Muscle-specific Ryanodine receptor 1 properties underlie limb-girdle muscular dystrophy 2B/R2 progression

Abstract

Ryanodine receptor 1 Ca²⁺ leak is a signal in skeletal muscle, but chronic leak can underlie pathology. Here we show that in healthy male mouse, limb-girdle muscle presents higher sympathetic input, elevated ryanodine receptor 1 basal phosphorylation, Ca²⁺ leak and mitochondrial Ca²⁺ content compared to distal leg muscles. These regional differences are consistent with heat generation in resting muscle to maintain core temperature. The dysferlin-null mouse develops severe pathology in the limb-girdle but not leg muscles. Absence of dysferlin disrupts dihydropyridine receptors' inhibitory control over ryanodine receptor 1 leak, synergistically increasing leak through the already phosphorylated channel of limb-girdle muscle. This alters Ca²⁺ handling and distribution leading to reactive oxygen species production prior to disease onset. With age, oxidation of Ca²⁺ -handling proteins in dysferlin-null limb-girdle muscle alters basal Ca²⁺ movements. Our results show that muscle-specific pathology in dysferlin-null mice is linked to increased ryanodine receptor 1 Ca²⁺ leak.

Fig. 1. Dysferlin deficiency exacerbates intrinsic differences in RyR1 leak between Psoas and TA prior to the development of dystrophic phenotype.

a H&E staining of WT and BlaJ muscles (8 and 36 weeks-old mice). b Fibrosis area calculated as percentage of total cross-sectional muscle area. n = 3–8. c Muscle weights from WT and BlaJ mice. n = 6–14. d Diagram showing t-system-trapped rhod-5N to measure RyR1 Ca²⁺ leak. RyR1 Ca²⁺ leak sets the [Ca²⁺]_JS above the bulk cytosol. [Ca²⁺]_JS directly influences PMCA activity and [Ca²⁺]_t-sys. When RyR1 leak is inhibited, [Ca²⁺]_t-sys drops proportional to the drop in [Ca²⁺]_JS. e, f Representative [Ca²⁺]_t-sys (t) during exposure to 1 mM Tetracaine in a WT TA (e) and WT psoas (f) fibre. h Steady-state [Ca²⁺]_t-sys across a range of [Ca²⁺]_cyto in presence (g) and absence (h) of RyR1 leak. n = 5–17 fibres. i RyR1 Ca²⁺ leak by means of Δ[Ca²⁺]_t-sys ([Ca²⁺]_t-sys before – after 1 mM tetracaine). n = 5–13 fibres. j (Top) Bright field image of two fibres (TA and psoas, both WT) aligned for simultaneous imaging. Scale bar: 400 µm. (Bottom) Spatially averaged Rhod-2 (cytosolic) profiles from a TA (orange) and a psoas (blue) fibre upon exposure to increasing [Caffeine]. k Ca²⁺ wave frequency across a range of [Caffeine]. n = 7 preparations. l. RyR1 phosphorylation (Ser2844) relative to non-phosphorylated RyR1. Data normalized to WT TA. Data are presented as mean +/− SD (b, c, l) or +/− SEM (g, h, i, k). e–l 8 weeks-old groups. Statistical analysis: (b, c), one-way ANOVA with Tukey’s multiple comparison. i two-way ANOVA. (#: Two significant results; &: Five significant results). (k), paired T-test. (l), unpaired (multiple) t-tests. ****p < 0.0001, **p < 0.01, *p < 0.05. Source data are provided as a Source Data file.

Fig. 2. Lack of dysferlin synergistically increases RyR1 leak via DHPR destabilization and RyR1 phosphorylation.

a, b Representative [Ca²⁺]_t-sys(t) in WT (a) and BlaJ (b) psoas fibre during sequential perfusion of nifedipine (5 mM) and tetracaine (1 mM). c Percentage of nifedipine-sensitive (black) and nifedipine-insensitive RyR1 leak WT and BlaJ psoas fibres. n = 5–8. d T-system rhod-5N confocal image of two TA fibres placed in parallel to each other for simultaneous live imaging. Scale bar: 100 µm. e Representative simultaneous [Ca²⁺]_t-sys traces from WT and BlaJ TA fibres exposed to cAMP (100 mM) and tetracaine (1 mM) subsequently. f Tetracaine-induced Δ[Ca²⁺]_t-sys of cAMP-exposed WT and BlaJ TA fibres. Summary of results shown in (e). n = 7. a–f 8 weeks-old fibres. Statistical analysis in (c), unpaired t-test. Statistical analysis in (f), paired t-test. **p < 0.01. Source data are provided as a Source Data file.

