Application of proteomics to identify biomarkers of myopathy

Abstract

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.