Fig. 7
From: DRP1-mediated mitochondrial shape controls calcium homeostasis and muscle mass
Acute DRP1 inhibition alters calcium homeostasis. a Cytosolic calcium level at rest is not affected in KO muscles (40 days: WT, n = 24; KO, n = 28; 70 days, n = 19 fibers). b Amplitude of cytosolic Ca2+ transients induced by 0.5 Hz electrical pulses is lower in Drp1−/− mice (40 days, WT, n = 25; KO, n = 28 fibers; 70 days, WT, n = 33; KO, n = 29 fibers). c Cytosolic calcium transients induced by caffeine–thapsigargin are strongly reduced (40 days, WT, n = 10; KO, n = 14 fibers; 70 days, WT, n = 13 fibers; KO, n = 14 fibers). d Mitochondrial matrix-free calcium concentration is not affected at rest but is higher in Drp1−/− compared with control during a train of electrical stimulation at 60 Hz, 2 s duration (WT, n = 9; KO, n = 7 fibers; Peak, n = 6 each condition). e Immunoblot analysis of isolated mitochondria. MCU levels were normalized to TOM20 levels. f Quantitative RT-PCR analysis showing a significant decrease of miR1 levels in Drp1−/− skeletal muscle (n = 4 per condition). The data represent average ± SEM. g Ultrastructure and geometry of CRUs in DRP1 KO muscles. Inserts A and B show T-tubule staining in WT (A) and in DRP1 KO (B); the frequency of oblique/longitudinal tubules increases (stars) when compared with control samples. Inserts C–F are representative EM pictures showing the structural geometry of a normal, an oblique, a longitudinal triad and of a dyad respectively. In inserts A and B, T-tubules are stained in black while in inserts C–F, T-tubules has been false-labeled in green. h Cytosolic calcium transients induced by caffeine–thapsigargin after the reintroduction of DRP1 (Drp1-YFP) in muscle fibers. Cytosolic calcium transients in Drp1−/− in the presence of Drp1-YFP show no difference compared with control fibers (Drp1fl/fl, n = 22; Drp1fl/fl—Drp1-YFP, n = 7; Drp1−/−, n = 22; Drp1−/− – Drp1-YFP, n = 13). i Inhibition of mitochondria Ca2+ uptake via MCU knockdown restored cytosolic Ca2+ levels during caffeine–thapsigargin treatment (WT, n = 17 fibers; WT shMCU, n = 23 fibers; KO, n = 27 fibers; KO shMCU, n = 17 fibers). j MCU inhibition reduces centrally nucleated fibers in KO muscles. For all experiments a minimum of three mice was used for each condition. Two-tailed unpaired Student’s t test and two-way analysis of variant (ANOVA) were used. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
