Setsuya Miyata

Hypertrophic growth of cultured neonatal rat heart cells mediated by vasopressin V1A receptor

Primary cultures of neonatal cardiac myocytes were used to determine both the identity of second messengers that are involved in vasopressin receptor-mediated effects on cardiac hypertrophy and the type of vasopressin receptor that is involved in vasopressin-induced cell growth. Neonatal rat myocytes were plated at a density of 1x10(6) cells per 60 mm dish and were incubated with serum-free medium for 7 days. Treatment of myocytes with vasopressin significantly increased the RNA-to-DNA ratio, by 18-25%, at culture days 4-6 and the protein-to-DNA ratio by 18-20% at culture days 5-7. Rates of protein synthesis were determined to assess their contribution to protein contents during myocyte growth. Vasopressin significantly accelerated rates of protein synthesis by 25% at culture day 6. Intracellular free Ca(2+) ([Ca(2+)](i)) was transiently increased after vasopressin exposure. After the peak increase in [Ca(2+)](i) at less than 30 s, there was a sustained increase for at least 5 min. The specific activity of protein kinase C in the particulate fraction was increased rapidly after exposure to vasopressin, and its activity remained higher for 30 min, returning to its control level within 60 min. The activity of protein kinase C in the cytosol was significantly decreased at all times after exposure to vasopressin. After vasopressin treatment, the content of c-fos mRNA was increased. The stimulatory effects of vasopressin on these parameters were significantly inhibited by vasopressin V(1A) receptor antagonist, OPC-21268, but not by vasopressin V(2) receptor antagonist, OPC-31260. These results suggest that vasopressin directly induces myocyte hypertrophic growth via the V(1A) receptor in neonatal rat heart cells.

Renin-angiotensin system in stretch-induced hypertrophy of cultured neonatal rat heart cells.

Although it is well known that mechanical load to cardiac muscles causes cardiac hypertrophy, little is known about how mechanical load is transduced into the activation of intracellular signals which are linked to cell growth. We investigated whether the cardiac renin-angiotensin system was involved in stretch-induced hypertrophy of cultured neonatal rat heart myocytes. Myocytes were cultured with serum-free medium in a deformable silicon dish. Stretch of cardiac myocytes significantly increased the protein/DNA ratio at culture days 6 and 7, and the RNA/DNA ratio at culture days 4 and 5. Stretch significantly accelerated rates of protein synthesis by 15%. c-fos mRNA expression was significantly increased after stretch. The stimulatory effects of cell stretch on these parameters were significantly inhibited by the angiotensin converting enzyme inhibitor, captopril, or the type 1 angiotensin II receptor antagonist, losartan. The concentrations of angiotensin I and angiotensin II in culture media were significantly increased by stretch. Stretch did not change the angiotensin converting enzyme activity. These studies demonstrate that mechanical stretch activates the cardiac renin-angiotensin system in a autocrine and paracrine system which acts as an initial mediator of the stretch-induced hypertrophic growth.

Expression of c-fos mRNA by Acute Pressure Overload in Perfused Rat Heart

It has been demonstrated that several mechanical or pharmacological overload to the heart induces immediate early gene expression such as protooncogenes and heat shock protein genes and acceler¬ates the cardiac protein synthesis to lead to cardiac hypertrophy. Norepinephrine induces cardiac hypertrophy and specific gene expression in cultured rat cardiac myocytes via α1-adrenergic recep¬tor [1,2]. Mechanical stimuli (myocyte stretching) directly induce c-fos expression in cultured neonatal rat cardiac myocytes and this response was associated with protein kinase C activation[3,4]. In these experimental models the induction of specific protooncogene expression in response to several cardiac overload might be mediated by α1-adrenergic receptor or protein kinase C-dependent signal transduction system. On the other hand, Morgan et al[5] have reported that increases in tissue cAMP content by acute pressure overload results in acceleration of protein synthesis rates in perfused adult rat hearts and that the effects of ventricular wall stretch by acute elevation of aortic pressure to accelerate synthesis may involve a cAMP-dependent protein synthesis mechanism. Our hypothesis was that protooncogene c-fos expression enhanced by acute pressure overload in adult rat hearts would play a transducing role in the cAMP-dependent protein synthesis mechanism in addition to protein kinase C-dependent mechanism. In the present study cAMP content, c-fos mRNA expres¬sion, and rates of protein synthesis were examined in adult rat hearts that were perfused at elevated aortic pressure or exposed to hormone receptor binding (glucagon) in order to determine whether c-fos expression by acute pressure overload coupled with an increase in cAMP content and continuous¬ly accelerated rates of protein synthesis.

Stretch Induces Hypertrophic Growth Through Renin-Angiotensin System in Cultured Neonatal Rat Myocytes

Cardiac hypertrophy occurs in response to various mechanical or hormonal stimuli [1]. Recentry, Baker et al [2] and Schunkert et al [3] reported that angiotensin-converting enzyme (ACE) activity, ACE mRNA expression, angiotensinogen mRNA expression and rate of angiotensin II production in heart were increased by pressure overload. Angiotensin II (A II) has two different receptors, such as type 1 angiotensin II receptor (AT1) and type 2 angiotensin II receptor (AT2) in heart [4].

Does Angiotensin II Accelerate Protein Synthesis in Adult Rat Hearts

Cardiac hypertrophy in regulated by a number of different processes such as hemodynamic load and circulating neurohumoral factors [1]. Angiotensin effects on cardiac hypertrophy are potentially of major importance and inhibitors of the renin-angiotensin system are effective therapeautic agents [2,3,4]. However, the effects of angiotensin II to stimulate cardiac hypertrophy could have been mediated by direct and indirect actions of the peptide on cardiac tissue. We have reported that elevation of aortic pressure in perfused adult rat hearts increases cAMP content and accelerates ribosomal formation and protein synthesis [5,6]. This model was used to examine the direct effects of angiotensin II on cardiac hypertrophy. The purpose of these expriments were to: 1) determine if angiotensin II accelerated rates of protein synthesis; and 2) evaluate if angiotensin coverting enzyme (ACE) inhibitors prevented the effects of elevated aortic pressure on protein synthesis.