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Pirfenidone (5-methyl-1-phenyl-2-[(1)H]-pyridone) is an effective drug for idiopathic interstitial pneumonia that can prevent and reverse tissue fibrosis in several organs. Therefore, we investigated whether pirfenidone has a potential role in preventing angiotensin II (Ang II)-induced cardiac hypertrophy. A cardiac hypertrophic mouse model was created using an Ang II infusion (200 ng kg(-1) min(-1)) in wild-type mice for 2 weeks. Mice were divided into the following three groups: a saline-infused (control) group, an Ang II infusion (vehicle) group and an Ang II infusion+pirfenidone-treated (PFD) group, which received pirfenidone (300 mg kg(-1) per day) by gastric gavage during the Ang II infusion. At 2 weeks, we assessed hemodynamics and cardiac function and investigated tissue fibrosis of the myocardium histologically and genetically. Blood pressure in the vehicle group was significantly increased compared to the control group. Although blood pressure was not different between the vehicle and PFD groups, heart weight was significantly decreased in the PFD group. Echocardiography revealed that left ventricular hypertrophy was significantly increased in the vehicle group vs. the control group. Interestingly, pirfenidone significantly inhibited this effect. Continuous infusion of Ang II increased the perivascular and interstitial tissue fibrosis, and pirfenidone inhibited these fibrotic changes. Pirfenidone also inhibited Ang II-induced hypertrophy. In the vehicle group, the mRNA expressions of atrial natriuretic peptide, brain natriuretic peptide and transforming growth factor-β1 were increased, which was significantly inhibited by pirfenidone. Furthermore, the expression of mineralocorticoid receptors was attenuated by pirfenidone. These results indicate that pirfenidone might be effective as an antifibrotic drug in the treatment of cardiac hypertrophy induced by hypertension.

Osteopontin has been reported to have an important role in cardiac fibrosis. However, little is known about the effects of angiotensin-converting enzyme inhibitor (ACEI) and angiotensin type 1 receptor blockers (ARB) on osteopontin expression in infarcted myocardium. The purpose of this study was to elucidate the effects of an ACEI (perindpril) and an ARB (candesartan cilexitil) on cardiac function as assessed by Doppler echocardiography and cardiac osteopontin expression associated with cardiac remodeling in myocardial infarcted rats. ACEI or ARB was administered after myocardial infarction (MI). At 4 weeks after MI, cardiac function, and mRNAs in non-infarcted myocardium were analyzed. ACEI and ARB equally prevented left ventricular dilatation, reduction of ejection fraction, and the increase in E/A wave velocity ratio and the rate of E wave deceleration by MI. ACEI and ARB significantly suppressed increased mRNA expression of atrial natriuretic peptide, brain natriuretic peptide, osteopontin, and collagen I and III in the non-infarcted ventricle at 4 weeks. Immunohistochemically stained osteopontin was increased in interstitial fibrosis of non-infarcted myocardium. Both ACEI and ARB significantly prevented cardiac fibrosis and osteopontin expression. In conclusion, angiotensin blockade inhibits osteopontin expression in non-infarcted myocardium and prevents cardiac remodeling after MI.

