Excessive intracellular Ca accumulation (EICA) is a fundamental pathogenetic event in muscle degeneration which occurs in several human disorders such as muscular dystrophy and acute pancreatitis. EICA is associated with reduced cellular energy charge, (Mg) and (K) with increased (Na), (P) and (Zn); mitochondrial (MIT) & sarcoplasmic reticulum (SR) Ca- overloading with functional impairments; and cellular necrosis. The goals of this proposal are: (1) to characterize the CHF-148 strain dystrophic hamsters (DH) as an in vivo model for the study of membrane mediated EICA and associated pathobiology in muscle degeneration, (2) to elucidate the mechanism(s) of EICA at the cellular and subcellular levels, and (3) to evaluate the effects of ATP-MgCl2, Diltiazem (DTZM), and their combination in the regulation of EICA in DH. To achieve these goals, cellular and subcellular total (Ca), exchangeable Ca pool, uptake and release of 45Ca, and myoplasmic free (Ca) will be quantitated and correlated at different stages of muscle degeneration in DH. Cellular energy charge, Na, K, Mg, Zn, and P, and plasma P, CK, and LD will also be determined and correlated with the EICA. Mitochondrial coupling, integrity, and purity will be evaluated by the measurement of RCR, ANT and ATP synthetic activities, long- chain FFA accumulation, and citrate synthase activity. SR purity will be assessed by the rotenone-insensitive NADPH-cytochrome C-reductase activity. Evaluation of ultrastructural and membranous changes in the MIT, SR, and tissue preparations will be accomplished by EM. Histologic correlations of tissues such as % of Ca2+-positive fibers, centronucleated fibers, necrotic fibers, and fiber splitting will be determined by morphometric analysis. The pharmacologic regulation of EICA and associated degenerative changes will be studied for 14 and 56 days in well- characterized DH by continuous (0.5 ul/hr) subcutaneous infusion of ATP-MgCl2 (membrane stabilizer and vasodilator), DTZM (Ca- channel blocker), and by their combination via implanted osmotic minipumps. Papavarine, dopamine, and saline infused DH will serve as the vasodilatory, inotropic, and Ca-channel blocker controls respectively. Changes in EICA and all of the above variables will be correlated during muscle degeneration and pharmacologic regulation. The long-term objectives of this study are to provide new information on the mechanism of membrane mediated EICA; and more vitally the pharmacologic regulation of intracellular Ca accumulation and associated pathobiology during muscle degeneration.
