Coupling of excitation to Ca2+ release is modulated by dysferlin

Abstract

Key points: Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca²⁺ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin-null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L-type Ca²⁺ channels protect voltage-induced Ca²⁺ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca²⁺ -free medium and S107, which inhibits SR Ca²⁺ leak, suggest the SR as the primary source of Ca²⁺ responsible for the loss of the Ca²⁺ transient upon injury. Ca²⁺ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury-induced SR Ca²⁺ leak.

Abstract: Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca²⁺ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage-induced Ca²⁺ transients elicited in dysferlin-null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus-dysferlin chimeras in A/J fibres restored the full amplitude of the Ca²⁺ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L-type Ca²⁺ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca²⁺ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI-induced damage suggests that damage is mediated by increased Ca²⁺ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca²⁺ sparks and Ca²⁺ waves. S107 (a stabilizer of RyR1-FK506 binding protein coupling that reduces Ca²⁺ leak) or local expression of Venus-dysferlin prevented OSI-induced Ca²⁺ waves. Our data suggest that dysferlin modulates SR Ca²⁺ release in skeletal muscle, and that in its absence OSI causes increased RyR1-mediated Ca²⁺ leak from the SR into the cytoplasm.

Keywords: dysferlin; excitation-contraction coupling; skeletal muscle.

Figure 1. Amplitude of the voltage‐induced Ca²⁺ transients in A/J and in A/W fibres with and without transfection

A and B, representative line‐scan confocal images of voltage‐induced Ca²⁺ transients (Rhod‐2 fluorescence) in A/W (A) and A/J (B) FBD fibres. Bars, 100 μm (vertical) and 250 ms (horizontal). C, corresponding profiles of voltage‐induced Ca²⁺ transients from the images A and B presented as (F _max − F _o)/F _o. Black line, A/W; grey line, A/J. D, averaged amplitude of voltage‐induced Ca²⁺ transients in A/W and A/J fibres, presented as (F _max − F _o)/F _o. Number of experiments is in white; ^* P < 0.001 compared to A/W. E and F, representative x–y confocal fluorescence images of A/J FBD fibres after transfection with Venus (E) or V‐Dysf (F). G, representative profiles of voltage‐induced Ca²⁺ transients from fibre areas transfected with V‐Dysf (black line) or non‐transfected (grey line). The voltage‐induced Ca²⁺ transients are normalized to F _max in the area transfected with V‐Dysf. H, averaged amplitude of voltage‐induced Ca²⁺ transients in A/W and A/J fibres presented as (F _max − F _o)/F _o. Number of experiments is in white; ^* P < 0.05 compared to V‐Dysf; bars, 10 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2. Effects of Ca²⁺ channel inhibitors on voltage‐induced Ca²⁺ transients in A/W and A/J fibres

A, effects of diltiazem on voltage‐induced Ca²⁺ transients in A/W (black circles) and A/J (grey circles) FDB fibres. B, effects of diltiazem on voltage‐induced Ca²⁺ transients in A/J (grey circles) fibres and A/J muscle fibres transfected with wild‐type dysferlin (black open circles). C–F, dose‐dependent effects of verapamil (C), nifedipine (D), dantrolene (E) and tetracaine (F) on voltage‐induced Ca²⁺ transients in A/W (black circles) and A/J (grey circles) fibres. Number of experiments are given for corresponding colour; ^* P < 0.05 to corresponding value at the same concentration.

Figure 3. Effect of osmotic shock on Ca²⁺ transients in FBD fibres

A, C and E, representative line‐scan confocal images of voltage‐induced Ca²⁺ transients in A/W (A) and A/J (C) FBD fibres and A/J fibres transfected with V‐Dysf (E) before and 5 min after OSI. Distribution of V‐Dysf, introduced by electroporation and seen as Venus fluorescence, is shown on x–y images located on the right of panel E. B and D, corresponding profiles of voltage‐induced Ca²⁺ transients from the images presented in A and C for pre‐OSI (black line) and 5 min after OSI (grey line). Transients are normalized to F _max for pre‐OSI conditions. F, averaged data for recovery of Ca²⁺ transients in FBD fibres under the different conditions used. Number of experiments in white; ^* P < 0.005 compared to A/W; bars, 100 μm (vertical) and 250 ms (horizontal).

