Takashi Haneda

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.

Arginine vasopressin increases the rate of protein synthesis in isolated perfused adult rat heart via the V1 receptor.

Arginine vasopressin (AVP) is known to contribute significantly to the pathogenesis of congestive heart failure and hypertension. However, little is known about its effect on the myocardium. The present study was conducted to determine whether AVP directly increases the rate of protein synthesis in isolated, perfused rat heart, and, if so, the mechanism involved. Elevation of the aortic pressure from 60 to 120 mmHg in perfused rat heart accelerated the rate of protein synthesis which was associated with increases in cAMP levels and Ca2+ uptake. AVP (100 μM) increased Ca2+ uptake and accelerated the rate of protein synthesis without a change in cAMP concentration. The latter events were inhibited by OPC-21268 (100 μM), a selective V1 receptor antagonist, or amiloride (100 μM), an inhibitor of the Na+/H+ exchange system. However, increases in cAMP concentrations, Ca2+ uptake, and rates of protein synthesis associated with the elevated aortic pressure were not inhibited by amiloride. Thus, AVP directly increased the rate of protein synthesis via the V1 receptor that is sensitive to amiloride, a mechanism that differs from the cAMP-dependent mechanism that is responsible for the cardiac hypertrophy induced by pressure overload.

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.

The effect of pericardium on the end-systolic pressure-segment length relationship in canine left ventricle in acute volume overload.

The effect of the pericardium on the end-systolic pressure-segment length relationship in the left ventricle was examined with an ultrasonic miniature gauge in open-chest dogs. In 12 dogs, blood was infused until left ventricular (LV) end-diastolic pressure reached about 20 mm Hg, and then the pericardium was opened widely. In the other 12 dogs a pericardiectomy was performed without blood infusion. Stroke volume was measured in six dogs in the former group and in seven dogs in the latter group. After blood infusion, LV systolic, end-systolic, and end-diastolic pressures increased from 120 +/- 14 to 162 +/- 16 mm Hg (mean +/- SD), from 106 +/- 13 to 146 +/- 17 mm Hg, and from 8 +/- 2 to 19 +/- 2 mm Hg, respectively (all p less than .01). End-systolic and end-diastolic segment lengths increased from 8.9 +/- 2.1 to 10.6 +/- 2.2 mm and from 11.6 +/- 2.5 to 14.9 +/- 2.7 mm, respectively (both p less than .01). After pericardiectomy, the segments were further lengthened by 8.9 +/- 4.4% and by 10.0 +/- 6.2%, respectively (both p less than .01). Heart rate, LV systolic and end-systolic pressures, and peak positive dp/dt did not change, although end-diastolic pressure fell from 19 +/- 2 to 18 +/- 2 mm Hg (p less than .01). Stroke volume rose from 13.1 +/- 3.7 to 23.9 +/- 5.0 ml due to volume loading and further increased by 26.7 +/- 9.0% after pericardiectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Methylenetetrahydrofolate Reductase Gene Polymorphism, Hyperhomocysteinemia, and Cardiovascular Diseases in Chronic Hemodialysis Patients

Cardiovascular disease (CVD) is the principle cause of death in patients with end-stage renal disease. Some gene polymorphisms and hyperhomocysteinemia have been implicated in the pathogenesis of CVD. The aim of this study was to assess the relationships between angiotensin-converting enzyme genotype, endothelial nitric oxide synthase genotype, and methylenetetrahydrofolate reductase (MTHFR) genotype and CVD in patients on hemodialysis and to clarify the determinants of plasma homocysteine level. One hundred and sixty-eight patients on hemodialysis (87 males and 81 females, mean age 60.7 ± 13.1 years) were included. A history of CVD was present in 25% of the patients. There was a significant difference in the distributions of MTHFR non-VV genotype and MTHFR VV genotype between patients with a CVD history and patients without a CVD history, but no difference in the distributions of angiotensin-converting enzyme genotypes and endothelial nitric oxide synthase genotypes. The plasma homocysteine concentration was significantly higher in patients with MTHFR VV genotype than in patients with MTHFR non-VV genotype. The plasma homocysteine concentration was negatively correlated with plasma vitamin B12 concentration and plasma folate concentration. On stepwise multiple-regression analysis for the predictors of plasma homocysteine concentration, MTHFR VV genotype and gender were significant. In conclusion, MTHFR polymorphism may be a risk factor for CVD in patients on hemodialysis, and MTHFR VV genotype and gender may be strong determinants of the plasma homocysteine level.

Stimulation of ribosomal RNA synthesis during hypertrophic growth of cultured heart cells by phorbol ester

Primary cultures of neonatal cardiac myocytes were used to determine the effects of tumor-promoting phorbol esters on ribosomal RNA (rRNA) synthesis during myocyte growth. Treatment of myocytes with phorbol-12,13-dibutyrate (PDBu) increased protein accumulation by 25% and RNA content by 20%. Rates of rRNA synthesis were measured to assess the mechanism by which rRNA accumulated during myocyte growth. Rates of rRNA synthesis were determined from the incorporation of [3H]uridine into UMP of purified rRNA and the specific radioactivity of the cellular UTP pool. After 24 h of PDBu treatment, cellular rates of 18S and 28S rRNA synthesis were accelerated by 67% and 64%, respectively. The increased rate of rRNA synthesis accounted for the net increase in myocyte rRNA content after PDBu treatment.

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.