Sarcolipin overexpression impairs myogenic differentiation in Duchenne muscular dystrophy

Abstract

Reduction in the expression of sarcolipin (SLN), an inhibitor of sarco(endo)plasmic reticulum (SR) Ca²⁺-ATPase (SERCA), ameliorates severe muscular dystrophy in mice. However, the mechanism by which SLN inhibition improves muscle structure remains unclear. Here, we describe the previously unknown function of SLN in muscle differentiation in Duchenne muscular dystrophy (DMD). Overexpression of SLN in C₂C₁₂ resulted in decreased SERCA pump activity, reduced SR Ca²⁺ load, and increased intracellular Ca²⁺ (${\text{Ca}}_{\text{i}}^{\text{2+}}$) concentration. In addition, SLN overexpression resulted in altered expression of myogenic markers and poor myogenic differentiation. In dystrophin-deficient dog myoblasts and myotubes, SLN expression was significantly high and associated with defective ${\text{Ca}}_{\text{i}}^{\text{2+}}$ cycling. The dystrophic dog myotubes were less branched and associated with decreased autophagy and increased expression of mitochondrial fusion and fission proteins. Reduction in SLN expression restored these changes and enhanced dystrophic dog myoblast fusion during differentiation. In summary, our data suggest that SLN upregulation is an intrinsic secondary change in dystrophin-deficient myoblasts and could account for the ${\text{Ca}}_{\text{i}}^{\text{2+}}$ mishandling, which subsequently contributes to poor myogenic differentiation. Accordingly, reducing SLN expression can improve the ${\text{Ca}}_{\text{i}}^{\text{2+}}$ cycling and differentiation of dystrophic myoblasts. These findings provide cellular-level supports for targeting SLN expression as a therapeutic strategy for DMD.

Keywords: Duchenne muscular dystrophy; calcium; differentiation; myoblast fusion; sarcolipin.

Fig. 1.

Sarcolipin (SLN) overexpression inhibits sarco(endo)plasmic reticulum (SR) Ca²⁺-ATPase (SERCA) function and alters SR Ca²⁺ handling in C₂C₁₂ cells. A: representative Western blots and box-and-whisker plots showing the protein levels of SLN, SERCA isoforms, and calsequestrin (CSQ) in C₂C₁₂ myoblasts and in myotubes after 5 days of differentiation infected with either adenovirus expressing LacZ gene encoding β-galactosidase (Ad.LacZ) or adenovirus expressing SLN (Ad.SLN). The protein levels of SERCA2a and CSQ are significantly increased in Ad.SLN compared with that of Ad.LacZ controls; n = 6 samples per group. B: Ca²⁺-dependent Ca²⁺ uptake is decreased in the Ad.SLN-infected C₂C₁₂ myotubes (n = 5 experiments) compared with that of Ad.LacZ-infected controls (n = 4 experiments). C–F: box-and-whisker plots showing the summary data for twitch Ca²⁺ transients (elicited by field stimulation at 0.5 Hz; C), SR Ca²⁺ content measured as the height of the caffeine (10 mmol/L)-induced Ca²⁺ transient (D), time taken for 50% decay of Ca²⁺ (T₅₀; E), and fractional SR Ca²⁺ release, calculated by dividing the height of the last twitch transient by the height of the caffeine transient (F), in control (Ad.LacZ) and SLN-overexpressing C₂C₁₂ myotubes; n = 12 myotubes per group. Statistical significance was determined by unpaired Student’s t test with Welch’s correction. P values are shown within the histograms. F/F₀, ratio of the fluorescence (F) to the basal diastolic fluorescence (F₀); NS, not statistically significant.

Fig. 2.

Sarcolipin (SLN) overexpression prevents myoblast fusion and differentiation. A: representative immunofluorescence images of C₂C₁₂ myotubes after 5 days of differentiation infected with adenovirus expressing LacZ gene encoding β-galactosidase (Ad.LacZ) or adenovirus expressing SLN (Ad.SLN), stained with the myosin heavy chain antibody MF20. Original magnification is ×10. Scale bars = 100 μm. B: box-and-whisker plots showing the percent fusion index calculated as the percentage of cells containing at least 3 nuclei within a differentiated myotube. A minimum of 8 different nonoverlapping areas from each coverslip slide were used for quantitation. Here, n = 4 experiments per group. C–F: representative Western blots and box-and-whisker blots showing the protein levels of myogenin, myoblast determination protein-1 (MyoD), caveolin 3 (Cav3), stabilin 2, calcineurin, utrophin, and dystrophin as fold changes in myoblasts and in C₂C₁₂ cells after 5 days of differentiation infected with either Ad.LacZ or Ad.SLN; n = 6 samples per group. Statistical significance was determined by unpaired Student’s t test with Welch’s correction. P values are shown within the histograms. NS, not statistically significant.

Fig. 3.

