Fig 2: Role of SLC25A46 in mitochondrial recruitment of phosphorylated forms of DRP1Immunoblot analysis of the localization of phosphorylated forms of DRP1 in control and subject fibroblasts after subcellular fractionation. Upper panel: DRP1 phosphorylated on serine 637. Bottom panel: DRP1 phosphorylated on serine 616. ITPR1 was used as an ER marker, VDAC1 as a mitochondrial marker, and alpha-tubulin as a cytosolic marker.Source data are available online for this figure.

Fig 3: Muscle-specific reduction in Drp1 reduces maximal running speed and muscle endurance capacity in female mice. (A–B) Immunoblots and densitometric analysis of skeletal and cardiac muscle Drp1 total protein in Control and mDrp1+/- mice (representative immunoblots N = 4 per genotype; densitometry N = 8; two-way unpaired t-test). (C) Maximum running speed (m/min), and (D) latency to fall (seconds; N = 3 with three independent trials/per animal) in SED Control (open bars) and mDrp1+/- (closed bars) mice (N = 7–9; two-tailed unpaired t-test). (E) Plasma lactate (mmol/l) and (F) muscle glycogen (mg/g) from quadriceps muscle of control (open bars) and mDrp1+/- (closed bars), SED vs. EX90 mice using repeated measures ANOVA and two-way ANOVA with Tukey's post hoc analysis respectively. (G–H) Quadriceps gene expression in Control (open bars) and mDrp1+/- (closed bars) mice after EX90 (two-tailed unpaired t-test). (I) Mitochondrial DNA copy number in quadriceps muscle of SED and EX90, Control (open bars) and mDrp1+/- (closed bars) mice (two-way ANOVA with Tukey post hoc analysis). (J–O) Inmmunoblots and densitometric analysis of protein and phospho-protein expression of select fission, fusion, and autophagy markers from quadriceps muscle of SED and EX90, Control (open bars) and mDrp1+/- (closed bars) mice (two-way ANOVA with Tukey's post hoc analysis). Data are means ± SEM. N = 6–8/group. *, P < 0.05 vs. control SED. #, P < 0.05 SED vs. EX90 for mDrp1+/-.

Fig 4: Orai1 might contribute to HG-induced cardiomyocyte hypertrophy via activation of CnA, ERK-Drp1 pathways.A Changes in CnA levels in NRCMs treated with 10 µM BTP2 (n = 4). B Changes of p-ERK1/2 and total ERK1/2 protein levels in NRCMs treated with BTP2 10 µM (n = 3). C Western blotting images and summarized data showing p-Drp1S616 (n = 4) and p-Drp1S637 (n = 3) protein levels in NRCMs treated with 10 µM BTP2. D Changes of p-Drp1S616 (n = 4) and p-Drp1S637 (n = 4) levels in NRCMs infected with sgOrai1 at 80 MOI for 72 h. The protein levels of phospho-Drp1 (S616/S637) were normalized by total Drp1. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; n.s: no significant statistical difference.

Fig 5: Chronic exercise training increases pro-fusion phosphorylation of Drp1 in post-exercise recovery period (Experiment 2). (A) Body and tissue weights (quadriceps, gastrocnemius, gonadal WAT, inguinal WAT, and heart) in SED and TRN female mice (N = 6/group; two-way repeated measures ANOVA with Tukey post hoc analysis for left panel and; two-tailed unpaired t-test for right panel). (B) Quadriceps mRNA abundance (presented in a heat map), representing mitochondrial fission, fusion, and autophagy signaling, in SED vs. TRN (N = 6/group; two-tailed unpaired t-test). Muscles were harvested from TRN mice 30 h following the cessation of exercise including a 6 h fast. (C–E) Immunoblot densitometry of quadriceps protein and phospho-protein abundance representing mitochondrial fission, fusion, and autophagy signaling, in SED (open bars) and TRN (closed bars) mice (N = 6/group) (representative immunoblots presented in Supplemental Figure 1). (F) Mitochondrial proteomics determined by mass spectrometry analysis of gastrocnemius muscle and presented as a volcano plot (dashed line indicates significance threshold; blue dots indicate significance, N = 4/group; two-tailed unpaired t-test with Bonferroni correction). (G) Mitochondrial proteomics pathway analysis (two-tailed unpaired t-test), and (H) mitochondrial functional annotation of significantly impacted protein clusters. Data are means ± SEM. *, P < 0.05 vs. SED.