Hideaki Kawaguchi

Can Exercise Training With Weight Loss Lower Serum C-Reactive Protein Levels?

Objective—C-reactive protein (CRP), an obesity-related inflammatory marker, is a promising predictor for cardiovascular disease and may be a mediator for atherogenesis. It has been reported that diet-induced weight loss lowered CRP levels. However, the effect of exercise training, another therapy that can reduce weight, on CRP is still unclear. We examined effects of exercise training with weight loss on CRP levels and conventional cardiovascular risks. Methods and Results—A total of 227 apparently healthy women were recruited, and 199 subjects (average age 52 years) completed a 2-month weight reduction program consisting of supervised aerobic exercises. After the program, weight was reduced from 65.8 to 62.8 kg (P<0.0001), and all conventional variables were remarkably improved. Similarly, CRP levels were significantly decreased, from 0.63 (0.28 to 1.19) to 0.41 (0.18 to 0.80) mg/L (P<0.0001). However, in contrast to other variables, the changes in CRP levels were not proportionally associated with the extent of weight reduction. In the quartile analysis of % weight reduction, the largest weight reduction quartile did not show significant decreases in CRP levels, whereas moderate quartile showed remarkable CRP decreases. Conclusions—Exercise training with weight reduction disproportionately lowered CRP levels. Considering inflammatory status, there might be an optimal pace of exercise with weight loss.

Endothelin stimulates angiotensin I to angiotensin II conversion in cultured pulmonary artery endothelial cells.

We studied the effects of endothelin on the conversion of angiotensin I to angiotensin II in pulmonary artery endothelial cells. Endothelin had a novel effect on angiotensin I conversion. When endothelin was added to pulmonary artery endothelial cells, the conversion of angiotensin I to angiotensin II was enhanced about two-fold. The maximum stimulation was achieved at 10(-8) M of endothelin. This stimulatory effect was suppressed by angiotensin converting enzyme inhibitors such an enalapril. When the calcium antagonist, nifedipine, was incubated with 10(-8) M of endothelin, the conversion of angiotensin I to angiotensin II stimulated with endothelin was slightly suppressed by nifedipine. Enalapril (10(-6) M) completely inhibited the conversion of angiotensin I to angiotensin II in the presence of endothelin. These results suggest that endothelin may play an important role in regulating vascular tone by modulating the conversion of angiotensin I to angiotensin II.

Expression of the angiotensinogen gene and localization of its protein in the human heart.

BackgroundThere have been no reports on the presence of the tissue renin-angiotensin system in the human heart, although the presence of angiotensinogen has been described in the animal heart. Methods and ResultsTo determine whether angiotensinogen is synthesized in the human heart, we examined angiotensinogen messenger RNA (mRNA) synthesis in autopsy hearts by using ribonuclease protection assay. As a result, angiotensinogen mRNA was detected in the atrial muscle, muscles of the conduction system, and the left ventricular wall. In the left ventricular wall, mRNA expression was more prominent in the subendocardial muscles than in the midcardial or epicardial muscles. Using a monoclonal antibody to human angiotensinogen in immunoblotting experiments, we detected two closely spaced bands at approximately 70 kd in the heart, which was quite consistent with the human angiotensinogen molecule. Immunohistochemical studies with this monoclonal antibody demonstrated intense immunoreactivity in the atrial muscles, the muscles of the conduction system, and those of the subendocardial layers. ConclusionsWe conclude that angiotensinogen was synthesized in the human heart. It was evident that the localization of angiotensinogen was not ubiquitous in the cardiac muscles, showing its predilection for the atrial muscles, muscles of the conduction system, and subendocardial layer of the left ventricle.

Effect of endothelin on angiotensin converting enzyme activity in cultured pulmonary artery endothelial cells.

We studied the effects of endothelin on angiotensin converting enzyme (ACE) in cultured pulmonary artery endothelial cells. ACE activity was increased 2.5-fold by the addition of 1 x 10(-8) mol/l endothelin-1. Endothelin-1 also stimulated calcium influx and phospholipase C activity in a dose-dependent manner. Calcium influx, phospholipase C and ACE activity were suppressed 60-70% in the presence of endothelin-1 (10(-10) to 10(-6) mol/l) by 50 microliters neomycin. These results suggest that ACE was stimulated by endothelin-1 and that its activity may be closely related to phosphatidylinositol turnover stimulated by endothelin-1.

