Hyperglycemia triggers RyR2-dependent alterations of mitochondrial calcium homeostasis in response to cardiac ischemia-reperfusion: Key role of DRP1 activation

Figures

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  Fig. 1. Effect of hyperglycemia on ROS production and mitochondrial calcium retention capacity. (A) Schematic illustration of the experimental protocol and representative traces of left ventricular (LV) pressure recording during IR on hearts perfused in normoglycemic (NG; 11 mM) or hyperglycemic (HG; 33 mM) buffer. Stabilization (30 min) in NG or HG buffer was followed by global no-flow ischemia (25 min) and reperfusion in NG or HG buffer (10 min). (B) LV developed pressure (devP) recovery at 10 min post-IR (n = 9 NG hearts, n = 6 HG hearts; *p < 0.05 NG vs. HG; non-parametric t-test). Values were normalized to the baseline level. (C–D) Evaluation of ROS production by staining heart tissue sections with DHE (10 μM) following 10 min of IR (n = 3 hearts/group; *p < 0.05 NG vs. HG; non-parametric Mann-Whitney test). (E) Cardiomyocytes isolated from 5 hearts/group were loaded with the ROS-sensitive fluorescent DHR123 probe (5 μM) and then incubated in NG (6 mM) or HG (33 mM) buffer. Fluorescence intensity was measured every 10 s and normalized to the baseline fluorescence (F/F0). Cardiomyocytes were stimulated with H2O2 (100 μM) to mimic IR-induced oxidative stress (*p < 0.05 NG vs. HG;…

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  Fig. 2. Effect of hyperglycemia during IR on mitochondrial fission and interactions between mitochondria and sarcoplasmic reticulum. (A) Schematic illustration of the experimental protocol. Hearts were stabilized in NG (11 mM) or HG (33 mM) buffer for 30 min, using a Langendorff apparatus. This was followed by global no-flow ischemia for 25 min and reperfusion in NG or HG buffer for 10 min. Heart tissues were used after 10 min of reperfusion. (B) Representative transmission electron microscopy images of NG- and HG-perfused heart tissue sections. Quantification of the mitochondrial aspect ratio (C) and area (D) of NG and HG mitochondria (n ≈ 600 mitochondria from 4 hearts/group; *p < 0.05 NG vs. HG; non-parametric t-test). (E–F) Representative western blots of total DRP1 and GAPDH expression in cytosolic fractions (E) and of phosphorylated DRP1 (DRP1S616), total DRP1 (DRP1) and VDAC1 (VDAC) levels in mitochondrial subfractions (F) of NG- and HG-perfused hearts after IR. (G) Mitochondrial DRP1 (relative to VDAC1) to cytosolic DRP1 (relative to GAPDH) ratio in NG- (n = 4 in duplicate) and HG-perfused hearts (n = 5 in duplicate) after IR (*p < 0.05 NG vs. HG; parametric t-test). (H) DRP1…

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  Fig. 3. Effect of hyperglycemia on mitochondrial calcium uptake. (A) Quantification of mitochondrial Ca2+ content using the o-Cresolphthalein-Complexone method in mitochondria isolated from NG- (11 mM) and HG- (33 mM)-perfused hearts (n = 3 NG hearts; n = 4 HG hearts, in triplicate; *p < 0.05 NG vs. HG; parametric t-test). (B) Mean traces of mitochondrial Ca2+ uptake by isolated mitochondria, using Ca2+ Green-5N (1 μM) as an extra-mitochondrial Ca2+ indicator. Mitochondrial Ca2+ uptake was induced by adding 2.5 μM of CaCl2 to mitochondria isolated from NG- or HG-perfused hearts (n = 12 experiments with 3 hearts/group). Fluorescence was measured every 7 s and normalized to the peak fluorescence (F/Fpeak) (*p < 0.05 NG vs. HG; repeated two-way ANOVA). (C) Mean traces of mitochondrial Ca2+ uptake by mitochondria isolated from control rat hearts (NG; n = 35 experiments with 7 independent rats) and STZ-treated (HG) rat hearts (STZ; 40 mg/kg; n = 45 experiments with 9 independent rats). Mitochondrial Ca2+ uptake was induced by adding 2.5 μM of CaCl2 to mitochondria. Fluorescence was measured every 10 s and normalized to the peak fluorescence (F/Fpeak) (*p < 0.05 NG vs. HG; repeated two-way…

