Masaaki Honda

Redox regulation of MAPK pathways and cardiac hypertrophy in adult rat cardiac myocyte

OBJECTIVES We analyzed the regulatory function of reactive oxygen species (ROS) on the hypertrophic signaling in adult rat cardiac myocytes: BACKGROUND The ROS regulate mitogenic signal transduction in various cell types. In neonatal rat cardiac myocyte, antioxidants have been shown to inhibit cardiac hypertrophy, and ROS are suggested to modulate the hypertrophic signaling. However, the conclusion may not reflect the situation of mature heart, because of the different natures between neonatal and adult cardiac myocytes. METHODS Cultured adult rat cardiac myocytes were stimulated with endothelin-1 (ET-1) or phenylephrine (PE), and intracellular ROS levels, the activities of mitogen-activated protein kinases (MAPKs; ERK, p38, and JNK), and 3H-phenylalanine incorporation were examined. We also examined the effects of antioxidant pretreatment of myocytes on MAPK activities and cardiac hypertrophy to analyze the modulatory function of redox state on MAPK-mediated hypertrophic signaling. RESULTS The ROS levels in ET-1- or PE-stimulated myocytes were maximally increased at 5 min after stimulation. The origin of ROS appears to be from NADH/NADPH oxidase, because the increase in ROS was suppressed by pretreatment of myocytes with NADH/NADPH oxidase inhibitor diphenyleneiodonium. Extracellular signal-regulated kinase (ERK) activity was increased by the stimulation of ET-1 or PE. In contrast, p38 and c-Jun-N-terminal protein kinase (JNK) activities did not change after these stimulations. Antioxidant treatment of myocytes suppressed the increase in ROS and blocked ERK activation and the subsequent cardiac hypertrophy induced by these stimuli. CONCLUSIONS These data demonstrate that ROS mediate signal transduction of cardiac hypertrophy induced by ET-1 or PE in adult rat cardiac myocytes.

Prominent inhibitory effects of tranilast on migration and proliferation of and collagen synthesis by vascular smooth muscle cells

To obtain some ideas about prevention of restenosis after percutaneous transluminal coronary angioplasty (PTCA), we examined the effects of transilast (anti-allergic agent) on migration and proliferation of, and collagen synthesis by, cultured vascular smooth muscle cells (VSMC) from the thoracic aorta of WKY rats. Tranilast was added to culture medium containing 10% fetal calf serum (FCS). The cultures were pulse-labeled with 3H-thymidine (TdR) or 3H-proline (Pro). TdR and Pro uptake into VSMC were measured. The effect of tranilast on migration of VSMC was examined by using culture dishes of an original design. We also examined the inhibitory effects of various drugs, such as a Ca antagonist, an angiotensin converting enzyme (ACE) inhibitor, a phosphodiesterase inhibitor, elastase, colchicine, and mitomycin C, on proliferation and migration of VSMC. Our data showed that the inhibitory effects of tranilast on migration and proliferation of, and collagen synthesis by, VSMC were prominent. Maximal percentage inhibition of proliferation, migration and collagen synthesis was 60.8 +/- 2.3%, 52.7 +/- 14.7% and 62.1 +/- 8.1%, respectively. On the other hand, the inhibitory effects of other drugs, with the exception of colchicine and mitomycin C, on proliferation and/or migration of VSMC were not very strong. Although the inhibitory effects of colchicine and mitomycin C were strong in vitro, their clinical usefulness may be limited by systemic side-effects. These results indicate the potential usefulness of tranilast for prevention of restenosis of coronary arteries after PTCA.

ANTI-APOPTOTIC EFFECT OF ATORVASTATIN, A 3-HYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE INHIBITOR, ON CARDIAC MYOCYTES THROUGH PROTEIN KINASE C ACTIVATION

1. Pleiotropic effects of statins, which are independent of lipid lowering, have been reported. In the present study, we examined the effect of a statin on apoptosis of adult rat cultured cardiac myocytes. We used the protein kinase C (PKC) inhibitors staurosporine (1 µmol/L), chelerythrine (10 µmol/L) and rottlerin (5 µmol/L) to induce myocyte apoptosis. The effect of atorvastatin (10−7 g/mL), a statin, on myocyte apoptosis induced by these PKC inhibitors was examined.

AN ANGIOTENSIN-CONVERTING ENZYME INHIBITOR PROTECTS AGAINST DOXORUBICIN-INDUCED IMPAIRMENT OF CALCIUM HANDLING IN NEONATAL RAT CARDIAC MYOCYTES

1. The effects of doxorubicin (DOX) on intracellular calcium transients were examined in neonatal rat cultured cardiac myocytes, as were the cardioprotective effects of an angiotensin‐converting enzyme (ACE) inhibitor on DOX‐induced impairment of calcium handling.

Effects of ACE inhibition and beta-blockade on collagen remodelling in the heart of Bio 14.6 hamsters.

