ABSTRACT
Damage to skeletal muscle occurs following excessive exercise, upon reperfusion following ischaemia and in disease states, such as muscular dystrophy. Key mechanisms by which damage is thought to occur include a loss of intracellular calcium homeostasis, loss of energy supply to the cell, increased activity of oxidising free radical-mediated reactions and activation of apoptosis pathways. An increased cellular content of heat shock proteins (HSPs) has been shown to protect skeletal muscle against some forms of damage, although the mechanistic basis of this protection is not clearly understood. The aim of this study was to establish a cell culture-based model of damage to C2C12 skeletal muscle cells using the calcium ionophore, A23187 and the mitochondrial uncoupler, 2,4-dinitrophenol (DNP) as damaging agents. Treatment of cells with 1 mM DNP for 60 min resulted in the release of 63.5 % of intracellular creatine kinase (CK) activity over the 3 h experimental period. Treatment of cells with 10 microM A23187 for 30 min resulted in the release of 47.9 % of CK activity. Exposure of myotubes to a period of hyperthermia resulted in a significant increase in their content of HSP25, HSP60, HSC70 (heat shock cognate) and HSP70. This increase in HSPs was associated with significant protection against both DNP-induced and A23187-induced damage to the myotubes. These results indicate that an increased content of HSPs may provide protection against the muscle damage that occurs by a pathological increase in intracellular calcium or uncoupling of the mitochondrial respiratory chain.
Subject(s)
Heat-Shock Proteins/physiology , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/pathology , Animals , Calcimycin/pharmacology , Calcium/metabolism , Cell Line , Cell Survival/drug effects , Cells, Cultured , Chaperonin 60/physiology , Cold Temperature , Creatine Kinase/metabolism , Energy Metabolism , HSP70 Heat-Shock Proteins/physiology , Heat-Shock Proteins/biosynthesis , Homeostasis , Kinetics , Mice , Models, Biological , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiologyABSTRACT
Several recent studies report endonuclease-mediated DNA degradation as evidence of apoptotic degeneration of skeletal muscle in the muscular dystrophies and other muscle disorders. Interpretation of the results of such studies is complicated by the ubiquitous presence of non-muscle cells within muscle in vivo and by a lack of knowledge concerning the nature of the process of apoptosis in postmitotic, multinucleated skeletal muscle and the potential mechanisms involved. Staurosporine treatment of C2C12 skeletal muscle myotubes induced several classic features of apoptosis, including cell and nuclear shrinkage with initial preservation of cellular membranes. Externalization of phosphatidylserine occurred within 2 hours of treatment, and myotubes contained procaspase 3, which seemed to be activated within 4 hours. DNA degradation was identified by transferase uridine triphosphate nick-end labeling within 4 hours, and DNA ladders were identified on agarose electrophoresis of genomic DNA within 8 hours. Thus, the process of apoptosis in postmitotic multinucleated skeletal muscle shares many of the characteristics of apoptosis in mononuclear mitotic cells. However, the pattern of degeneration does not seem to be compatible with that seen in the muscular dystrophies.