Angiotensinogen gene polymorphism in Japanese patients with hypertrophic cardiomyopathy

To examine the contribution of the renin-angiotensin system to hypertrophic cardiomyopathy (HCM), we studied 96 patients with HCM (mean age 50 years, 55% male), 105 of their unaffected siblings and offspring, and 160 healthy subjects without known hypertension and left ventricular hypertrophy (LVH) who were frequency matched to cases by age and sex. Patients were divided into familial or sporadic HCM (FHCM or SHCM) groups with or without affected members of their family. The region of interest in the angiotensinogen (AGT) gene, the missense mutation with methione-to-threonine amino acid substitution at codon 235 in angiotensinogen (M235T), was amplified by polymerase chain reaction with the use of allele-specific oligonucleotide primers flanking the polymorphic region of the AGT gene to amplify template deoxyribonucleic acid prepared from peripheral leukocytes. The T allele frequency was higher in the SHCM group than in unaffected siblings and offspring (88% vs 78%, X2 = 4.6, p < 0.05). The M allele frequency was higher in unaffected siblings and offspring than in patients with SHCM (23% vs 12%, X2 = 4.6, p < 0.05). The T allele frequency among unaffected siblings and offspring was similar to that observed in healthy subjects (78% vs 78%). We conclude that HCM, especially in sporadic cases, is partially determined by genetic disposition. The molecular variant of angiotensinogen T235 seems to be a predisposing factor for cardiac hypertrophy in HCM and carries an approximately twofold increased risk.

Phosphatidylinositol metabolism in hypertrophic rat heart.

The accumulation of inositol 1,4,5-trisphosphate (IP3) after hormonal stimulation has a physiological role, possibly by alteration of Ca2+ levels in cardiac myocyte. However, this accumulation has not been studied under pathophysiological conditions. In this report, we examine phosphatidylinositol metabolism during cellular response to norepinephrine in pressure-overloaded hypertrophic rat heart. After stimulation with norepinephrine, the accumulations of IP3 and diacylglyceride significantly increased in isolated myocytes from stroke-prone spontaneously hypertensive rat (SHRSP) heart, indicating phosphatidylinositol-specific phospholipase C activity increased in SHRSP heart cells. Protein kinase C activity was also enhanced in SHRSP, with a marked increase in particulate activity. We determined the intracellular calcium concentration and found it to be higher in SHRSP than in Wistar-Kyoto (WKY) rats at 30-40 weeks of age. Ca2+ influx was also elevated in SHRSP stimulated by norepinephrine. In SHRSP heart, cytosolic Ca2+ concentration may rise quickly in response to some stimuli, such as alpha 1-adrenergic stimulation, which is shown to be one of the pathways that increases cytosolic Ca2+ levels in hypertrophied rat heart. These data suggest that a part of the phosphatidylinositol-turnover pathway, such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway, may play an important role in the development of hypertrophy in SHRSP heart.

Chronic effects of enalapril and amlodipine on cardiac remodeling in cardiomyopathic hamster hearts.

This study examined the effects of long-term treatments with the angiotensin-converting enzyme inhibitor, enalapril, and the calcium antagonist, amlodipine, on the morphologic changes, progressive left ventricular dysfunction, and gene expression of the ryanodine receptor (RyR) and phospholamban (PLN) in dilated cardiomyopathy. From the ages of 5 through 20 weeks, dilated cardiomyopathic hamsters, BIO53.58 (BIO), and control hamsters, F1b, orally received either enalapril or amlodipine. Cardiac function was assessed by echocardiography. At the age of 20 weeks, the collagen volume fractions were analyzed by the stereologic method. RyR and PLN messenger RNAs (mRNAs) were examined by Northern blot in the amlodipine group. In BIO, the reduction of left ventricular percentage of fractional shortening was attenuated in the enalapril group (p < 0.05) and amlodipine group (p < 0.001), and the increase in the collagen volume fraction and the loss of myocytes were suppressed in the amlodipine group compared with the untreated group. RyR mRNA level decreased in BIO (p < 0.01) compared with F1b, but PLN mRNA level was unchanged. RyR and PLN mRNA levels were unaffected by the treatment with amlodipine. Enalapril and amlodipine prevent progressive remodeling and reduce cardiac dysfunction in BIO. Amlodipine prevents fibrosis and cell death without modifying RyR and PLN mRNA levels in BIO.

