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 isolated cardiomyocytes (n = 15–30 cells/condition from 3 to 6 hearts; *p < 0.05 NG vs. HG, $p < 0.05 Mdivi-1 effect; repeated two-way ANOVA). (F–G), Mean traces of Ca²⁺ uptake by mitochondria isolated from NG rat hearts (n = 35 experiments from N = 7 hearts) or STZ-treated rat (NG) hearts (STZ; 40 mg/kg; n = 45 experiments from N = 9 hearts), treated or not with Mdivi-1, 24 h and 1 h before the experiment (NG Mdivi-1: n = 19 experiments from N = 4 hearts. HG Mdivi-1: n = 20 experiments from N = 3 hearts; $p < 0.05 Mdivi-1 effect, repeated two-way ANOVA). Mitochondrial Ca²⁺ uptake was stimulated by a single CaCl₂ pulse (2.5 μM) in isolated mitochondria in the presence of Ca²⁺ Green-5N (1 μM). Fluorescence was measured every 7 s and normalized to the peak fluorescence. (H) Mean CRC traces in mitochondria isolated from NG rats (n = 20 experiments from N = 5 hearts) or STZ-treated rats (HG; n = 32 experiments from 8 hearts), treated or not with Mdivi-1 (50 μM) (NG Mdivi-1: n = 16 experiments from N = 4 hearts; HG Mdivi-1: n = 12 experiments from N = 3 hearts). CRC was monitored using the Ca²⁺ Green-5N probe (1 μM). Each peak corresponds to Ca²⁺ addition (3 CaCl₂ pulses at 1 μM followed by pulses at 2.5 μM; changes in concentration are represented by black arrows). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)