Fig. 3. Differences in intracellular Ca²⁺ distribution across WT TA and psoas increase in absence of dysferlin.

a Representative BAPTA-lysis experimental trace. A mechanically skinned mouse psoas fibre segment with endogenous resting Ca²⁺ content was depleted of SR calcium and then equilibrated with known free [BAPTA]. TX-Oil-induced lysis produces ~45% of the maximal force. Force response (relative to maximal response) in conjunction with pCa and known [BAPTA] employed, enables quantification of total Ca²⁺ released upon lysis. b, c Endogenous Ca²⁺ content (SR, b; mitochondrial, c) in TA and psoas fibres from WT and BlaJ muscles. n = 9–13. Data presented as Mean +/− SD. d Estimation of resting [Ca²⁺]_cyto across TA and psoas (WT and BlaJ) derived from total mitochondrial Ca²⁺. e Estimation of total cytosolic Ca²⁺ bound to cytosolic Ca²⁺ buffers. Mathematical model obtained from Lamboley et al.. f Ca²⁺ content (per fibre volume) of the major subcellular calcium compartments in TA and psoas (WT and BlaJ), shown as cumulative histograms. a–f 8 weeks-old. Statistical analysis in b and c were performed with one-way ANOVA with Tukey’s multiple comparison. ****p < 0.0001, *p < 0.05. Source data are provided as a Source Data file.

Fig. 4. Psoas exhibits a distinctive thermogenic program compared to TA.

a Western blot against p-PKA substrates (left) and Ponceau-S signal (right) in TA and psoas muscle homogenates (8 w/o, WT). b Densitometry of anti p-PKA substrate antibody signal normalised to Ponceau-S signal. n = 9. c Quantification of norepinephrine concentration in TA and psoas (8 w/o, WT) n = 4. d (Top) Venn diagram showing identified proteins in WT psoas and WT TA by Data-Dependent Acquisition (DDA) workflow. (Bottom) Kegg pathways represented by the WT psoas and WT TA-specific proteins identified through DDA workflow. Terms were filtered by the criteria of ≥ 10 proteins and adjusted p-value (Benjamini) < 0.05. e TA and psoas-exclusive biological processes revealed by Gene Ontology (GO) enrichment analysis of 180 most abundant proteins in WT TA (top) and WT psoas (bottom) (8 weeks old) revealed by SWATH workflow. Terms were filtered by the criteria of ≥ 6 proteins and adjusted p-value (Benjamini) < 0.05. f Volcano plot of total quantified proteins showing significantly increased (blue) protein content in WT psoas relative to WT TA. g GO biological process analysis of upregulated proteins in WT psoas relative to WT TA. Terms filtered by adjusted p-value (Benjamini) < 0.05. h, i Metabolites ratio (Lactate/Pyruvate, h Glutamate/a-ketoglutarate, i present in WT TA and psoas (8 weeks old). n = 5. j Simultaneous confocal imaging of 150 nM Tetramethylrhodamine, Ethyl Ester (TMRE) uptake in a WT TA and a psoas fibre. Scale bar = 50 µm. k TMRE fluorescence profile over time in TA (orange) and psoas fibre (blue) (derived from j). l TMRE intensity after plateau in TA and psoas fibres (normalised to TA). m TMRE halftime in TA and psoas fibres (normalised to TA). n = 11. Statistical analysis in (b, c, l, m) were performed with paired T-test. **p < 0.01, *p < 0.05. Statistical analysis in h&i were performed with an unpaired one-tailed T-test. Source data are provided as a Source Data file.