Adaptor Protein Crk Induces Src-Dependent Activation of p38 MAPK in Regulation of Synovial Sarcoma Cell Proliferation

The adaptor protein Crk mediates intracellular signaling related to cell motility and proliferation and is implicated in human tumorigenesis. The role of Crk in the growth of human sarcoma has remained unclear, however. The present study shows that Crk-induced activation of Src and subsequent signaling by p38 mitogen-activated protein kinase (MAPK) contribute to the enhanced proliferation of human synovial sarcoma cells. Depletion of Crk by RNA interference markedly inhibited proliferation of the synovial sarcoma cell lines HS-SYII, SYO-1, and Fuji as well as prevented anchorage-independent growth. Conversely, reconstitution with CrkII by authentic small interfering RNA–resistant Crk gene restored proliferation in Crk-silenced SYO-1 cells. Crk-depleted synovial sarcoma cells manifested enhanced transcriptional activity and expression of the p16INK4A gene, resulting in their accumulation in G1 phase of the cell cycle. In response to hepatocyte growth factor stimulation, Crk prominently induced the tyrosine phosphorylation of Grb2-associated binder 1 through activation of Src and focal adhesion kinase, and the Src family kinase inhibitor PP2 almost completely inhibited the proliferation of SYO-1 cells. Crk also induced the phosphorylation of p38 MAPK, and SB203580, a p38 MAPK–specific inhibitor, increased expression of p16INK4A gene in SYO-1 cells. Furthermore, SB203580 or depletion of p38 MAPK by small interfering RNA suppressed both the phosphorylation of Akt triggered by hepatocyte growth factor and the proliferation of SYO-1 cells. These results suggest that Crk promotes proliferation of human synovial sarcoma cells through activation of Src and its downstream signaling by a novel p38 MAPK-Akt pathway, with these signaling molecules providing potent new targets for molecular therapeutics. (Mol Cancer Res 2009;7(9):1582–92)

Effect of palmitic acid and fatty acid binding protein on ventricular fibrillation threshold in the perfused rat heart

SummaryThe effects of increased free fatty acid (FFA) levels on ventricular arrhythmias remain controversial. Using ventricular fibrillation threshold (VFT), we examined the relationship between FFA levels and ventricular arrhythmias. Isolated rat hearts were perfused with palmitate bound to either albumin or fatty acid binding protein (FABP) by Langendorf’s method. The VFT was determined by electrical stimulation. Perfusion with 0.12 mM albumin alone, 0.12 mM palmitate bound to 0.12 mM albumin, and 0.36 mM palmitate bound to 0.12 mM albumin did not lower the VFT significantly. However, 0.60 mM palmitate bound to 0.12 mM albumin lowered VFT from 2.19 ± 0.20 mA to 1.56 ± 0.13 mA. The perfusion of 0.36 mM palmitate bound to 0.12 mM FABP lowered the VFT from 2.05 ± to 0.19 mA to 1.47 ± 0.23 mA, but 0.12 mM FABP alone did not affect the VFT. Perfusion with 0.36 mM palmitate bound to 0.12 mM FABP caused the VFT to fall more than perfusion with 0.36 mM palmitate bound to 0.12 mM albumin. Then the effects of verapamil perfusion or a low concentration of perfusate Ca2+ on VFT were examined. VFT was determined by electrical stimulation. Palmitate (0.6 mM) bound to 0.12 mM albumin lowered VFT. Verapamil 10−7 perfusion and a low concentration of Ca2+ (Ca2+ 1.67 mM) suppressed the FFA-induced fall of VFT. These results suggested that the arrhythmogenic action of FFA was related to Ca2+ overload in myocardial cells.

Diffication of high affinity fatty acid receptors in rat myocardial sarcolemmal membranes

High affinity receptors for fatty acid were purified from rat cardiac sarcolemmal membrane using gel filtration, DEAE-cellulose chromatography and affinity chromatography. The purified protein was homogeneous on polyacrylamide gel electrophoresis with the molecular weight of 60 kDa. Binding studies revealed the presence of a single class of high affinity binding sites with an apparent dissociation constant of 1.0 μM and a maximal binding capacity of 12.1 pmol/μg protein.