1 The effects of angiotensin converting enzyme (ACE) inhibition and beta‐blockade on collagen in the heart and on plasma catecholamines and tissue angiotensin (Ang) I and II were examined in Bio 14.6 Syrian hamsters. Male hamsters (76–79 days old) were given low‐dose enalapril (3 mg/kg per day), high‐dose enalapril (30 mg/kg per day), atenolol (50 mg/kg per day) or vehicle for 65 days. Age and sex matched healthy F1b hamsters were used as controls. Collagen concentration was determined by measuring hydroxyproline content and the relative proportion of type I, III, and V collagens was obtained by non‐interrupted sodium dodecyl polyacrylamide gel electro‐phoresis (SDS‐PAGE). Per cent collagen area (PCA) was measured by pixel counting in myocardial tissue by a personal computer. 2 Although heartweight (HW) and bodyweight (BW) in F1b controls were significantly higher compared with drugtreated groups and vehicles, the HW/BW ratio in cardiomyopathic Bio 14.6 hamsters tended to be high compared with F1b controls and was decreased by each drug treatment. 3 Collagen concentration, total collagen content and PCA in the heart of Bio 14.6 hamsters were significantly higher than F1b controls. Collagen concentration and total collagen content were significantly decreased in all drug‐treated groups compared with vehicles. 4 The proportion of type I collagen tended to decrease while that of type III collagen tended to increase in all drugtreated groups compared with vehicles. Type V collagen in vehicle‐treated group was significantly higher than in F1b controls, while it tended to decrease in all drug‐treated groups compared with vehicles. 5 Plasma concentrations of catecholamines (adrenaline and noradrenaline) were decreased significantly by atenolol and high‐dose enalapril, but not by low‐dose enalapril. Tissue Angl remained unaltered in any of the drug‐treated hamsters. Tissue Angll was decreased by the high‐dose enalapril and beta‐blockade, and tended to be decreased by low‐dose enalapril treatment. 6 These results reveal that enalapril and atenolol produced similar beneficial effects on collagen remodelling in Bio 14.6 hamsters by decreasing the total amount of collagen, and also by changing collagen phenotypes through the inhibition of the renin‐angiotensin system. Both drugs also improved myocardial morphological integrity.

Different effects of an angiotensin converting enzyme inhibitor and a calcium antagonist on protein metabolism in rats with right ventricular hypertrophy.

Objective We examined the effects of a calcium antagonist and an angiotensin converting enzyme (ACE) inhibitor on contractile and non-contractile protein metabolism and cardiac function in a monocrotaline-induced right ventricular hypertrophy model, in order to define the effects of these drugs on cardiac hypertrophy. Methods One week after monocrotaline injection, male Sprague-Dawley rats were given either a calcium antagonist (nilvadipine; 3mg/kg per day) or an ACE inhibitor (delapril-HCI; 30mg/kg per day) for 2 weeks. Right ventricular pressure, the right ventricle:(left ventricle + interventricular septum) ratio, myosin isoenzymes, collagen concentration, collagen types and contractility of right ventricular free wall were examined. Results In untreated rats significant monocrotaline-induced right ventricular hypertrophy with an increase in the proportion of collagen types III and V was observed. There were no significant changes in collagen concentration. Both drugs reduced right ventricular pressure to the same degree and decreased right ventricular hypertrophy. However, the inhibitory effect of delapril on right ventricular hypertrophy was stronger than that of nilvadipine. Nilvadipine reduced the collagen concentration and reversed changes in collagen types, whereas delapril did not have any significant effect on collagen concentration or collagen types. Cardiac contractility was improved by delapril, but not by nilvadipine. Conclusions The results show that a calcium antagonist disproportionately inhibited contractile and non-contractile protein metabolism, whereas an ACE inhibitor proportionally inhibited them and improved cardiac function in a model of right ventricular hypertrophy. The improvement in cardiac function may be due partly to the proportional inhibition of contractile and non-contractile proteins elicited by an ACE inhibitor.

CALCIUM TRANSIENTS IN SINGLE MYOCYTES AND MEMBRANOUS ULTRASTRUCTURES DURING THE DEVELOPMENT OF CARDIAC HYPERTROPHY AND HEART FAILURE IN RATS

1. We examined changes in intracellular calcium transients of separated single myocytes from the right ventricle (RV) of the rat heart during the change from adaptation to maladaptation in response to a pressure overload.

CONTRASTING EFFECTS OF ISOPROTERENOL AND PHOSPHODIESTERASE I11 INHIBITOR ON INTRACELLULAR CALCIUM TRANSIENTS IN CARDIAC MYOCYTES FROM FAILING HEARTS

1. Effects of a newly developed phosphodiesterase (PDE) I11 inhibitor, E‐1020, on intracellular calcium transients were compared with those of isoproterenol (ISO) in isolated single myocytes from failing hearts secondary to pulmonary hypertension induced by monocrotaline injection. Myocytes were isolated by enzyme digestion using a Langendorff apparatus. Changes in intracellular calcium concentrations ([Ca2+]i,) were recorded using a fura‐2 fluorescence microscopic technique. Cyclic AMP contents of the hearts were measured by radio‐immunoassay.

ANGIOTENSIN CONVERTING ENZYME INHIBITOR, CAPTOPRIL, INHIBITS CARDIAC HYPERTROPHY WITHOUT CHANGING COLLAGEN TYPES AND CONCENTRATION IN SPONTANEOUSLY HYPERTENSIVE RATS

1. The effects of the ACE inhibitor, captopril, on collagen metabolism in spontaneously hypertensive rats (SHR) with cardiac hypertrophy was examined. Captopril (100 mg/kg per day) was administered in drinking water to 20 week old male SHR for 12 weeks. Collagen concentration was calculated from hydroxyproline content, and relative proportions of types I, III and V collagen were determined by non‐interrupted SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). These parameters were examined in age and sex matched Wistar‐Kyoto (WKY) rats, as well as in non‐treated SHR, and compared with those of captopril‐treated SHR.

Different biventricular remodelling of myosin and collagen in pulmonary hypertension

1. To clarify the metabolism of contractile and non‐contractile proteins of the ventricles during the development of right ventricular hypertrophy (RVH) and accompanying congestive heart failure (CHF) in response to a pressure overload, monocrotaline was injected subcutaneously into Sprague‐Dawley (SD) rats. Myosin isoenzymes (MIE) were analysed by pyrophosphate gel electrophoresis under non‐dissociating conditions. Acid‐soluble collagens were analysed by an improved, non‐interrupted sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Tissue collagen content was also measured after the estimation of hydroxyproline concentration in tissues.