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Damage to developing mouse skeletal muscle myotubes in culture: protective effect of heat shock proteins
A A Maglara1, A Vasilaki, M J Jackson
1Department of Medicine, University of Liverpool, Liverpool L69 3GA, UK.
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.
Insights
Increased heat shock proteins (HSPs) protect skeletal muscle cells from damage caused by calcium imbalance or mitochondrial dysfunction. This study demonstrates HSPs
Area of Science:
- Cellular and Molecular Biology
- Muscle Physiology
- Biochemistry
Background:
- Skeletal muscle damage arises from exercise, ischemia-reperfusion, and diseases like muscular dystrophy.
- Mechanisms include calcium dysregulation, energy depletion, oxidative stress, and apoptosis.
- Heat shock proteins (HSPs) are known to confer protection, but the underlying mechanisms require elucidation.
Purpose of the Study:
- To develop a cell culture model for skeletal muscle damage using C2C12 cells.
- To investigate the protective role of heat shock proteins (HSPs) against specific damaging agents.
Main Methods:
- C2C12 skeletal muscle cells were treated with 2,4-dinitrophenol (DNP) or calcium ionophore A23187 to induce damage.
- Creatine kinase (CK) release was measured as an indicator of cell damage.
- Hyperthermia was used to increase the cellular content of HSPs (HSP25, HSP60, HSC70, HSP70).
Main Results:
- DNP treatment (1 mM, 60 min) caused 63.5% CK release over 3 hours.
- A23187 treatment (10 µM, 30 min) resulted in 47.9% CK release.
- Elevated HSP levels significantly protected myotubes against both DNP- and A23187-induced damage.
Conclusions:
- Increased HSP content confers protection against skeletal muscle damage induced by calcium overload or mitochondrial uncoupling.
- This study provides evidence for the protective role of HSPs in models of muscle injury.
