Control of Skeletal Muscle Fiber Types by Calcium Signaling Pathways

Different patterns of motor nerve activity drive distinctive programs of gene expression in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Previous studies have demonstrated that calcineurin activity is required to maintain slow myofiber identity. I am interested in determining the transcription factors downstream of calcineurin and other calcium-regulated signaling pathways in the control of myofiber specialization. By analyzing two fiber type-specific enhancers, I was able to demonstrate that there are functional NFAT (nuclear factor of activated T cells) and MEF2 (myocyte-specific enhancer factor 2) binding sites within the enhancer of troponin I slow, and both sites are required for slow fiber specific activity of this enhancer. Next, I identified MEF2 as a target of calcineurin in cultured myogenic cells. Calcineurin physically interacts with MEF2 and dephosphorylates MEF2. C-terminal transactivation domain, but not N-terminal DNA binding domain of MEF2, responds to calcineurin activation. The use of "MEF2 indicator" transgenic mice that harbor a MEF2-dependent lacZ transgene enabled us to monitor the endogenous activities of MEF2 transcription factors. MEF2 is selectively active in slow and oxidative myofibers. Calcineurin is both necessary and sufficient for MEF2 activation in skeletal muscles. I also found a dose-response relationship between calcineurin activity and expression level of slow, oxidative fiber-specific and MEF2 target genes. Furthermore, I observed that functional activity of MEF2 transcription factors was stimulated by sustained periods of endurance exercise or low-frequency motor nerve pacing in a calcineurin-dependent manner. In addition to calcineurin, CaMKs (calcium, calmodulin-dependent kinases) also transduce their signaling through MEF2. CaMKIV synergistically activates MEF2-dependent gene expression together with calcineurin. Transgenic mice expressing constitutively active CaMKIV in their skeletal muscles showed increased percentage of slow and oxidative myofibers, which was accompanied by increased mitochondrial biogenesis mediated through the upregulation of PGC-1 (PPARg co-activator). Taken together, these findings delineate a molecular pathway in which MEF2 and NFAT integrate signaling inputs from multiple calcium-regulated pathways in the control of skeletal muscle fiber types.