Characterizing RyR and SERCA function in the C57 and D2 mdx mouse models of Duchenne Muscular Dystrophy

Abstract

Duchenne Muscular Dystrophy (DMD) is a male-affected muscle wasting disease caused by the complete loss of the sarcolemmal protein dystrophin. No cure exists and patients typically succumb to cardiorespiratory issues in the third or fourth decade of life. Dystrophin loss also leads to dysfunction in other pathways; including impaired sarcoplasmic reticulum (SR) calcium (Ca2+) handling, further perpetuating the disease. This thesis examined potential differences in SR Ca2+ handling in two mouse models of DMD. The D2.B10-Dmdmdx/J (D2 mdx) mouse has emerged as a more pathologically representative model of DMD than the C57BL/10ScSn-Dmdmdx/J (C57 mdx) mouse model, showing greater muscle weakness, wasting and earlier disease onset. However, SR Ca2+ has not yet been characterized in the D2 mdx mouse. Thus, the aim of this study was to compare SR Ca2+ handling in the D2 mdx and C57 mdx mice. Using age-matched (9-10 week-old) mice, we found that D2 mdx mice had less mass, smaller gastrocnemius muscles, and were less ambulant. The D2 mdx mice had significantly higher energy expenditure and respiratory exchange ratio compared with the D2 WT mice. Two separate SR Ca2+ uptake assays revealed that D2 mdx mice have less Ca2+ uptake and leak, and higher starting myoplasmic Ca2+. SERCA activity (ATP hydrolysis) was lower in D2 mdx mice while higher in C57 mdx mice. These dramatic impairments in SR Ca2+ handling were not attributed to differences in SERCA isoform content or changes in its regulator, sarcolipin. However, under reducing conditions, protein nitration and nitrosylation content were significantly higher in D2 mdx gastrocnemius muscles. Further, pre-treatment with dithiothreiotol (DTT) did not improve SR Ca2+ handling in these muscles, suggestive of irreversible reactive oxygen/nitrogen post-translational modifications. Finally, calpain proteolytic activity was examined to determine the consequence of the impaired SR Ca2+ handling in the D2 mdx mouse. While D2 WT mice already had higher levels of calpain activity, the D2 mdx mouse had significantly higher calpain activity vs the C57 mdx mouse. Altogether, the results from this thesis suggest that impaired SR Ca2+ handling may be partially responsible for more severe pathology found in the D2 mdx mice.