Fig 2: Characterization of caveolae and Cav3 expression in WT, Cav3 P28L, and Cav3 R26Q myotubes. a Electron micrographs of WT, Cav3 P28L, and Cav3 R26Q myotubes. Caveolae, interconnected caveolae, and aberrant sized caveolae are indicated with black arrowheads, asterisks, and white arrowheads, respectively. b Quantification of the number of caveolae/µm2 in a. c Immunoblot analysis (lower panel) and quantification (upper panel) of total levels of Cav3 in WT, Cav3 P28L, and Cav3 R26Q differentiated myotubes. Tubulin serves as a loading control. Quantification of the expression of Cav3 by calculating the ratio between Cav3 and tubulin expression. d Immunofluorescent labeling of Cav3 and GM130 in WT, Cav3 P28L, or Cav3 R26Q myotubes analyzed by confocal microscopy. Arrows in inset indicate the plasma membrane and arrowheads indicate the Golgi complex. Cav1 staining is shown in Supplementary Fig. 1c. a Scale bar = 200 nm. Representative cells quantified in b (number of regions analyzed: WT = 115, P28L = 154, R26Q = 146; total area screened: WT = 1140 µm2, P28L = 1187 µm2, R26Q = 1216 µm2). Reproducibility of experiments: a Representative cells. a, c, d Representative data. b, c Quantification was done on 3 independent experiments. d Quantification was done on 3 independent experiments (WT n = 24 cells, P28L n = 27 cells, R26Q n = 17 cells). Mean value ± SEM. b, c Statistical analysis with a two-tailed unpaired t test. d Statistical analysis with a one-way ANOVA *P < 0.05; ***P < 0.001; ****P < 0.0001

Fig 3: Forced expression of caveolin-3 restores mitophagy and mitochondrial function after knockdown of RORα.A, quantitative RT-PCR for Cav-3 in H9c2-Flag/RORα4/HA cells. B, immunoblotting of cell lysates with (C) summary densitometry from H9c2-Flag/RORα4/HA cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h in the absence or presence of doxycycline (Dox) induction of RORα. D, H9c2-Flag/RORα4/HA cells with m-Raspberry-mito-7 with or without EGFP-Cav-3 plasmid transfection. E, citrate synthase assay for function mitochondrial mass. F, MitoSOX red for oxidative stress. G, caspase-3 activity assay for apoptosis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by unpaired t test (A) or one-way ANOVA with Tukey's post hoc test (C–G). Cav-3, caveolin-3; DAPI, 4′,6-diamidino-2-phenylindole; Dox, doxycycline; LC3, microtubule-associated protein 1A/1B-light chain 3.

Fig 4: Popdc1 density sedimentation in discontinuous sucrose gradients.(a) Western blot analysis of gradient fractions. Fraction 1 and fraction 12 represent the lowest and highest density, respectively. [+/+], WT; [−/−], Popdc1-null; MβCD, methyl-β-cyclodextrin; I/R, ischemia/reperfusion; Cx43, connexin43. The molecular weights of the reactive bands were ∼68 kDa, ∼22 kDa, and ∼43 kDa, for Popdc1, Cav3, and Cx43, respectively. (b) The relative distribution of cholesterol and Cav3 throughout gradient fractions 1–9. *P<0.05, Cav3 in fraction 5, Popdc1-null vs. WT; #P<0.05, cholesterol in fraction 5, Popdc1-null vs. WT.

Fig 5: Impairment of caveolar invagination upon syndapin III KO in heart tissue has no consequences on cardiac integrity.(a–c) Wheat germ agglutinin stainings of 8 µm cryosections of hearts of 20 weeks old syndapin III KO mice (b) show no signs of cellular damage or alteration of cross-sectional areas of cardiac myocytes when compared to WT (a). Bars, 50 µm. Quantitative data (c) represent mean ± SEM. WT, 273 cells, 6 animals; KO, 378 cells, 7 animals. Statistical significance was tested using two-tailed Student’s t test. p=0.7462 (n.s.). (d,e) Ratios (x1000) of heart and body weights of about 4-month-old female (d) and male (e) mice. WT, eight female and male mice each; KO, seven female and nine male mice. Statistical significance, two-tailed Student’s t test. Female mice, p=0.3132 (n.s.); male mice, p=0.1377 (n.s.). (f–h) H&E stainings of WT and syndapin III KO heart cryosections show no left ventricular wall thickening upon syndapin III KO. Bars, 500 µm. Quantitative data represent mean ± SEM. WT, seven animals; KO, nine animals. Statistical significance, two-tailed Student’s t test. p=0.9451 (n.s.). (i–l) H&E stainings of paraffin sections of aged WT and syndapin III KO myocard (three animals each) display no signs of cellular damage or of necrosis irrespective of whether mice were trained or not. Bar, 50 µm. (m,n) Details of wide-field views of P-faces of freeze-fractured heart tissues double-immunogold labeled for cav3 and syndapin III. Note that, also in heart tissue, the formation of caveolae is impaired upon syndapin III KO. In WT, syndapin III and cav3-immunopositive caveolae are marked (long black arrows; shallow, cav3-positive indentation, short grey arrow). Bar, 100 nm. (o,p) Quantitative determinations of caveolae (o) and of shallow, cav3-positive membrane indentations (p) in WT and syndapin III KO hearts. (q) Quantitative assessment of the anti-cav3 labeling densities at membranes of freeze-fractured syndapin III KO hearts relative to WT (n.s.). (r) Quantitative western blotting of TritonX-100-resistant membrane fractions from WT and syndapin III KO hearts showing unchanged cav3 levels. 25 µg protein loaded each. n = 6 each genotype. (s,t) Quantitative assessment of the cav3 immunolabeling at caveolae (s) and shallow indentations (t). Note that the observed decreases are in accordance with the reduced abundance of these structures (compare s,t) vs. (o,p). (o–q, s,t) WT, 72.7 µm2 membrane from 21 images; KO, 72.7 µm2 membrane from 21 images from two independent cardiomyocyte preparations (from two hearts) each genotype. In total, 107 cav3-positive structures scored. Data, mean ± SEM. Statistical significance, two-tailed Student’s t test.10.7554/eLife.29854.029Figure 9—source data 1.This spreadsheet contains the data for all quantitative evaluations shown in the different panels of Figure 9 (i.e. of Figure 9c–e,h and o–t).