Signal transduction pathways involved in skeletal muscle fibre type regulation

Abstract

Mature skeletal muscle fibres can be classified as type 1, type Ha, type IIx or type IE[b fibres according to the myosin heavy chain (MIHQ isoform that they express. More broadly, type I fibres are classified are slow fibres and type Ha, IIx and 11b fibres as fast fibres. However, the phenotype of an adult skeletal muscle fibre is not fixed: it displays plasticity being capable of adapting to changing activity and loading levels by either transition towards a slower phenotype or transition towards a faster phenotype.
Overall, the aims of these studies were to further investigate and define the signal transduction pathways implicated in the control of skeletal muscle fibre phenotype. The ability of a fast muscle to undergo a transition towards a slower phenotype in
response to chronic low-frequency stimulation (CLFS) was assessed, via metabolic enzyme activity assays and NADH-TR staining, following blockade of the calcineurin signalling pathway. Metabolic enzyme assays and northern blots were employed to assess the changes in enzyme activities and MEC isoform expression levels following blockade of the calcineurin and ERKI/2 signalling pathways in primary cultures of rat myotubes. Differences in the levels of various signal transduction proteins/transcription factors between slow and fast muscle were investigated using western blotting. The nuclear translocation kinetics of NFAT and NF-icB in response to treatment with the calcium ionophore A23187 were investigated in L6 myotubes using immunocytochernistry.
Calcineurin blockade using cyclosporin A failed to prevent a decrease in lactate dehydrogenase activity and an increase in NADH-TR staining intensity, both characteristics of a transition towards a slower phenotype, following CLFS of the fast
rat tibialis anterior muscle. Blockade of the ERKI/2 pathway in primary cultures Of rat myotubes using U0126 significantly decreased MHC Ip mRNA levels and significantly increased MIHC Ux, MEC IIb and perinatal MEC mRNA levels.
Calcineurin blockade significantly decreased MHC Ip and embryonal mRNA levels and significantly increased MEC IN mRNA levels. Calcineurin blockade also significantly increased the activities of lactate dehydrogenase and creatine kinase
while ERKI/2 blockade significantly increased the activities of lactate dehydrogenase, creatine kinase, hexokinase, malate dehydrogenase and 0-hydroxyacyl-CoA cleydrogenase. ERKI/2 and NF-KB levels were found to be higher in slow muscle compared to fast muscle while calcineurin and p38a, p levels were higher in fast muscle compared to slow muscle. No nuclear translocation of NF-KB and only limited evidence for NFAT nuclear translocation was seen in L6 myotubes following treatment with A23187.
Overall these studies further characterize the roles of the ERKI/2 and calcineurin pathways in the regulation of muscle phenotype suggesting that each pathway controls some, but not all, of the genes that are differentially expressed between slow and fast muscle fibres. Western blotting suggests further signal transduction protein/transcription factor targets, the functions of which may be explored in the future.