Nobuki Takahashi

Endothelial Nitric Oxide Synthase Gene Is Positively Associated With Essential Hypertension

Essential hypertension has a genetic basis. Accumulating evidence, including findings of elevation of arterial blood pressure in mice lacking the endothelial nitric oxide synthase (eNOS) gene, strongly suggests that alteration in NO metabolism is implicated in hypertension. There are, however, no reports indicating that polymorphism in the eNOS gene is associated with essential hypertension. We have identified a missense variant, Glu298Asp, in exon 7 of the eNOS gene and demonstrated that it is associated with both coronary spastic angina and myocardial infarction. To explore the genetic involvement of the eNOS gene in essential hypertension, we examined the possible association between essential hypertension and several polymorphisms including the Glu298Asp variant, variable number tandem repeats in intron 4 (eNOS4b/4a), and two polymorphisms in introns 18 and 23. We performed a large-scale study of genetic association using two independent populations from Kyoto (n=458; 240 normotensive versus 218 hypertensive subjects) and Kumamoto (n=421; 223 normotensive versus 187 hypertensive subjects), Japan. In both groups, a new coding variant, Glu298Asp, showed a strong association with essential hypertension (Kyoto: odds ratio, 2.3 [95% confidence interval, 1.4 to 3.9]; Kumamoto: odds ratio, 2.4 [95% confidence interval, 1.4 to 4.0]). The allele frequencies of 298Asp in hypertensive subjects were significantly higher than those in normotensive subjects in both groups (Kyoto: 0.103 versus 0.050, P<0.0017; Kumamoto: 0.120 versus 0.058, P<0.0013, respectively). No such disequilibrium between genotypes was significantly associated with any other polymorphisms we examined; the Glu298Asp variant was also not linked to any other polymorphisms. In conclusion, the Glu298Asp missense variant was significantly associated with essential hypertension, which suggests that it is a genetic susceptibility factor for essential hypertension.

NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function

Reactivation of the fetal cardiac gene program is a characteristic feature of hypertrophied and failing hearts that correlates with impaired cardiac function and poor prognosis. However, the mechanism governing the reversible expression of fetal cardiac genes remains unresolved. Here we show that neuron‐restrictive silencer factor (NRSF), a transcriptional repressor, selectively regulates expression of multiple fetal cardiac genes, including those for atrial natriuretic peptide, brain natriuretic peptide and α‐skeletal actin, and plays a role in molecular pathways leading to the re‐expression of those genes in ventricular myocytes. Moreover, transgenic mice expressing a dominant‐negative mutant of NRSF in their hearts exhibit dilated cardiomyopathy, high susceptibility to arrhythmias and sudden death. We demonstrate that genes encoding two ion channels that carry the fetal cardiac currents If and ICa,T, which are induced in these mice and are potentially responsible for both the cardiac dysfunction and the arrhythmogenesis, are regulated by NRSF. Our results indicate NRSF to be a key transcriptional regulator of the fetal cardiac gene program and suggest an important role for NRSF in maintaining normal cardiac structure and function.

THE EFFECTS OF THE SELECTIVE ROCK INHIBITOR, Y27632, ON ET-1-INDUCED HYPERTROPHIC RESPONSE IN NEONATAL RAT CARDIAC MYOCYTES : POSSIBLE INVOLVEMENT OF RHO/ROCK PATHWAY IN CARDIAC MUSCLE CELL HYPERTROPHY

A small GTPase, Rho, participates in agonist‐induced cytoskeletal organization and gene expression in many cell types including cardiac myocytes. However, little is known about the functions of Rhos downstream targets in cardiac myocytes. We examined the role of ROCK, a downstream target of Rho, in ET‐1‐induced hypertrophic response. Y27632, a selective ROCK inhibitor, inhibited ET‐1‐induced increases in natriuretic peptide production, cell size, protein synthesis, and myofibrillar organization. In addition, a dominant‐negative mutant of p160ROCK suppressed ET‐1‐induced transcription of the BNP gene. These findings suggest that the Rho/ROCK pathway is an important component of ET‐1‐induced hypertrophic signals in cardiac myocytes.

Involvement of Cardiotrophin-1 in Cardiac Myocyte-Nonmyocyte Interactions During Hypertrophy of Rat Cardiac Myocytes In Vitro