Background: Because the effects of an aldosterone receptor antagonist on transcriptional factors and mRNA expression have not been fully examined in myocardial infarction (MI), the present study examined the effects of spironolactone (SPIRO) and candesartan cilexitil (CAN) on activation of activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB) and mRNA expression in the non-ischemic myocardium after MI., Methods and Results: MI was induced by ligation of the coronary artery in Wistar rats, which were separated into (1) vehicle-treated group, (2) CAN-treated group (10 mg/kg per day), (3) SPIRO-treated group (100 mg/kg per day) and (4) CAN + SPIRO-treated group. The activity of both AP-1 and NF-kappaB was significantly increased at 4 weeks after MI. CAN or SPIRO significantly prevented the cardiac remodeling and activity of AP-1 and NF-kappaB. Furthermore, CAN + SPIRO prevented the cardiac remodeling and activation of AP-1 and NF-kappaB, compared with CAN or SPIRO alone. Myocardial atrial natriuretic peptide, brain natriuretic peptide, collagen I and III mRNAs were enhanced by MI, and CAN or SPIRO alone significantly reduced the mRNAs. CAN + SPIRO significantly prevented these mRNAs, compared with CAN or SPIRO alone., Conclusions: Both aldosterone and angiotensin II are involved in the myocardial transcriptional activation of AP-1, NF-kappaB and the cardiac remodeling-related mRNAs. The combination of CAN and SPIRO may be a potent therapeutic strategy for preventing cardiac remodeling after MI.

As one of the signal transduction pathways related to myocardial remodeling, mitogen-activated protein kinases (MAPKs) possibly play an important role in ischemic heart disease, but it is still unknown whether myocardial MAPKs are activated in the non-ischemic region of an acute myocardial infarction (AMI). Therefore, the present study investigated the myocardial activity of extracellular signal-regulated kinases (ERKs), c-Jun NH2 terminal kinases (JNKs) and p38MAPK during the acute phase of an infarction of the rat heart, and measured the geometrical ventricular changes by echocardiography. All MAPKs were significantly activated in the ischemic myocardium (IM), non-ischemic septal wall (SW), and right ventricular wall (RV). Furthermore, the activation patterns of MAPKs differed in each region. The activation of p44ERK, JNKs and p38MAPK in the IM occurred rapidly after myocardial ischemia, followed by those in the SW and RV. The activator protein-1 DNA binding activities of the IM, SW and RV increased significantly at I day after coronary ligation. Echocardiography showed increased SW motion and RV dilatation. In conclusion, this is the first in vivo evidence that myocardial MAPKs are activated in the non-ischemic region of an AMI. Echocardiographic results suggest that acceleration of workload and/or stretch may partially induce the activation of MAPKs.

The effect of taurine on angiotensin II-induced changes in cell morphology and biochemistry of the cultured neonatal cardiomyocyte was examined. Angiotensin II (1-100 nM) alone caused a slow increase in the surface area of the myocyte accompanied by an induction of the expression of atrial natriuretic peptide (ANP) and an upregulation of transforming growth factor beta(1) gene (TGF-beta(1)). The signaling pathway of angiotensin II (1-100 nM) was found to proceed through protein kinase C and the rapid activation of mitogen-activated protein (MAP) kinases. Pretreatment of the myocyte with taurine (20 mM) in the absence of angiotensin II had no visible effect on cell size or growth rate. However, the cells that were pretreated with taurine (20 mM) for 24 h exhibited reduced responsiveness to angiotensin II (100 nM) relative to surface cell area enlargement and the upregulation of the late and growth factor genes(ANP, TGF-beta(1)). Angiotensin II-mediated activation of the MAP kinases (extracellular signal-regulated protein kinase 1/2: ERK1/2) was not blocked by taurine. Taurine reduced the phosphorylation of a 29-kDa protein, a reaction which was enhanced by angiotensin II and appears to involve protein kinase C step. The results indicate that taurine is an effective inhibitor of certain aspects of angiotensin II action.