Fig. 5. Lack of dysferlin drives oxidation-dependent t-system Ca²⁺ handling changes in a muscle-specific fashion.

a T-system-trapped rhod-5N signal over time during fibre Ca²⁺ extrusion and SOCE. b [Ca²⁺]_t-sys(t) profile from fibre displayed in (a). c, d Representative Ca²⁺ extrusion transients from 8 w/o WT and BlaJ TA (c) and psoas (d) fibres. e, f Representative SOCE transients from 8 w/o WT and BlaJ TA (e) and psoas (f) fibres. (c–f) transients are superposed for visual comparison). g Summary of Ca²⁺ extrusion rate constants derived from exponential fitting. h Summary of SOCE rate constants derived from exponential fitting. i Rhod-5N_t-sys confocal image of two psoas fibres (WT and BlaJ) positioned in parallel. j Rhod-5N profile from WT (black) and BlaJ (magenta) psoas fibre during exposure to 30 mM caffeine and calcium removal (release solution). k transients shown in (j) on an expanded timescale. Rhod-5N signal converted to standard score values derived from 5 s baseline. l. Summary of experiments presented in (k). Delay in SOCE activation; comparison between BlaJ vs WT (n = 18) against WT vs WT fibres (n = 19). m Fluo-5N_SR confocal image of two psoas fibres (WT and BlaJ) positioned in parallel. n Real-time [Ca²⁺]_SR in WT (black) and BlaJ (green) psoas fibre during exposure to release solution. o transients shown in n on an expanded timescale. Fluo-5N signal converted to standard score values derived from 5 s of baseline. p Comparison of BlaJ-to-WT delay in SR depletion (o) (n = 8) vs Ca²⁺ entry (n = 18) (k). q Rhod-5N_t-sys confocal image of three fibres (BlaJ, WT and BlaJ (NAC)) positioned in parallel. r Rhod-5N profile from WT (black) and BlaJ (pink, -NAC; yellow, +NAC) psoas fibres during exposure to release solution. s transients shown in r on an expanded timescale. t Summary of experiments shown in (s) (n = 15). SOCE activation threshold in WT served as reference value to define the effect of NAC on BlaJ-to-WT SOCE activation delay. Frame acquisition: 37 fps (j, n), 20 fps (r). I, m, q Scale bar: 100 µm. Statistical analysis: (g, h), one-way ANOVA with Tukey’s multiple comparison. l, p Unpaired t-test. (t), paired t-test. ****p < 0.0001,**p < 0.01, *p < 0.05. Source data are provided as a Source Data file.

Fig. 6. Lack of dysferlin drives age-dependent, muscle-specific Ca²⁺-handling alterations.

a, b Steady-state [Ca²⁺]_t-sys in 36 weeks-old TA and psoas fibres across a range of [Ca²⁺]_cyto in presence (black) and absence (red) of RyR1 leak. c 36 weeks-old BlaJ psoas [Ca²⁺]_t-sys (t) showing the effect of tetracaine on [Ca²⁺]_t-sys in absence and presence of BTP2 (5 mM). d Effect tetracaine on [Ca²⁺]_t-sys in absence and presence of BTP2. Data is presented as Mean +/− SEM. n = 5–11. e Diagram displaying relationship between RyR1 leak and chronic SOCE observed in 36 weeks-old BlaJ psoas fibres. Upon extensive RyR1 leak, chronic SOCE is activated. Inhibition of this leak inactivates chronic SOCE, leading to accumulation of Ca²⁺ in the t-system. f Effect of N-acetyl cysteine (NAC) on RyR1 leak of 36 weeks-old BlaJ psoas fibres. n = 7–11. g Ratio of oxidized/reduced RyR1 peptides in psoas. Data derived from data-dependent acquisition MS (Supplementary Table 9). h Endogenous Ca²⁺ content (per fibre volume) of the major subcellular compartments in 8 and 36-weeks-old WT and BlaJ TA and psoas fibres, shown as cumulative histograms. (see Supplementary Figure 5). Statistical analysis in d&f were performed with an unpaired T-test. ****p < 0.0001. Statistical analysis in (g) were performed with a one-way ANOVA with Tukey’s multiple comparison. Source data are provided as a Source Data file.