Reduction in sarcolipin (SLN) expression improves the sarco(endo)plasmic reticulum (SR) Ca²⁺ handling in primary myoblasts from the dog model of Duchenne muscular dystrophy (DMD) after 4 days of differentiation. A: representative Western blots showing the protein levels of SLN, SR Ca²⁺-ATPase 1 (SERCA1), calsequestrin (CSQ), myogenin, and myoblast determination protein-1 (MyoD) in normal and DMD dog myoblasts. B: quantitation showing that SLN is upregulated in the primary myoblasts from the DMD dog; n = 4 samples per group. C: Alamar blue assay showing the viability in normal (N) myoblasts, normal myoblasts treated with adeno-associated virus expressing short hairpin RNA specific for canine SLN (AAV.cshSLN; N/shSLN), DMD myoblasts, and DMD myoblasts treated with AAV.cshSLN (DMD/shSLN) after 4 days of differentiation. Values shown are percent relative fluorescence units relative to normal controls; n = 10 experiments per group. D–F: representative Western blots (D) and box-and-whisker plots (E and F) showing the protein levels of SLN and SERCA isoforms in normal and DMD (D) myoblasts treated with AAV.cshSLN after 4 days of differentiation; n = 4 samples per group. G–J: box-and-whisker plots showing the summary data for twitch Ca²⁺ transients (elicited by field stimulation at 0.5 Hz; G), time taken for 50% decay of Ca²⁺ (T₅₀; H), SR Ca²⁺ content measured as the height of the caffeine (10 mmol/L)-induced Ca²⁺ transient (I), and fractional SR Ca²⁺ release, calculated by dividing the height of the last twitch transient by the height of the caffeine transient (J), in normal and DMD myotubes; n = 14 myotubes per group. Statistical significance was determined by unpaired Student’s t test with Welch’s correction. P values are shown within the histograms. F/F₀, ratio of the fluorescence (F) to the basal diastolic fluorescence (F₀); NS, not statistically significant.

Fig. 4.

Reduction in sarcolipin (SLN) expression improves dystrophic myoblast fusion. A: representative Western blots showing the protein levels of myogenic markers and utrophin in normal (N) and Duchenne muscular dystrophy (DMD; D) myoblasts treated with adeno-associated virus expressing short hairpin RNA specific for canine SLN (shSLN; AAV.cshSLN) after 4 days of differentiation. B: quantitation showing that myogenin is downregulated in the AAV.cshSLN-treated DMD myotubes; n = 4 samples per group. *P < 0.05 for myogenin in the DMD/shSLN group vs. other groups. C: representative immunofluorescence images of control and AAV.cshSLN-treated primary myoblasts after 4 days of differentiation from the normal and DMD dogs, stained with the myosin heavy chain antibody MF20. Original magnification is ×10. Scale bars = 100 μm. Arrows indicate the multinucleated myotubes; n = 4 experiments per group. D: box-and-whisker plots showing the myosin-positive cells containing ≤3 and >3 nuclei in DMD myoblasts and in DMD myoblasts treated with AAV.cshSLN. A minimum of 5 different nonoverlapping areas from each coverslip slide were used for quantitation; n = 4 experiments per group. Cav3, caveolin 3.

Fig. 5.

Reduction in sarcolipin (SLN) expression restores autophagy in differentiated dystrophic myotubes. A: representative Western blots showing the microtubule-associated protein 1A/1B-light chain 3-II (LC3II) levels in normal and dystrophic myotubes with or without chloroquine treatment. Quantitation showing that the LC3II-to-LC3I ratio was significantly reduced in dystrophic myotubes after 4 days of differentiation. Chloroquine treatment (Chloro) significantly increased the LC3II levels and the LC3II-to-LCI ratio in normal (N) and Duchenne muscular dystrophy (DMD) myotubes; n = 4 samples per group. **P < 0.005 vs. respective untreated group. B: representative Western blots showing the LC3II, sequestosome 1 (SQSTM1), mitofusin 2 (Mfn2), dynamin-related protein-1 (Drp1), and phospho-Drp1 (pDrp1; S616) protein levels in normal myoblasts, normal myoblasts treated with adeno-associated virus expressing short hairpin RNA specific for canine SLN (AAV.cshSLN; N/shSLN), DMD (D) myoblasts, and DMD myoblasts treated with AAV.cshSLN (D/shSLN) after 4 days of differentiation. C: quantitation showing that the LC3II-to-LC3I ratio is significantly reduced in differentiated DMD myoblasts, whereas AAV.cshSLN treatment restores these changes; n = 4 samples per group. Statistical significance was determined by unpaired Student’s t test with Welch’s correction. *P < 0.05 vs. all other groups. D: representative Western blots showing LC3I and LC3II levels in normal, normal/shSLN, DMD, and DMD/shSLN myotubes with or without chloroquine treatment. E–G: box-and-whisker plots showing the SQSTM1, Mfn2, and Drp1 protein levels as fold changes in control and AAV.cshSLN-infected normal and DMD myoblasts differentiated for 4 days; n = 4 samples per group. Statistical significance was determined by unpaired Student’s t test with Welch’s correction. *P < 0.05 vs. N and N/shSLN groups. H: representative Western blots showing the protein levels of oxidative phosphorylation complex I (CI) through complex V (CV) subunits in control and AAV.cshSLN-infected primary myoblasts from normal and DMD dogs after 4 days of differentiation; n = 4 samples per group. I: oxygen consumption rate (OCR) obtained from normal, normal/shSLN, DMD, and DMD/shSLN myoblasts after 4 days of differentiation; n = 5 experiments per group. ATP5A, ATP synthase subunit-α; MTCO1, cytochrome c oxidase subunit I; NDUFB8, NADH-ubiquinone oxidoreductase 1 β-subcomplex 8; SDHB, succinate dehydrogenase complex, subunit B, iron sulfur protein; UQCRC2, ubiquinol-cytochrome c reductase core protein II. NS, not statistically significant.