In non-infarcted myocardium after myocardial infarction, the change of cardiac phenotypic modulation of contractile protein, extracellular matrix and intracellular Ca2+ transport protein, such as sarcoplasmic reticulum Ca2+(SR-Ca2+)-ATPase, Na+-Ca2+ exchanger, have a important role during cardiac remodeling. However, the time course in this gene expression in the adjacent and remote left ventricular, or right ventricular myocardium after myocardial infarction has not been well examined. The purpose of this study was to examine the left ventricular function and regional cardiac gene expression after myocardial infarction. Myocardial infarction was produced in Wistar rats by the ligation of the left anterior descending coronary artery. After 3 weeks, 2 months and 4 months from myocardial infarction, we performed Doppler echocardiography and measured the systolic and diastolic function. Then, we analyzed the contractile protein, extracellular matrix and intracellular Ca2+ transport protein mRNAs of cardiac tissues in the adjacent and the remote noninfarcted myocardium, and right ventricular myocardium by Northern blot hybridization. Fractional shortening of infarcted heart progressively decreased. Peak early diastolic filling wave (E wave) velocity increased, and the deceleration rate of the E wave velocity was more rapid in myocardial infarction areas. Atrial filling wave (A wave) velocity decreased, resulting in a marked increase in the ratio of E wave to A wave velocity. Expression of myocardial alpha-skeletal actin, beta-MHC and ANP mRNA, or collagen I and III mRNA were higher at 3 weeks after myocardial infarction. SR Ca2+-ATPase mRNA in the adjacent non-infarcted myocardium was decreased at 2 months, and that in remote myocardium was decreased at 4 months after infarction. Na+-Ca2+ exchanger mRNA levels were increased at 3 weeks, but was decreased at 2 months in the adjacent non-infarcted myocardium and at 4 months in the remote myocardium. These findings suggest that the compensation for myocardial infarction by myocardial gene expression in non-infarcted myocardium may occur at an early phase after myocardial infarction, and myocardial dysfunction may begin from adjacent to remote non-infarcted myocardium during progressive cardiac remodeling.

The purpose of this study was to examine cardiac geometry and function by Doppler echocardiography and to analyze mRNA expression of cardiac phenotype and extracellular matrix in myocardial infarcted rats. Doppler echocardiograms and hemodynamics were measured 2 weeks after myocardial infarction (MI). mRNA levels in the non-infarcted left ventricle (LV) and infarct site were measured by Northern blot analysis. LV internal diastolic dimension was greater in infarcted (MI) than in sham-operated rats (control) (MI 7.2+/-0.3 mm vs control 4.6+0.3 mm, p<0.01). The fractional shortening decreased in MI rats (MI 32+4% vs control 61+/-3%, p<0.01). Peak early filling velocity increased in MI rats (MI 91+/-5 cm/sec vs control 72+/-4 cm/sec, p<0.05), and deceleration rate of the early filling wave was more rapid in rats with MI (MI 25.1+/-2.8 m/sec2 vs control 12.4+/-1.7 m/sec2, p < 0.01). Late filling velocity decreased (MI 16+/-3 cm/sec vs control 35+/-6 cm/sec, p <0.05), resulting in a marked increase in the ratio of early filling to late filling (MI 7.1+/-1.2 vs control 2.5+/-0.4, p<0.01). mRNA levels for beta-myosin heavy chain (beta-MHC), a-skeletal actin, atrial natriuretic polypeptide (ANP), collagen types I and III, and matrix metalloproteinase 2 (MMP-2) in the non-infarcted LV increased significantly by 1.8-, 2.4-, 4.7-, 2.6-, 2.1- (all p<0.01) and 1.4-fold (p<0.05), respectively, compared with sham-operated myocardium. In the infarct site, mRNA levels for transforming growth factor (TGF)-beta1, collagen types I and III, and MMP-2 significantly increased by 3.2-, 11.0-, 9.7-, and 6.3-fold (all p<0.01), respectively, compared with sham-operated myocardium. Myocardial infarcted rat was characterized by cavity dilation and marked abnormalities of systolic and diastolic function, accompanied by a shift of myocytes to fetal phenotype and activation of collagen genes in the non-infarcted myocardium.

