Programme Contents

Abnormal calcium transients and calcium handling protein expression in cardiomyocytes from mdx (dystrophic) mice

Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disorder caused by deficiency of the cytoskeletal protein dystrophin. DMD patients have extensive skeletal muscle degeneration and a dilated cardiomyopathy (DCM). The mdx mouse also lacks dystrophin and in skeletal muscle it exhibits membrane damage and an abnormal influx of Ca²⁺. The present study was aimed at characterising ventricular performance which may contribute to DCM in mdx mice. Ventricular myocytes were isolated from 8-week old wild-type and mdx mice and intracellular Ca²⁺ measured with the fluorescent indicator fluo-4 during electrical and caffeine (10 mM) induced stimulation. Protein expression of the ryanodine receptor (RyR), the sarco endoplasmic reticulum calcium ATPase (SERCA) and phospholamban were analysed using immunoblotting techniques. The peak of the electrically stimulated Ca²⁺ transient was significantly greater in mdx mice, but the time to peak was significantly shorter. These findings were not the result of increased sarcoplasmic reticulum (SR) Ca²⁺ loading as the caffeine-induced Ca²⁺ transient peak was unchanged in mdx mice. The increase in peak calcium transient and the decreased time to peak could be due to the significantly increased levels of RyR protein expression (4-fold), allowing more rapid Ca²⁺ release from the SR during excitation. However, this is not usually found in established DCM (Kubo et al., 2001). It was found that the rate of decline of the electrically stimulated Ca²⁺ transient was significantly slower in mdx mice, but the rate of decline of the caffeine-induced Ca²⁺ transient was unchanged, suggesting that the slower removal of Ca²⁺ from the intracellular milieu was a result of decreased SERCA activity, and not decreased sodium-calcium exchanger activity. SERCA protein levels were unchanged, but phospholamban levels were increased significantly (2-fold). The slower rate of decline of the Ca²⁺ transient in mdx mice is therefore possibly a result of increased inhibition of SERCA by phospholamban, a finding which is consistent with other studies of DCM (Meyer et al., 1995). This is further supported by the finding that the SERCA/phospholamban ratio was significantly smaller in mdx mice. It is concluded that dystrophin deficiency causes impairment in the Ca²⁺ handling properties of mdx ventricular myocytes, which may play a role in the development of DCM. Future work will test whether the increase in peak Ca²⁺ transients in the mdx mouse is an early compensatory mechanism that reverses as the cardiomyopathy progresses, by investigating myocyte Ca²⁺ handling in older mice.

Kubo, H., Margulies, K.B., Piacentino, V 3^rd., Gaughan, J.P. & Houser, S.R. (2001) Circulation 104:1012-8.

Meyer, M., Schillinger, W., Pieske, B., Holubarsch, C., Heilmann, C., Posival, H., Kuwajima, G., Mikoshiba, K., Just, H. & Hasenfuss, G. (1995) Circulation 92, 778-784.