BACKGROUND The mechanism responsible for cardiac hypertrophy is currently conceptualized as having 2 components, mediated by cardiac myocytes and nonmyocytes, respectively. The interaction between myocytes and nonmyocytes via growth factors and/or cytokines plays an important role in the development of cardiac hypertrophy. We found that cardiac myocytes showed hypertrophic changes when cocultured with cardiac nonmyocytes. Cardiotrophin-1 (CT-1), a new member of the interleukin-6 family of cytokines, was identified by its ability to induce hypertrophic response in cardiac myocytes. In this study, we used the in vitro coculture system to examine how CT-1 is involved in the interaction between cardiac myocytes and nonmyocytes during the hypertrophy process. METHODS AND RESULTS RNase protection assay revealed that CT-1 mRNA levels were 3. 5 times higher in cultured cardiac nonmyocytes than in cultured cardiac myocytes. We developed anti-CT-1 antibodies and found that they significantly inhibited the increased atrial and brain natriuretic peptide secretion and protein synthesis characteristic of hypertrophic changes of myocytes in the coculture. In addition, non-myocyte-conditioned medium rapidly elicited tyrosine phosphorylation of STAT3 and induced an increase in natriuretic peptide secretion and protein synthesis in cultured cardiac myocytes; these effects were partially suppressed by anti-CT-1 antibodies. Finally, the hypertrophic effects of CT-1 and endothelin-1, which we had previously implicated in the hypertrophic activity in the coculture, were additive in cardiac myocytes. CONCLUSIONS These results show that CT-1 secreted from cardiac nonmyocytes is significantly involved in the hypertrophic changes of cardiac myocytes in the coculture and suggest that CT-1 is an important local regulator in the process of cardiac hypertrophy.

Blockade of the natriuretic peptide receptor guanylyl cyclase-A inhibits NF-κB activation and alleviates myocardial ischemia/reperfusion injury

Acute myocardial infarction (AMI) remains the leading cause of death in developed countries. Although reperfusion of coronary arteries reduces mortality, it is associated with tissue injury. Endothelial P-selectin-mediated infiltration of neutrophils plays a key role in reperfusion injury. However, the mechanism of the P-selectin induction is not known. Here we show that infarct size after ischemia/reperfusion was significantly smaller in mice lacking guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. The decrease was accompanied by decreases in neutrophil infiltration in coronary endothelial P-selectin expression. Pretreatment with HS-142-1, a GC-A antagonist, also decreased infarct size and P-selectin induction in wild-type mice. In cultured endothelial cells, activation of GC-A augmented H2O2-induced P-selectin expression. Furthermore, ischemia/reperfusion-induced activation of NF-kappaB, a transcription factor that is known to promote P-selectin expression, is suppressed in GC-A-deficient mice. These results suggest that inhibition of GC-A alleviates ischemia/reperfusion injury through suppression of NF-kappaB-mediated P-selectin induction. This novel, GC-A-mediated mechanism of ischemia/reperfusion injury may provide the basis for applying GC-A blockade in the clinical treatment of reperfusion injury.

The Neuron-Restrictive Silencer Element–Neuron-Restrictive Silencer Factor System Regulates Basal and Endothelin 1-Inducible Atrial Natriuretic Peptide Gene Expression in Ventricular Myocytes

ABSTRACT Induction of the atrial natriuretic peptide (ANP) gene is a common feature of ventricular hypertrophy. A number of cis-acting enhancer elements for several transcriptional activators have been shown to play central roles in the regulation of ANP gene expression, but much less is known about contributions made by transcriptional repressors. The neuron-restrictive silencer element (NRSE), also known as repressor element 1, mediates repression of neuronal gene expression in nonneuronal cells. We found that NRSE, which is located in the 3′ untranslated region of the ANP gene, mediated repression of ANP promoter activity in ventricular myocytes and was also involved in the endothelin 1-induced increase in ANP gene transcription. The repression was conferred by a repressor protein, neuron-restrictive silencer factor (NRSF). NRSF associated with the transcriptional corepressor mSin3 and formed a complex with histone deacetylase (HDAC) in ventricular myocytes. Trichostatin A (TSA), a specific HDAC inhibitor, relieved NRSE-mediated repression of ANP promoter activity, and chromatin immunoprecipitation assays revealed the involvement of histone deacetylation in NRSE-mediated repression of ANP gene expression. Furthermore, in myocytes infected with recombinant adenovirus expressing a dominant-negative form of NRSF, the basal level of endogenous ANP gene expression was increased and a TSA-induced increase in ANP gene expression was apparently attenuated, compared with those in myocytes infected with control adenovirus. Our findings show that an NRSE-NRSF system plays a key role in the regulation of ANP gene expression by HDAC in ventricular myocytes and provide a new insight into the role of the NRSE-NRSF system outside the nervous system.

Guanylyl Cyclase-A Inhibits Angiotensin II Type 1A Receptor-Mediated Cardiac Remodeling, an Endogenous Protective Mechanism in the Heart

Background—Guanylyl cyclase (GC)-A, a natriuretic peptide receptor, lowers blood pressure and inhibits the growth of cardiac myocytes and fibroblasts. Angiotensin II (Ang II) type 1A (AT1A), an Ang II receptor, regulates cardiovascular homeostasis oppositely. Disruption of GC-A induces cardiac hypertrophy and fibrosis, suggesting that GC-A protects the heart from abnormal remodeling. We investigated whether GC-A interacts with AT1A signaling in the heart by target deletion and pharmacological blockade or stimulation of AT1A in mice. Methods and Results—We generated double-knockout (KO) mice for GC-A and AT1A by crossing GC-A-KO mice and AT1A-KO mice and blocked AT1 with a selective antagonist, CS-866. The cardiac hypertrophy and fibrosis of GC-A-KO mice were greatly improved by deletion or pharmacological blockade of AT1A. Overexpression of mRNAs encoding atrial natriuretic peptide, brain natriuretic peptide, collagens I and III, transforming growth factors &bgr;1 and &bgr;3, were also strongly inhibited. Furthermore, stimulation of AT1A by exogenous Ang II at a subpressor dose significantly exacerbated cardiac hypertrophy and dramatically augmented interstitial fibrosis in GC-A-KO mice but not in wild-type animals. Conclusions—These results suggest that cardiac hypertrophy and fibrosis of GC-A-deficient mice are partially ascribed to an augmented cardiac AT1A signaling and that GC-A inhibits AT1A signaling-mediated excessive remodeling.

