Mitochondrial biogenesis, oxygen consumption, and energy production in muscle depend on regulatory mechanisms which control aerobic gene expression. A focal point of such mechanisms is the control of cytochrome c oxidase (COX) expression. The transcription of COX genes is dramatically upregulated by increases in muscular activity via chronic nerve stimulation. In addition, abnormalities in COX gene expression can result in fatal mitochondrial myopathies. Thus, a determination of the mechanisms which govern COX expression will provide insight into processes which result in disease and adaptive changes in muscle. Of the 13 subunits which compose the COX complex, 3 have been shown to exhibit tissue-specific isoforms. Two of these, COXVIa and COXVIII are present in rats and their muscle-specific isoforms, COXVIa-H and COXVIII-H, have been cloned and will be examined by transfection analysis to identify muscle-specific, stimulation-responsive, and denervation-sensitive regulatory elements. Available information suggests that cis-acting elements controlling expression of these genes in cultured cells are distinct. No data exists regarding the elements governing responses to stimulation or denervation. Experiments will include in vitro transfection of cultured cells and in vivo transfection of regenerating muscle. Regulatory elements will be initially mapped using deletion analysis and further define using site-directed mutagenesis and gel shift assays. A novel technique of in vivo transfection in conjunction with chronic nerve stimulation or denervation will be utilized to identify the cis-acting regulatory elements which confer changes in transcriptional activity with changes in muscular activity. Novel sequences which are identified as response elements will be used as probes in expression cloning techniques to obtain cDNAs for potentially novel transcription factors identified by these aims. These elements will also be characterized for functional capabilities by assessing their influence as regulatory motifs in chimeric promoters. Expression of transcription factors (novel or known) will be examined by Northern blotting of mRNA levels and evaluation of the response of aerobic gene promoters to forced expression of these regulatory factors.
