Junzo Osaki

Lovastatin prevents angiotensin II-induced cardiac hypertrophy in cultured neonatal rat heart cells.

Angiotensin II activates p21ras, and mediates cardiac hypertrophic growth through the type 1 angiotensin II receptor in cardiac myocytes. An inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase has been shown to block the post-translational farnesylation of p21ras and inhibit protein synthesis in several cell types. Primary cultures of neonatal cardiac myocytes were used to determine whether HMG-CoA reductase inhibitors, lovastatin, simvastatin and pravastatin inhibit the angiotensin II-induced hypertrophic growth. Angiotensin II (10(-6) M) significantly increased protein-DNA ratio, RNA-DNA ratio, ratios of protein synthesis and mitogen-activated protein (MAP) kinase activity. Lipid-soluble HMG-CoA reductase inhibitors, lovastatin (10(-6) M) and simvastatin (10(-6) M) partially and significantly inhibited the angiotensin II-induced increases in these parameters, but a water-soluble HMG-CoA reductase inhibitor, pravastatin (10(-6) M) did not. Mevalonate (10(-4) M) overcame the inhibitory effects of lovastatin and simvastatin on angiotensin II-induced increases in these parameters. A selective protein kinase C inhibitor, calphostin C (10(-6) M) partially and significantly prevented angiotensin II-induced increases in these parameters, and treatment with both lovastatin and calphostin C inhibited completely. Angiotensin II increased p21ras activity and membrane association, and lovastatin inhibited them. These studies demonstrate that a lipid-soluble HMG-CoA reductase inhibitor, lovastatin, may prevent angiotensin II-induced cardiac hypertrophy, at least in part, through p21ras/MAP kinase pathway, which is linked to mevalonate metabolism.

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.

Roles of calcineurin and calcium/calmodulin-dependent protein kinase II in pressure overload-induced cardiac hypertrophy

Calcineurin and calcium/calmodulin-dependent protein kinase (CaMK) II have been suggested to be the signaling molecules in cardiac hypertrophy. It was not known, however, whether these mechanisms are involved in cardiac hypertrophy induced by pressure overload without the influences of blood-derived humoral factors, such as angiotensin II. To elucidate the roles of calcineurin and CaMK II in this situation, we examined the effects of calcineurin and CaMK II inhibitors on pressure overload-induced expression of c-fos, an immediate-early gene, and protein synthesis using heart perfusion model. The hearts isolated from Sprague-Dawley rats were perfused according to the Langendorff technique, and then subjected to the acute pressure overload by raising the perfusion pressure. The activation of calcineurin was evaluated by its complex formation with calmodulin and by its R-II phosphopeptide dephosphorylation. CaMK II activation was evaluated by its autophosphorylation. Expression of c-fos mRNA and rates of protein synthesis were measured by northern blot analysis and by 14C-phenylalanine incorporation, respectively. Acute pressure overload significantly increased calcineurin activity, CaMK II activity, c-fos expression and protein synthesis. Cyclosporin A and FK506, the calcineurin inhibitors, significantly inhibited the increases in both c-fos expression and protein synthesis. KN62, a CaMK II inhibitor, also significantly prevented the increase in protein synthesis, whereas it failed to affect the expression of c-fos. These results suggest that both calcineurin and CaMK II pathways are critical in the pressure overload-induced acceleration of protein synthesis, and that transcription of c-fos gene is regulated by calcineurin pathway but not by CaMK II pathway.

Pressure-induced expression of heat shock protein 70 mRNA in adult rat heart is coupled both to protein kinase A-dependent and protein kinase C-dependent systems

Background Production of heat shock protein 70 (HSP70) in the heart is induced by hemodynamic stress, but its intracellular signal transduction system has not been elucidated well. Objective To investigate the hypothesis that protein kinase A (PKA)-dependent and protein kinase C (PKC)-dependent systems are involved in the pressure-induced expression of HSP70 mRNA in perfused adult rat heart. Methods Isolated tetrodotoxin-arrested Sprague–Dawley rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. Aortic pressure in rats of the pressure-overloaded group was elevated from 60 to 120 mmHg for 2–120 min. cAMP contents and rates of synthesis of protein were measured by radioimmunoassay and the incorporation of [14C]phenylalanine into total heart protein, respectively. Expression of HSP70 mRNA was determined by Northern blot analysis. Results Elevation of aortic pressure significantly increased cAMP content after 2 min of perfusion (by 41%), significantly increased rates of synthesis of protein during the second hour of perfusion (by 41%), and induced expression of HSP70 mRNA maximally after 60 min of perfusion (2.7-fold the control value). Exposure to glucagon, forskolin or 1-methyl-3-isobutylxanthine mimicked increases in these parameters caused by elevation of aortic pressure. Administration of a selective PKA inhibitor, H-89, significantly prevented induction of increases in expression of HSP70 mRNA and rates of synthesis of protein by a high pressure overload and exposure to agents that increase cAMP content. Furthermore, administration of phorbol ester induced expression of HSP70 mRNA. Administration of a PKC inhibitor, calphostin C, significantly prevented induction of increases in expression of HSP70 mRNA by a pressure overload and by exposure to phorbol ester. Conclusions These results suggest that the pressure-induced induction of production of HSP70 is regulated both by PKA-dependent and by PKC-dependent systems during periods of active synthesis of protein in adult rat heart.