Overexpression of Brain Natriuretic Peptide Facilitates Neutrophil Infiltration and Cardiac Matrix Metalloproteinase-9 Expression After Acute Myocardial Infarction

Background—Recent clinical trials have shown that systemic infusion of nesiritide, a recombinant human brain natriuretic peptide (BNP), improves hemodynamic parameters in acutely decompensated hearts. This suggests that BNP exerts a direct cardioprotective effect and might thus be a useful therapeutic agent with which to treat acute myocardial infarction (MI). In the present study, we used BNP-transgenic (BNP-Tg) mice with elevated plasma BNP to determine whether and how BNP contributes to left ventricular remodeling and healing after MI. Methods and Results—We examined the accumulation of neutrophils and the expression and activation of matrix metalloproteinase (MMP)-9 in the ventricles of male BNP-Tg mice and their nontransgenic (non-Tg) littermates during the early phase after acute MI. The numbers of neutrophils infiltrating the infarcted area were significantly increased in BNP-Tg mice 3 days after MI. In addition, both the gene expression and zymographic activity of MMP-9, but not MMP-2, were significantly higher in BNP-Tg than non-Tg mice. Double immunostaining revealed that neutrophils are the main source of the MMP-9, although doxycycline, an MMP inhibitor, had no effect on neutrophil infiltration of the infarcted area in BNP-Tg mice. Conclusions—These results demonstrate that elevated plasma BNP facilitates neutrophil infiltration of the infarcted area after MI and increases the activity of the MMP-9 they produce. This suggests that BNP plays a key role in the processes of extracellular matrix remodeling and wound-healing during the early phase after acute MI.

Angiotensin II type 2 receptor deficiency exacerbates heart failure and reduces survival after acute myocardial infarction in mice.

Background—Angiotensin II plays a prominent role in the progression of heart failure after acute myocardial infarction (AMI). Although both angiotensin type 1 (AT1) and type 2 (AT2) receptors are known to be present in the heart, comparatively little is known about the latter. We therefore examined the role played by AT2 receptors in post-AMI heart failure. Methods and Results—In wild-type mice subjected to AMI by coronary artery ligation, AT2 receptor immunoreactivity is upregulated in the infarct and border areas. Among AT2 receptor-null (−/−) mice, the 7-day survival rate after AMI was significantly lower than among wild-type mice (43% versus 67%;P <0.05). All sham-operated animals of both genotypes survived through the study. Ventricular mRNA levels for brain natriuretic peptide were elevated in both genotypes 24 hours after coronary occlusion, with levels in AT2−/− significantly higher than in wild-type mice, as were their lung weights, and histological examination revealed marked pulmonary congestion in the AT2−/− mice. Cardiac function was significantly decreased in AT2−/− mice 2 days after AMI. Conclusions—AT2 receptor deficiency exacerbates short-term death rates and heart failure after experimental AMI in mice. The AT2 receptor may thus exert a protective effect on the heart after AMI.

Interaction of myocytes and nonmyocytes is necessary for mechanical stretch to induce ANP/BNP production in cardiocyte culture

In cardiac hypertrophy or ventricular remodeling, enlargement of myocytes and interstitial or perivascular fibrosis are observed simultaneously, which suggests an interaction between cardiac myocytes and fibroblasts. In this study we examined the mechanism of cyclic mechanical stretch-induced myocytic hypertrophy, focusing on the interaction between myocytes and cardiac nonmyocytes, mostly fibroblasts. Ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs) were separately extracted from neonatal rat ventricles by the discontinuous Percoll gradient method and primary cultures of cardiac cells were prepared. When MCs were co-cultured with NMCs, the size of MCs and the ANP/BNP secretion were significantly increased. This hypertrophic change of MCs in the co-culture was significantly suppressed by BQ-123, an endothelin-A (ETA) receptor antagonist. Cyclic stretch did not induce hypertrophic responses in MC culture. However, it further increased ANP/BNP production in MC-NMC co-culture (2.2-fold and 2.1-fold increases vs. non-stretch group after 48-h incubation). This increase in ANP/BNP production in the co-culture was significantly suppressed by CV-11974, an angiotensin II (Ang II) type 1 receptor antagonist. This study raises the possibility that NMCs regulate cardiocyte hypertrophy via secretion of endothelin-1 and that Ang II is involved in the interaction between MCs and NMCs during the course of hypertrophic response of cardiocytes to mechanical stretch.