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  Fig. 4. Hyperglycemia effect on calcium homeostasis in response to stress. (A) Schematic illustration of the experimental protocol and representative traces of left ventricular (LV) pressure recording during ischemia and reperfusion. The blue arrows represent measurements of ischemic and early-reperfusion contracture. (B) Maximum ischemic and (C) early-reperfusion LV contracture in isolated hearts undergoing IR in NG (11 mM) or HG (33 mM) conditions (n = 8 NG hearts; n = 6 HG hearts; *p < 0.05 NG vs. HG; parametric t-test). (D) Representative trace of a Ca2+ transient obtained in paced (0.5 Hz, Ionoptix System) isolated cardiomyocytes loaded with the Fluo-4 Ca2+ sensitive dye (10 μM). (E) Amplitude of Ca2+ transients measured in isolated cardiomyocytes pre-incubated in NG (6 mM) or HG (33 mM) buffer, paced at 0.5 Hz for 30 s, and stimulated or not with ISO (1 μM) (n = 20–30 cells/condition from 4 hearts). (F) Ca2+ transient exponential decay time (Tau) measured in isolated cardiomyocytes incubated in NG (6 mM) or HG (33 mM) buffer for 45 min, paced at 0.5 Hz for 30 s and stimulated or not with ISO (1 μM) (n = 20–30 cells/condition from 4 hearts; #p < 0.05 ISO effect; repeated two-way…

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  Fig. 5. Effect of the DRP1 inhibitor Mdivi-1 on hyperglycemia-induced alterations of ROS production, calcium homeostasis, and mPTP opening in cardiomyocytes. (A) Cardiomyocytes isolated from 4 hearts/group were loaded with the ROS-sensitive fluorescent probe dihydro-rhodamine 123 (DHR123; 5 μM) and then incubated or not with Mdivi-1 (50 μM, 30 min), in NG (6 mM) or HG (33 mM) buffer, for 45 min. Cells were stimulated with H2O2 (100 μM) to mimic IR-induced oxidative stress. Fluorescence was measured every 10 s and normalized to baseline fluorescence (F/F0) (*p < 0.05 NG vs. HG; repeated two-way ANOVA). (B) Representative confocal line scan recording of Ca2+ transients and spontaneous Ca2+ waves obtained in isolated cardiomyocytes loaded with the intracellular Ca2+ indicator Fluo-4 AM (10 μM) and incubated in NG (6 mM) or HG (33 mM) buffer for 45 min. Cardiomyocytes were paced at 0.5 Hz (30s) and 2 Hz (30s). The stimulation was then stopped to record SCaW for 30 s. Cardiomyocytes were pre-incubated or not with Mdivi-1 (50 μM) for 30 min and stimulated or not with ISO (1 μM). Quantification of the amplitude of Ca2+ transients (C), exponential decay time (Tau) (D) and number of SCaW (E) in…

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  Fig. 6. Effect of the DRP1 inhibitor Midiv-1 on the hyperglycemia-induced heart sensitivity to IR. (A) Control rats (NG) and STZ-treated rats (HG) were treated or not with Mdivi-1 (1.2 mg/kg; intraperitoneal injection), 24 h and 1 h before in vivo IR (30 min of ischemia followed by 2 h of reperfusion). (B) Bood glucose levels before IR (N = 4–6 rats/group; *p < 0.05 NG vs. HG; two-way ANOVA). (C) Animal survival rate (no cardiac arrest before the reperfusion end) during IR (N = 4–6 rats/group). (D) Representative ECG recording before ischemia (baseline), after 30 min of ischemia (coronary artery ligation), and after reperfusion (90 min) in NG and HG rats pre-treated or not with Mdivi-1 (1.2 mg/kg; intraperitoneal injection) 24 h and 1 h before coronary artery ligation. (E) QTc values recorded at baseline and after 90 min reperfusion (N = 4–6 rats/group; *p < 0.05 NG vs. HG post-IR; repeated two-way ANOVA). (F) Representative transverse sections of triphenyl tetrazolium chloride-stained hearts from NG or HG rats treated or not with Mdivi-1 (1.2 mg/kg). The deep red visible area represents the area at risk (AAR), the white area is the infarcted area, and the blue area indicates…

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  Fig. 7. Hyperglycemia-induced DRP1 activation plays a central role in the deleterious impact of hyperglycemia on the heart sensitivity to IR. Hyperglycemia prior and during ischemia-reperfusion, increases interactions between the sarcoplasmic reticulum and mitochondria (1). This promotes both Ca2+ and ROS exchanges between these organelles (2), and reinforces the vicious circle between mitochondrial ROS production and RyR2-dependent Ca2+leak (3), to finally drives excessive mitochondrial fragmentation and early mPTP activation (4). Finally, DRP1, through its canonical and non-canonical roles, is proposed as a key trigger of these phenomena and plays then a key role in the higher sensitivity of the hyperglycemic heart to ischemia-reperfusion.