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Sarcolipin (SLN) is an inhibitor of sarco/endoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA) and expressed at high levels in the ventricles of animal models for and patients with Duchenne muscular dystrophy (DMD). The goal of this study was to determine whether the germline ablation of SLN expression improves cardiac SERCA function and intracellular Ca 2+ (Ca 2+ i ) handling and prevents cardiomyopathy in the mdx mouse model of DMD. Wild-type, mdx , SLN-haploinsufficient mdx ( mdx:sln +/- ), and SLN-deficient mdx ( mdx:sln -/- ) mice were used for this study. SERCA function and Ca 2+ i handling were determined by Ca 2+ uptake assays and by measuring single-cell Ca 2+ transients, respectively. Age-dependent disease progression was determined by histopathological examinations and by echocardiography in 6-, 12-, and 20-mo-old mice. Gene expression changes in the ventricles of mdx:sln +/- mice were determined by RNA-Seq analysis. SERCA function and Ca 2+ i cycling were improved in the ventricles of mdx:sln +/- mice. Fibrosis and necrosis were significantly decreased, and cardiac function was enhanced in the mdx:sln +/- mice until the study endpoint. The mdx:sln -/- mice also exhibited similar beneficial effects. RNA-Seq analysis identified distinct gene expression changes including the activation of the apelin pathway in the ventricles of mdx:sln +/- mice. Our findings suggest that reducing SLN expression is sufficient to improve cardiac SERCA function and Ca 2+ i cycling and prevent cardiomyopathy in mdx mice. NEW & NOTEWORTHY First, reducing sarcopolin (SLN) expression improves sarco/endoplasmic reticulum Ca 2+ uptake and intracellular Ca 2+ handling and prevents cardiomyopathy in mdx mice. Second, reducing SLN expression prevents diastolic dysfunction and improves cardiac contractility in mdx mice Third, reducing SLN expression activates apelin-mediated cardioprotective signaling pathways in mdx heart.

Sarcolipin (SLN) is an inhibitor of the sarco/endoplasmic reticulum (SR) Ca 2+ ATPase (SERCA) and is abnormally elevated in the muscle of Duchenne muscular dystrophy (DMD) patients and animal models. Here we show that reducing SLN levels ameliorates dystrophic pathology in the severe dystrophin/utrophin double mutant (mdx:utr -/- ) mouse model of DMD. Germline inactivation of one allele of the SLN gene normalizes SLN expression, restores SERCA function, mitigates skeletal muscle and cardiac pathology, improves muscle regeneration, and extends the lifespan. To translate our findings into a therapeutic strategy, we knock down SLN expression in 1-month old mdx:utr -/- mice via adeno-associated virus (AAV) 9-mediated RNA interference. The AAV treatment markedly reduces SLN expression, attenuates muscle pathology and improves diaphragm, skeletal muscle and cardiac function. Taken together, our findings suggest that SLN reduction is a promising therapeutic approach for DMD.

The functional importance of threonine 5 (T5) in modulating the activity of sarcolipin (SLN), a key regulator of sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pump was studied using a transgenic mouse model with cardiac specific expression of threonine 5 to alanine mutant SLN (SLNT5A). In these transgenic mice, the SLNT5A protein replaces the endogenous SLN in atria, while maintaining the total SLN content. The cardiac specific expression of SLNT5A results in severe cardiac structural remodeling accompanied by bi-atrial enlargement. Biochemical analyses reveal a selective downregulation of SR Ca2+ handling proteins and a reduced SR Ca2+ uptake both in atria and in the ventricles. Optical mapping analysis shows slower action potential propagation in the transgenic mice atria. Doppler echocardiography and hemodynamic measurements demonstrate a reduced atrial contractility and an impaired diastolic function. Together, these findings suggest that threonine 5 plays an important role in modulating SLN function in the heart. Furthermore, our studies suggest that alteration in SLN function can cause abnormal Ca2+ handling and subsequent cardiac remodeling and dysfunction.