cAMP-mediated c -fos expression in pressure-overloaded acceleration of protein synthesis in adult rat heart

OBJECTIVES The aim was to determine whether proto-oncogene c-fos expression and acceleration of protein synthesis by acute pressure overload to the heart were coupled with a cAMP- and protein-kinase-A-dependent system in adult rat heart. METHODS Isolated adult rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. In the pressure-overloaded group, aortic pressure was raised from 60 to 120 mmHg for the time indicated. Agents that increase cAMP were added to the perfusate at an aortic pressure of 60 mmHg. Furthermore, a selective protein kinase A inhibitor (H-89) or a selective protein kinase C inhibitor (calphostin C) was administered before the elevation of aortic pressure or the addition of the agents. cAMP content or rates of protein synthesis were measured by RIA or the incorporation of [14C]phenylalanine into total heart protein, respectively. c-fos mRNA expression was determined by Northern blot analysis. RESULTS Elevation of aortic pressure in beating hearts and arrested hearts increased cAMP content at 2 min of perfusion by 36 and 41%, induced c-fos mRNA expression at 30-60 min of perfusion by 4.8- and 2.0-fold, and accelerated rates of protein synthesis during the 2nd hour of perfusion by 39 and 41% over control levels, respectively. Glucagon, forskolin or IBMX mimicked increases in these parameters by elevated aortic pressure. H-89 prevented these changes by elevated pressure overload or exposure to forskolin or IBMX in arrested hearts. On the other hand, calphostin C prevented the pressure-induced increases in c-fos expression and protein synthesis rates in arrested hearts. CONCLUSIONS These results suggest that c-fos expression induced by acute pressure overload may be coupled with increased cAMP content and protein kinase A activity in addition to increased protein kinase C activity in adult rat heart.

Differential Effects of Amiloride on the Basal Rate and the Pressure Overload-induced Increase in Protein Synthesis in Perfused Rat Heart

The purpose of the present study were to determine the contribution of Na+/H+ exchange to pressure overload-induced cardiac hypertrophy and to examine its potential interaction with cAMP-dependent signaling pathway. Isolated rat hearts were perfused as Langendorff preparations with aortic pressure of 60 mmHg. In pressure overload group, aortic pressure was increased to 120 mmHg. cAMP contents in the heart perfused at 2 min were examined by RIA. Rates of protein synthesis were examined by 14C-phenylalanine incorporation into myocardial protein during the second hour of perfusion. Expression of c-fos mRNA in the heart perfused at 1 hour was analyzed by Northern blotting. Elevation of aortic pressure from 60 mmHg to 120 mmHg in perfused rat hearts increased cAMP contents from 4.89 +/- 0.09 to 6.30 +/- 0.28 pmol/mg protein and accelerated rates of protein synthesis from 644 +/- 13 to 860 +/- 49 mmol Phe/g dry heart/hr. Expression of c-fos mRNA was induced by elevated aortic pressure. Amiloride, an inhibitor of Na+/H+ exchange, decreased rates of protein synthesis in a concentration-dependent manner (12.5, 25, 50, 100 microM) but did not change cAMP content (5.25 +/- 0.11 pmol/mg protein) or expression of c-fos mRNA. Furthermore, amiloride did not prevent the increases in cAMP (6.99 +/- 0.34 pmol/mg protein), protein synthesis rates (476 +/- 18 to 689 +/- 31 nmolPhe/g dry heart/hr) and expressions of c-fos mRNA that were induced by elevation of aortic pressure. These results indicate that amiloride, an inhibitor of Na+/H+ exchange system, while influencing rates of protein synthesis, does not play an important role in pressure overload-induced cardiac hypertrophy. The mechanism by which amiloride influences cardiac protein synthesis is independent of the cAMP-dependent mechanism by which pressure overload induces cardiac hypertrophy.

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.