Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation

Abstract

Electrical stimulation of neonatal cardiac myocytes produces hypertrophy and cellular maturation with increased mitochondrial content and activity. To investigate the patterns of gene expression associated with these processes, cardiac myocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation [c-fos, c-jun, JunB, nuclear respiratory factor 1 (NRF-1)], mitochondrial proliferation [cytochrome c (Cyt c), cytochrome oxidase], and mitochondrial differentiation [carnitine 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 (0.5-3 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 fidelity of this pattern of gene regulation. Consistent with a transcriptional mechanism, electrical stimulation increased c-fos, beta-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element, and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the NRF-1 and CRE sites inhibited the induction by electrical stimulation (5-fold and 2-fold, respectively) whereas mutation of the Sp-1 site maintained or increased the fold induction. This finding is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c and suggests that induction of these transcription factors is a prerequisite for the transcriptional activation of Cyt c expression. These results support a regulatory role for NRF-1 and possibly AP-1 in the initiation of mitochondrial proliferation.

Figure 1

Increases in β-MC and COXVa mRNA following electrical stimulation in neonatal rat cardiac myocytes. Total RNA was extracted from cardiac in the absence (control cells; ▾, β-MHC; •, COX) and in the presence of electrical stimulation (▿, β-MHC; ○, COX). Quantitation of Northern blots was carried out by densitometric scanning as described. The results represent the mean ± SE of determinations on three different cultures.

Figure 2

Induction of mRNA for the muscle isoform of CPT-I and decreased mRNA for the liver CPT-I isoform following electrical stimulation. RNA was isolated in the absence and presence of electrical stimulation. Northern blots for the liver and muscle CPT-I mRNA were quantified by densitometric scanning and normalized to 18s RNA for statistical analysis. “C” and “S” are results from control, nonstimulated myocytes and from electrically stimulated myocytes. The results are representative of RNA isolated from at least three different cultures. RNA loading controls are presented.

Figure 3

Increases in luciferase activity using promoter constructs for c-fos, β-MHC and Cyt c following electrical stimulation of neonatal rat cardiac myocytes. Plasmids pFOS.Luc, pβ-MHC.Luc, and pRC4Luc/−326 were cotransfected with RSV β-gal expression plasmid. After 18 hr in serum-free medium, luciferase expression was measured in control (nonstimulated; □, ○, and ▵, c-fos, β-MHC, and Cyt c, respectively) and in parallel cultures of electrically stimulated cells (▪, •, and ▴, c-fos, β-MHC, and Cyt c, respectively). The inset histograms are luciferase/β-gal ratios at the 24-hr time period after electrical stimulation (▪) and directly compared with control expression (□), where ★ = P < 0.01. The results represent three separate cultures each for luciferase and β-gal determinations (mean ± SE).

Figure 4

Sequential induction of mRNA for c-fos, NRF-1, and Cyt c after electrical stimulation. RNA was isolated from neonatal rat cardiac myocytes cultured in serum-free medium either in the absence or presence of electrical stimulation. Northern blots for c-fos and Cyt c mRNA were quantified by densitometric scanning. Quantitation of NRF-1 expression was carried out using densitometric scanning with normalization to 18s RNA following generation of the riboprobe and separation of the RNA/RNA hybrids. “C” and “S” represent results from control, nonstimulated myocytes and from electrically stimulated myocytes. The results are representative of RNA isolated from three different cultures. RNA loading controls for each time point are summarized in Fig. 2.

Figure 5

Sequential induction of mRNA for c-jun and JunB after electrical stimulation. RNA was isolated from neonatal rat cardiac myocytes cultured in serum-free medium either in the absence or presence of electrical stimulation. Northern blots for c-jun and JunB expression were quantitated by densitometric scanning. “C” and “S” represent results from control, nonstimulated myocytes and from electrically stimulated myocytes.

Figure 6

Luciferase expression of the transfected Cyt c promoter and its mutant constructs in control myocytes and following electrical stimulation. After 18 hr in serum-free culture, plasmids containing the wild type (RC4Luc/−326) and the mutated constructs (dCRE-1 mt, NRF-1 mt, and Sp1 mt) were cotransfected with β-gal. Cell extracts were isolated from stimulated cells (□) 24 hr after electrical stimulation, and after 24 hr from parallel cultures of nonstimulated myocytes (▪). The results (mean ± SE) represent activities from three different cultures. ∗, P < 0.05 compared with wild-type stimulated/nonstimulated ratio (S/N).