0.05, Student’s t-test

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  Fig. 11. COX7RP modulates oxygen consumption in H9C2 myoblasts. A Representative western blot demonstrating efficiency of COX7RP overexpression and COX7RP shRNA constructs in H9C2 cells infected with adenoviruses for 48 h. Hsp60 and GAPDH were used as loading controls. B Representative Seahorse Analyzer recordings of oxygen consumption in H9C2 cells in control cells and cells expressing COX7RP or COX7RP shRNA. C Pooled respirometry data, Mean ± SEM, n ≥ 18, N = 3 independent experiments, *p < 0.05, one way ANOVA

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  Fig. 12. COX7RP expression levels regulate formation of mitochondrial supercomplexes in rat ventricular myocytes. A, B Transfection of rat ventricular cardiomyocytes with Ad-COX7RP-FLAG (male (M)) and with shRNA COX7RP (female (F)) alters expression levels of COX7RP protein 48 h after infection (10 MOI). Hsp60 was used as loading control. B Pooled data for (A), Mean ± SEM, N = 4 M and N = 5 F, *p < 0.05, Student’s t-test. C Representative BN-page experiments demonstrating that COX7RP overexpression increases formation of multimolecular ETC supercomplexes in Ms, while COX7RP shRNA decreases it in Fs. Antibodies used: Complex I—anti-NDUFA9; Complex III—anti-UQCRFS1; Complex IV—anti-COX IV; anti-GAPDH all from Abcam. Hsp60 was used as loading control (inset, Anti-Hsp60 from Cell Signaling). Supercomplexes (SCs) are indicated by black bars. D Pooled normalized optical density data for (C) mean ± SEM, N = 4 M and N = 5 F animals, *p < 0.05, Student’s t-test