The assessment of skeletal muscle toxicity is important in the safety evaluation of
new chemical entities, particularly those used as lipid-lowering agents. As current
strategies for assessing myopathy are based on in vivo measurements, which
sometimes lack sensitivity and specificity, there is a need for biomarkers that better
predict myopathy and also for an approach that is more amenable to highthroughput
screening. Here, the use of cultured rat L6 myoblasts and myotubes
was explored in combination with a variety of techniques to identify potential
biomarkers, following exposure to a known experimental myotoxicant, 2,3,5,6
tetramethyl-p-phenylenediamine (TMPD), or a highly selective peroxisome
proliferator-activated receptor δ agonist, 4-[2-(3-fluoro-4-trifluoromethyl-phenyl)-4-
methyl-thiazol-5-ylmethylsulfanyl]-2-methyl-phenoxy-acetic acid (GWδ).
The use of candidate biomarkers, based on the utilisation of a variety of established
and putative markers of myopathy, proved to be of little additional value in
myoblasts treated with low concentrations of TMPD. In myotubes, however,
intracellular levels of aldolase and creatine kinase appeared to be responsive
markers. Proteomic analysis of TMPD and GWδ-treated skeletal muscle cells using
surface-enhanced laser desorption/ionization-time of flight-mass spectrometry
(SELDI-TOF-MS) showed changes in the levels of 16 protein ions in myoblasts and
12 changes in myotubes, although SELDI-TOF-MS did not lend itself to the
conclusive identification of these ions. Analysis of the muscle cell proteins using
label-free quantitative proteomics was able to simultaneously quantify TMPD-induced
changes and identify the proteins involved. In this way, 8 proteins in
myoblasts and 10 proteins in myotubes were discovered to be the most responsive
proteins to treatment. Changes in the levels of Rho GDP dissociation inhibitor (GDI)
alpha in myoblasts and Myosin light chain 2 in myotubes were confirmed by
immunoblotting, and these proteins were also responsive to treatment with GWδ
and pravastatin.
In conclusion, cultured skeletal muscle cells were responsive to the effects of
potentially myotoxic agents, and proteomics was successful in identifying and
quantifying proteins that may act as biomarkers of effect.