Fig 2: TIRF measurements of Ca2+ influx induced by 60 mM KCl in human myotubes. A: Myotubes from a control patient were visualized by brightfield (top left panel), with a surface reflection interference contrast (SRIC) filter to visualize and fix the focal plane of the coverglass/cell membrane interphase (top central panel). Next panels show pseudocolored ratiometric images (peak fluorescence after addition of KCl/ resting fluorescence) of fluo-4 fluorescence changes at the indicated time-points after application of KCl. Fluorescence was monitored through a 60 × TIRF objective and analyzed using Metamorph as detailed in the Methods section. Bar indicates 30 µm. B: Representative traces from ECCE showing changes in fluo-4 fluorescence in a myotube from a control individual (_____) and a patient harbouring a CAV3 mutation (…….) stimulated with 60 mM KCl in the presence of 2 mM Ca2+ or myotubes from a control in the absence of added Ca2+ and in the presence of 100 µM La3+ (.-.-.-.-.). TIRF measurements were performed as indicated in the Methods section in myotubes pretreated with 100 µM ryanodine. C: Bar graph depicting mean (± SEM) peak increase of fluo-4 fluorescence induced by 60 mM KCl in control and caveolin-3 deficient myotubes in the presence of 2 mM Ca2+. Insert shows the mean (± SEM) peak fluo-4 fluorescence increase of control myotubes in the presence of 100 µM La3+ (gray bar) or 2 mM Ca2+ (empty bar).

Fig 3: Voltage-dependence of Ca2+ transients in control and RMD myotubes. Cells were patch-clamped and held at a holding potential (VH) of - 80 mV. A: Paired sample traces of current (lower trace) and Ca2+ release recordings (upper trace) at different test potentials (from - 40 to 0 mV) in a fluo-3 loaded control myotube. Depolarizations (50 msec) to increasing membrane potentials activated Ca2+ release from the SR.B: Comparison of Ca2+ release during a depolarizing step to - 20 mV in control (black) and Cav-3 deficient RMD (red) myotubes. Linescan images and line profiles show the reduced Ca2+ transient amplitude in caveolin-3 deficient RMD myotubes at same trigger voltage when compared with control. C: Summary of the voltage-dependence of Ca2+ release in control (WT, n = 10) and caveolin-3-deficient RMD myotubes (n = 8). Ca2+ transient amplitudes have been normalized to the maximal release amplitude in each cell. Membrane potentials at half-maximal activation (V1/2) indicate a right shift of the voltage-dependence in RMD myotubes (P<0.05).

Fig 4: Characterization of cytoplasmic Ca2+ homeostasis in myotubes with CAV3 deficiency. Calcium imaging was performed in fura-2 loaded myotubes as described in the Methods section. A: Mean (± SEM of n = 58 and 92 for control and RMD, respectively) resting [Ca2+] (expressed as fluorescence intensity ratio 340/380 nm) was not different in control and RMD myotubes. B: Mean (± SEM) peak Ca2+ increase induced by the addition of 100 mM KCl (inducing depolarization) and 600 µM 4-chloro-m-cresol (4-cmc, RyR1 agonist) in the presence of Krebs-Ringer medium (KR) containing 100 µM La3+. Open boxes, control myotubes; gray boxes, myotubes from CAV3 deficient myotubes (n = 7–15 measurements). C: Traces showing fura-2 change in fluorescence (ratio 340/380 nm) of individual myotubes from a control and a CAV3 mutation-bearing patient in response to 100 mM KCl and 600 µM 4-cmc.