The Caveolin-3 G56S sequence variant of unknown significance: Muscle biopsy findings and functional cell biological analysis

Figures

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  Figure 1. Analysis of patient material. Microscopical investigations (1A): (I) M. vastus lateralis biopsy of patient 1 showing a myopathic pattern with minor muscle fiber atrophy and hypertrophy as well as frequent nonsubsarcolemmal nuclei (arrows). Cryostat section, H&E. Scale bar: 80 μm. (II) Low‐grade muscle fiber atrophy and solitary rimmed vacuole (arrow) in cryostat section of the rectus femoris muscle biopsy of patient 2. Cryostat section, H&E. Scale bar: 50 μm. (III) M. quadriceps femoris biopsy of patient 3 showing minor atrophic as well as hypertrophic muscle fibers. Cryostat section, H&E. Scale bar: 80 μm Electron microscopic studies (1B): (I) Subsarcolemmal membrane‐bound vacuoles, presumably corresponding to enlarged caveolae (arrows) in the muscle biopsy of patient 1. Scale bar: 250 nm. (II–VI) Patient 2; (II) subsarcolemmal membrane‐bound vacuoles (arrows) similar to the one depicted in Fig. 1A. Scale bar: 200 nm. (III) Further abnormal membrane bound subsarcolemmal vacuoles as well as tubular structures (arrows). Scale bar: 2 μm. (IV) Large vacuole (asterisk) associated with a myonucleus. Scale bar: 4 μm. (V) Large (asterisk) and small (arrowheads) vacuoles in the vicinity…

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  Figure 2. Analysis of human RCMH cells transfected with wild‐type Caveolin‐3, R26Q or G56S Caveolin‐3 constructs. After transient transfection of RCMH cells with the wt Caveolin‐3‐HA constructs, the HA antibody mainly detects Caveolin‐3 protein at the sarcolemma (green fluorescence, arrows). After transfection of the R26Q Caveolin‐3‐HA plasmid Caveolin‐3 is mainly detectable in the Golgi (arrow). The transfection of the G56S Caveolin‐3‐HA construct, in contrast, led to a mislocalization of the mutated protein to the Golgi (gm130: red fluorescence) while some of the protein is still located at the sarcolemma (arrows). Green: Caveolin‐3‐HA, red: Golgi. White arrows: localization of Caveolin‐3. Scale bar: 10 μm. The assays were performed at least for three times with similar results.

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  Figure 3. Electron microscopic findings in human RCMH myoblasts transfected with wild‐type or G56S Caveolin‐3 constructs. EM of RCMH cells transfected with wt Caveolin 3 (A–B) or the G56S mutant form (C–L): (A) A representative RCMH cell overexpressing wt Caveolin‐3 presents with proliferated, dilated endoplasmic reticulum (ER) partially filled with osmiophilic material. Scale bar: 2.0 μm. (B) ER structures at a higher magnification within another myoblast (black arrows). Golgi cisternae and vesicles are marked by white arrows. Scale bar: 0.5 μm. (C) RCMH myoblast transfected with G56S Caveolin‐3: Large subplasmalemmal vacuoles, one of them marked by an asterisk. Scale bar: 0.5 μm. (D) Osmiophilic, often membranous material localized within enlarged ER structures and autophagic vacuoles (arrows). Scale bar: 0.5 μm. (E) Proliferated and widened Golgi structures within the same RCMH cell. Scale bar: 0.5 μm. (F) Autophagic vacuoles also within the same cell. Scale bar: 0.5 μm (G) Widened Golgi structures within a different RCMH cell. Scale bar: 0.5 μm. (H) Autophagic vacuole (arrow) filled with membranous material adjacent to widened ER. Scale bar: 0.5 μm. (I) Widened ER containing membranous…

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  Figure 4. Analysis of EGFR‐ and downstream signaling after transfection of mutated Caveolin‐3 constructs into NR6W cells. (A) Immunoblots detecting phosphorylated EGFR (pEGFR), phosphorylated Akt (pAkt), and phosphorylated ERK1/2 (pERK1/2). Transfection of the R26Q and G56S constructs leads to lower phosphorylation levels of EGFR. Lysates of unstimulated NR6W cells transfected with the same constructs were used as negative controls. As expected, these cells show minimal levels of pEGFR. Transfection of R26Q and G56S has no influence on phosphorylation levels of Akt and ERK1/2. Loading control: Caveolin‐3 using the antibody against HA and tubulin. (B) Quantitative assessment of three Immunoblots. Statistical significance was determined by the Kruskal–Wallis test followed by the Dunn's comparison test. A p‐value ≤ 0.05 was considered statistically significant. Data were shown as mean values ± SEM. *Statistically significant (p‐value ≤ 0.05).

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  Figure 5. Analysis of RCMH cells transfected with wild‐type or G56S Caveolin‐3 and treated with osmotic stress. (A) RCMH myoblast cells were transfected with either the wt or G56S Caveolin‐3 and were treated with 30% hypoosmotic stress medium for 30 min or were left unstressed. The medium was replaced by normal DMEM containing 10% FCS. Both wt and G56S Caveolin‐3 cells started swelling during the 30 min of stress. After the shift to normal culture medium, wt cells remained distended within the analyzed 120 min. Shortly after the shift to normal culture medium, the G56S Caveolin‐3 cells started to rupture. One hundred twenty minutes after osmotic stress, cells were prepared for quantitative label‐free MS. Each condition was measured in triplicate in randomized order; four representative base peak chromatograms are shown. Only proteins that were quantified with at least two unique peptides (at 1% false discovery rate) between all samples were considered to represent proteins that show significant regulation. Among these, microtubule‐actin cross‐linking factor 1 (Q9UPN3) showed a significant regulation upon both, G56S Caveolin‐3 overexpression and osmotic stress burden. (B) logFC fold changes…