Objective: The purpose of this study was to examine the activation of mitogen-activated protein kinases (MAPK) plus activator protein-1 (AP-1) and nuclear factor-kB (NF-kB) DNA binding activities, all of which seem to be important in a signal transduction cascade upstream of the increased level of mRNA expression observed after myocardial infarction., Methods: Myocardial infarction was produced in Wistar rats. The activities of MAPKs in the ischemic region were measured using an in-gel kinase method or an in vitro kinase method. AP-1 and NF-kB binding was determined using an electrophoretic mobility shift assay. Levels of transforming growth factor beta-1(TGF-beta-1) and collagen I and III mRNAs were analyzed by Northern blot hybridization., Results: p42 Extracellular signal-regulated kinase (ERK), p44ERK and p38MAPK activities increased 5.2-fold, 4.3-fold and 1.9-fold (P < 0.01), respectively, at 5 min after coronary artery ligation but returned to normal levels by 30 min. p55c-Jun NH2-terminal kinase (JNK) and p46JNK activities increased 4.0-fold and 3.2-fold (P < 0.01), respectively, at 15 min and returned to normal levels by 24 h after ligation. AP-1 DNA and NF-kB binding activities increased 8.7-fold and 7.1-fold (P < 0.01), respectively, at 3 days but returned to normal levels by 7 days after ligation. Interestingly, analyses of the levels of TGF-beta-1, collagen I and III mRNAs revealed increases of 6.3-fold, 15.2-fold and 12.0-fold (P < 0.01), respectively, at 1 week after myocardial infarction., Conclusions: Myocardial ischemia increased MAPK activities, which were followed by enhancement of AP-1 and NF-kB DNA binding activity in areas of myocardial infarction in rats. These signal transduction mechanisms may contribute to the myocardial ischemia and injury associated with myocardial infarction by causing an increased expression of TGF-beta-1 mRNA, collagen I and III in the area.

Objective: The purpose of this study was to examine the effect of amlodipine, a long-acting calcium antagonist, on the left ventricular remodeling, including systolic and diastolic dysfunction, the change of cardiac gene expression in the myocardial infarcted rats (MI)., Methods: On the first day after myocardial infarction, the animals were randomly assigned to amlodipine treatment (n = 8) or untreated groups (MI; n = 9). We then performed Doppler-echocardiographic examinations and measured the hemodynamics at four weeks after myocardial infarction. Following these measurements, their cardiac mRNA was analyzed., Results: Left ventricular end-diastolic pressure (LVEDP) and central venous pressure (CVP) increased to 22 +/- 1 mmHg and 5 +/- 1 mmHg. Amlodipine reduced LVEDP and CVP to 15 +/- 1 mmHg (P < 0.01) and 3 +/- 0 mmHg (P < 0.01). The weight of right ventricle in MI was significantly larger than in the control rats (Control; 0.48 +/- 0.01 g/kg, MI; 0.79 +/- 0.04 g/kg, P < 0.01). Left ventricular end-diastolic dimension (LVDd) in MI increased to 10.3 +/- 0.3 mm (P < 0.01) (Control; 6.2 +/- 0.3 mm). Amlodipine prevented an increase of the weight of right ventricle (0.62 +/- 0.03 g/kg, P < 0.01) and LVDd (7.9 +/- 0.2 mm, P < 0.01 to MI). The rats in MI showed systolic dysfunction shown by the decreased fractional shortening (Control; 31 +/- 2% versus MI; 15 +/- 1%, P < 0.01), and diastolic dysfunction shown by E wave deceleration rate (Control; 18.1 +/- 2.0 m/s2, MI; 32.6 +/- 2.1 m/s2, P < 0.01). Amlodipine significantly prevented systolic and diastolic dysfunction. The increases in beta-MHC, alpha-skeletal actin, and ANP mRNAs in the non-infarcted left ventricle and right ventricle at four weeks after the myocardial infarction were all significantly suppressed by the treatment with amlodipine. On the other hand, depressed alpha-MHC was restored to normal levels by amlodipine in both regions., Conclusions: Amlodipine prevents the left ventricular remodeling process accompanied by systolic and diastolic dysfunction, and inhibits abnormal cardiac gene expression after myocardial infarction.

