ELECTRICAL-STIMULATION OF NEONATAL CARDIOMYOCYTES RESULTS IN THE SEQUENTIAL ACTIVATION OF NUCLEAR GENES GOVERNING MITOCHONDRIAL PROLIFERATION AND DIFFERENTIATION

Electrical stimulation of neonatal cardiac myocytes produces hypertrop hy and cellular maturation with increased mitochondrial content and ac tivity. To investigate the patterns of gene expression associated with these processes, cardiac myocytes were stimulated for varying times u p to 72 hr in serum-free culture. The mRNA contents for genes associat ed with transcriptional activation [c-fos, c-jun, JunB, nuclear respir atory factor 1 (NRF-1)], mitochondrial proliferation [cytochrome c (Cy t c), cytochrome oxidase], and mitochondrial differentiation [carnitin e palmitoyltransferase I (CPT-I) isoforms] were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0 .25-3 hr) and followed sequentially by c-jun (0.53 hr), JunB (0.5-6 hr ), NRF-1 (1-12 hr), Cyt c (12-72 hr), and muscle-specific CPT-I (48-72 hr), Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA, thus supporting the developmental fidel ity of this pattern of gene regulation. Consistent with a transcriptio nal mechanism, electrical stimulation increased c-fos, beta-myosin hea vy chain, and Cyt c promoter activities. These increases coincided wit h a rise in their respective endogenous gene transcripts, NRF-1, cAMP response element, and Sp-1 site mutations within the Cyt c promoter re duced luciferase expression in both stimulated and nonstimulated myocy tes. Mutations in the NRF-1 and CRE sites inhibited the induction by e lectrical stimulation (5-fold and 2-fold, respectively) whereas mutati on of the Sp-1 site maintained or increased the fold induction. This f inding is consistent with the appearance of NRF-1 and fos/jun mRNAs pr ior to that of Cyt c and suggests that induction of these transcriptio n factors is a prerequisite for the transcriptional activation of Cyt c expression. These results support a regulatory role for NRF-1 and po ssibly AP-1 in the initiation of mitochondrial proliferation.