J. Thorburn

We have investigated the role of the proto-oncogene HRas in cardiac cell growth and hypertrophy. By direct needle microinjection of activated Ras protein into primary neonatal rat ventricular cardiac myocytes, we find that, unlike many other cell types, Ras does not induce DNA synthesis in these cells. However, injection of activated Ras does induce expression of both the c-Fos and atrial natriuretic factor (ANF) genes. Expression of both these genes is associated with the hypertrophic response in ventricular myocytes suggesting that Ras is involved in the hypertrophic signalling pathway. Ras injection also causes morphological changes in the cells so that they increase in profile and show changes in the organization of the contractile apparatus. Further support for a role for Ras in the hypertrophic response was obtained from studies showing that activated Ras stimulates ANF promoter activity in transient transfection assays. We also show that a dominant interfering Ras mutant inhibits the hypertrophic stimulation of the ANF promoter by phenylephrine, indicating a role for Ras in the hypertrophic effect of an alpha-adrenergic agonist.

Shortly after birth, cardiac myocytes lose the ability to divide, and, in adult animals, heart muscle grows by a process of cellular hypertrophy where each individual cell gets larger. We have previously shown that activated Ras protein can induce markers of the hypertrophic phenotype, including atrial natriuretic factor (ANF) expression and organization of contractile proteins, and that Ras is at least partially required for the hypertrophic effect of phenylephrine. In the present study, we examine the requirement for the mitogen-activated protein kinases (MAP kinases) in the hypertrophic response induced by phenylephrine. We find that phenylephrine treatment results in the activation of the MAP kinases and that this activity is required for transactivation of the fos, ANF, and MLH promoters. However, inhibition of MAP kinases does not prevent phenylephrine-induced organization of actin. These results suggest that the signal transduction pathways leading to different hypertrophic responses diverge upstream of the MAP kinases but possibly downstream of Ras.

alpha 1-Adrenergic agonists activate a hypertrophic response in cultured neonatal ventricular myocytes, which include an increase in cell size, organization of contractile proteins into sarcomeric units, and the induction of the atrial natriuretic factor (ANF) gene. Previous findings have supported a role for ras in this signaling pathway. Utilizing microinjection techniques to delivery affinity-purified neutralizing antibodies to G alpha q,11 into cultured ventricular myocytes, the current studies demonstrate a functional requirement for the heterotrimeric G protein, Gq, in the alpha 1-adrenergic induction of the ANF gene, changes in cell size, organization of myofilaments, and phosphoinositide hydrolysis. Expression of a constitutively active mutant of G alpha q leads to the expression of ANF protein in these cells. Taken together, these data suggest that G q-dependent pathways are necessary and sufficient to activate defined features of the hypertrophic response. In attempts to further delineate the relative roles of ras and Gq in this pathway, we found that G alpha q is required for alpha 1-adrenergic phosphoinositide hydrolysis, though ras does not appear to be necessary for this response. In addition, we coexpressed an inhibitory ras mutant, along with the constitutively active G alpha q. Expression of ANF protein stimulated by the G alpha q mutant was not inhibited. Thus, both ras- and Gq-dependent pathways are necessary to fully transduce defined features of alpha 1-adrenergic-stimulated hypertrophy of neonatal cardiac ventricular myocytes, but activated Gq may be able to induce ANF expression independent of inhibitory ras.

Around the time of birth, cardiac muscle cells lose the capacity to divide and, from this time on, growth of the heart occurs by hypertrophy where each cells gets bigger. The hypertrophic response is characterized by changes in gene expression including expression of the atrial natriuretic factor (ANF) and myosin light chain-2 (MLC-2) genes. In cultured neonatal ventricular myocytes, hypertrophy also involves reorganization of contractile proteins into sarcomeric units. We have investigated the role of the Raf-1 kinase in this response. Activation of an estradiol-regulated Raf-1 protein kinase led to activation of mitogen-activated protein (MAP) kinase and activated expression from the ANF and MLC-2 promoters. Raf-1-induced activation of these genes was inhibited by a kinase deficient mutant of the 44-kDa MAP kinase, Erk1 indicating a requirement for MAP kinases in the Raf-1-induced response. However, activation of Raf-1 was not sufficient to induce the organization of actin into sarcomeric units. Transfection of dominant negative Raf-1 inhibited phenylephrine-induced activation of the ANF and MLC-2 promoters. Transactivation was rescued by the introduction of increased amounts of c-Raf suggesting a role for Raf-1 in the response to alpha-adrenergic agonists. These results suggest that activation of Raf-1 kinase is a critical component of the signal transduction pathway leading to changes in gene expression associated with hypertrophy but that Raf-1 is not sufficient for the regulation of actin organization during the hypertrophic response.