To examine chronic changes in mitogen-activated protein (MAP) kinases in cardiac hypertrophy, we determined the activities of two subfamilies of MAP kinases, including extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinases (JNKs), in the heart of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) aged 5, 8, 14, and 24 weeks. MAP kinases were determined by using in-gel kinase assay. In both the left and right ventricles of WKY, the activities of ERKs (p44ERK and p42ERK) and JNKs (p46JNK and p55JNK) decreased significantly with age, indicating that aging remarkably downregulated cardiac MAP kinase activities. In SHRSP, left ventricular ERK and JNK activities were already significantly higher at the mild hypertensive phase than they were in the same age of WKY, and they remained higher until development of left ventricular hypertrophy. On the contrary, the right ventricle of SHRSP, which did not exhibit cardiac hypertrophy, had no significant increase in ERK or JNK activities compared with WKY, except for the slight increase in p55JNK in 24-week-old SHRSP. Antihypertensive treatment of SHRSP with imidapril, an angiotensin-converting enzyme inhibitor, decreased the left ventricular JNK activities (P<.01) but did not affect ERK activities, suggesting the contribution of hypertension or the renin-angiotensin system to the increase in JNKs. Our observations provide the first evidence that both ERK and JNK activities are higher in the left ventricle of SHRSP than WKY. However, further study is needed to elucidate the mechanism and the significance of the increased cardiac MAP kinases in SHRSP.

The present study was undertaken to examine the effects of volume overload on cardiac gene expression and the possible role of angiotensin AT1 receptor in such expression. Cardiac volume overload was prepared by abdominal aortocaval shunt in rats. Rats with aortocaval shunt were treated with 1) vehicle, 2) an angiotensin AT1 receptor antagonist, CS-866 (10 mg/kg/d), or 3) an angiotensin-converting enzyme inhibitor, temocapril (10 mg/kg/d), for 7 days. Cardiac tissue mRNA was measured by Northern blot analysis with specific probes. Aortocaval shunt not only caused cardiac hypertrophy but also upregulated the gene expression of atrial natriuretic polypeptide, collagen III, and downregulated Ca(2+)-ATPase expression in the left ventricle. These changes were prevented by treatment with CS-866, while temocapril failed to normalize left ventricular Ca(2+)-ATPase expression. Unlike the left ventricle, the significant downregulation of alpha-myosin heavy chain and transforming growth factor-beta 3 by aortocaval shunt was observed in the right ventricle, and CS-866 normalized this decreased expression of transforming growth factor-beta 3. The left and right atria showed increased expression of collagen type I as well as of collagen type III and atrial natriuretic polypeptide, and these increases were more effectively prevented by CS-866 than by temocapril. Thus, the effects of cardiac volume overload on cardiac performance-related gene expression differ between the ventricles and atria. Our results suggest that AT1 receptor partially contributed to volume overload-induced changes in cardiac gene expression and that AT1 receptor antagonists and angiotensin-converting enzyme inhibitors have different effects in this model of cardiac hypertrophy.

