Damage to developing mouse skeletal muscle myotubes in culture: protective effect of heat shock proteins

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.

Figure 1

HSP25 (A), HSP60 (B) and HSC70 (C) content of C₂C₁₂ cells at 1, 2, 3, 5 and 7 days following treatment with HS. Data are expressed as a percentage of HSP content of myoblasts. D, representative Western blot showing HSP content of C₂C₁₂ cells prior to (lane 1) and at 1, 2, 3, 5 and 7 days following treatment with HS (lanes 2–6). E, specific CK activity in C₂C₁₂ cells prior to (0 time point) and at specific time points following treatment with HS. *P < 0.05 cf. myoblast content.

Figure 2

Time course of release of CK activity expressed as percentage of total CK activity in the well per 30 min (A) and cumulative release of activity (B), from control myotubes (▪) and myotubes treated with 0.25 (▴), 1 (▾) and 10 mm (♦) DNP. *P < 0.05 compared with untreated myotubes.

Figure 3

Live/Dead staining of control myotubes (A), and myotubes at 90 (B), 120 (C) and 150 min (D) following treatment with 1 mm DNP.

Figure 4

Time course of release of CK activity expressed as a percentage of total CK activity in the well per 30 min (A) and cumulative release of activity (B), from control myotubes(▪) and myotubes treated with 2 (•), 10 (▾), 20 (♦) and 50 μm (○) A23187. Data from control myotubes and myotubes treated with 2 μm A23187 overlap. *P < 0.05 compared with untreated myotubes.

Figure 5

HSP25 (A), HSP60 (B) and HSC70 (C) content of myotubes at 4, 8, 12, 18 and 24 h following hyperthermia. Data are expressed as a percentage of non-heated cells which were harvested at the same time point. D, representative Western blots showing HSP content of myotubes at 4 (lane 2), 8 (lane 4), 12 (lane 6), 18 (lane 8) and 24 h (lane 10) following hyperthermia and control cells harvested at the same time point (lanes 1, 3, 5, 7, 9). *P < 0.05 cf. control (non-heated) value.

Figure 6

Effect of prior hyperthermia on release of CK activity from myotubes following treatment with 1 mm DNP (A) or 10 μm A23187 (B). *P < 0.05 cf. control (non-heated) value.