Figure 4. Effects of pharmacological agents on recovery of voltage‐induced Ca²⁺ transients in A/J fibres after OSI

A, averaged data for recovery of Ca²⁺ transients in FBD fibres in the presence of 10 μm diltiazem (Dilt.), 10 μm verapamil (Verap.) or 5 μm nifedipine (Nifed). B, averaged data for recovery of Ca²⁺ transients after OSI in FBD fibres in the presence of 10 μm dantrolene (Dantr.), 1 μm tetracaine (Tetrac.) or 10 μm S107. Number of experiments is shown in white; ^* P < 0.05, compared to A/J. The two bars to the left duplicate those in A. C, averaged data for recovery of voltage‐induced Ca²⁺ transients during wash‐out of 10 μm tetracaine for A/W (no OSI; black circles) and A/J (no OSI; grey circles) fibres, and for A/J fibres after OSI (open circles). Data points are means of 6–27 independent experiments.

Figure 5. Effect of Ca²⁺‐free Tyrode solution on recovery of voltage‐induced Ca²⁺ transients from OSI in A/J fibres

A, representative line‐scan confocal images of the voltage‐induced Ca²⁺ transients in A/J FBD fibres before and after OSI in nominally Ca²⁺‐free Tyrode solution. B, corresponding profiles of transients from the images presented on A for pre‐OSI (black) and 5 min after OSI (grey) conditions. Voltage‐induced Ca²⁺ transients are normalized to F _max for pre‐OSI conditions. Bars, 100 μm (vertical) and 250 ms (horizontal).

Figure 6. Ca²⁺ waves in A/J fibres after OSI

A, representative line‐scan confocal images of the voltage‐induced Ca²⁺ transients and Ca²⁺ waves in A/J fibres before and after OSI. Time (in minutes) after OSI is shown between panels. B, enlarged images of Ca²⁺ signals, reoriented with the initial time of stimulation at the upper left of each panel. Bars: left panel, 100 μm (vertical) and 250 ms (horizontal); right panel, 100 ms (vertical) and 25 μm (horizontal).

Figure 7. Spontaneous Ca²⁺ waves and sparks in A/J fibres after OSI

A, representative x–y scans of confocal images of spontaneous Ca²⁺ wave in A/J fibres after OSI as a function of time (in seconds). Bar, 10 μm. B, line‐scan confocal images of the voltage‐induced Ca²⁺ transients, and voltage‐induced (yellow arrows) and spontaneous (white arrows) Ca²⁺ sparks and bursts (green arrows) visualized at 9 min after OSI. The arrowhead indicates a location at which sparks arise repeatedly. Bars, 20 μm (vertical) and 250 ms (horizontal). C, representative Ca²⁺ sparks and bursts shown at higher magnification at 15 min after OSI. On the right are plotted time‐dependent changes in [Ca²⁺], recorded by averaging a 2 μm line at sites indicated by arrows. Bars, 10 μm (vertical) and 300 ms (horizontal). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 8. V‐Dysf suppresses the development of Ca²⁺ waves in A/J fibres after OSI

Representative line‐scan confocal images of the voltage‐induced Ca²⁺ transients and Ca²⁺ waves in A/J fibres showing regional expression of V‐Dysf before and 5 min after OSI. Distribution of V‐Dysf, seen as Venus fluorescence, is shown on x–y images to the right. Bars, 100 μm (vertical) and 250 ms (horizontal). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 9. Evidence against proteolysis in A/J fibres

A, representative line‐scan confocal images of the voltage‐induced Ca²⁺ transients and Ca²⁺ waves before and 5 min after OSI in A/J fibres pretreated for 1 h with 50 μm calpeptin. B, averaged data for recovery of Ca²⁺ transients in A/J FBD fibres pretreated and not pretreated with 50 μm calpeptin in comparison to A/W FBD fibres. Number of experiments is shown in white; ^* P < 0.005 compared to A/W. Bars, 100 μm (vertical) and 250 ms (horizontal). C, representative SDS‐PAGE shows amount of junctophilins 1 or 2 (JPH1 or JPH2) in A/J and A/W muscle fibres. Molecular mass markers, in kDa, are indicated. Glyceraldehyde phosphate dehydrogenase is the loading control, at 36 kDa.