Background: The transgenic rat TGR(mRen2)27, carrying the mouse Ren-2 gene, is a new model to elucidate the role of the local renin-angiotensin system in vivo. However, the role of the local renin-angiotensin system in the heart remains to be determined in TGR(mRen2)27., Methods and Results: TGR(mRen2)27 were treated with various antihypertensive drugs for 6 weeks to examine the effects on cardiac hypertrophy and gene expression. Cardiac mRNAs were examined by Northern blot analysis. In TGR(mRen2)27, left ventricular hypertrophy was associated with a decrease in alpha-myosin heavy chain expression of 31% and an increase in skeletal alpha-actin and atrial natriuretic polypeptide expression by 2.6- and 21-fold, respectively (P < .05), thereby showing the shift of myocardium to a fetal phenotype. Furthermore, cardiac collagen and laminin expressions were increased in TGR(mRen2)27 (P < .05), suggesting the occurrence of cardiac remodeling. Although treatment of TGR(mRen2)27 with a high dose of TCV-116 (angiotensin AT1 receptor antagonist) or manidipine (calcium antagonist) combined with atenolol (beta 1-adrenergic receptor blocker) completely normalized blood pressure, TCV-116 regressed cardiac hypertrophy and suppressed the changes in cardiac mRNA levels of TGR(mRen2)27 much more potently than manidipine with atenolol. Furthermore, the inhibitory effects of a low dose of TCV-116 on cardiac hypertrophy and altered gene expressions of TGR(mRen2)27 were greater than those of doxazosin (alpha 1-adrenergic receptor blocker) combined with atenolol, despite their similar hypotensive effects., Conclusions: Our present observations provide evidence that the cardiac renin-angiotensin system in TGR(mRen2)27 is responsible for cardiac hypertrophy, phenotypic modulation, and remodeling.

1. Recent evidence indicates that cardiac hypertrophy induced by pressure overload is associated with a decrease in sarcoplasmic reticulum (SR) Ca2+ -ATPase of myocytes, which may contribute to a diastolic dysfunction of the heart by causing intracellular Ca2+ overload. To elucidate whether or not this is also the case in genetic hypertension, we examined cardiac mRNA levels of SR Ca2+ ATPase in 11 week old spontaneously hypertensive rats (SHR) by northern blot analysis. 2. Furthermore, to test the effects of short-term inhibition of the renin-angiotensin system on its expression, we treated 10 week old SHR with angiotensin-converting enzyme inhibitors (alacepril and imidapril) or an AT1 receptor antagonist (SC-52458) for 7 days. 3. Though the left ventricular weight of SHR was significantly higher than that of Wistar-Kyoto (WKY) rats (277 +/- 6 vs 237 +/- 4 mg/100 g bodyweight, respectively, P < 0.05), the level of SR Ca2+ -ATPase mRNA showed no difference between SHR and WKY at this age. 4. Moreover, the aforementioned three drugs did not at all affect the SR Ca2+ -ATPase expression of SHR. 5. Thus, the expression of SR Ca2+ -ATPase was not down-regulated in the heart of 11 week old SHR, and seemed not to be mediated by angiotensin AT1 receptor at this age. Since some evidence on pressure-overloaded cardiac hypertrophy indicate that the decrease in SR Ca2+ -ATPase expression occur in prominent hypertrophy and in the failured heart, further studies on cardiac SR Ca2+ -ATPase expression in more aged SHR will be required.

The effect of calcitonin gene-related peptide(CGRP) on the release of atrial natriuretic peptide(ANP) was studied in spontaneously beating, isolated rat atria. CGRP stimulated the ANP release in a dose-dependent manner. When the atria were incubated with a combination of phentolamine, propranolol, and atropine, these antagonists blocked neither the rise in ANP release nor the positive chronotropic and inotropic effects of CGRP. Therefore, we conclude that CGRP stimulates ANP release as well as cardiac contractility independently of adrenergic and cholinergic receptors.

We investigated the influences of captopril (CAP) and hydralazine (HYD) on the ANP mRNA level in the hypertrophied left ventricle (LV) of spontaneously hypertensive rats (SHR). Male SHRs (16 weeks of age) were given CAP (35 mg/kg/day) or HYD (3.5 mg/kg/day) for two weeks. Both drugs reduced blood pressure by a similar magnitude. Treatment with CAP caused a reduction in the ANP mRNA level in LV by 62%, and a reduction in the weight of the LV. The ANP mRNA level in LV of the HYD-treated rats was also decreased, but only by 31%. HYD did not affect LV hypertrophy. ANP gene expression in LV of SHR might be effectively suppressed by a reduction of blood pressure and also by the concomitant attenuation of hypertrophy.