Isoproterenol-induced Heart Failure Research Articles

Fasudil hydrochloride hydrate, a Rho‐kinase inhibitor, suppresses isoproterenol‐induced heart failure in rats via JNK and ERK1/2 pathways

The Rho-kinase (ROCK) plays an important role in the pathogenesis of heart injury. Recent cellular and molecular biology studies indicated a pivotal role of the RhoA/ROCK cascade in many aspects of cardiovascular function such as heart failure, cardiac hypertrophy, and ventricular remodeling following myocardial infarction. However, the signal transduction of RhoA/ROCK and its down-stream signaling pathways remains elusive, and the mechanism of ROCK-mediated isoproterenol (ISO)-induced heart failure is still not thoroughly understood. In the present study, we investigated the effect of the ROCK inhibitor, fasudil hydrochloride hydrate, on ISO-induced heart failure and the potential relationship of RhoA/ROCK to the extracellular signal-regulated kinases (ERK) and the c-jun NH 2-terminal kinase (JNK) pathways. Male Sprague-Dawley (SD) rats, maintained on a normal diet, were randomly divided into four groups given control, ISO alone, ISO with low-dose fasudil, or ISO with high-dose fasudil treatments. Fasudil effectively inhibited ISO-induced heart failure, as evaluated by biometric, hemodynamic, and histological examinations. Consistently, ISO-induced ROCK-1 mRNA expression and myosin phosphatase target subunit-1 (MYPT-1) phosphorylation were markedly suppressed by fasudil. In addition, fasudil significantly decreased ISO-induced JNK activation, ERK translocation to the nucleus and subsequent c-fos, c-jun expression and upregulated c-FLIP(L) expression. Taken together, these results indicate that the RhoA/ROCK pathway is essential for ISO induced heart failure, which can be effectively suppressed by fasudil.

Combined atorvastatin and coenzyme Q10 improve the left ventricular function in isoproterenol-induced heart failure in rat

The effect of atorvastatin on cardiac remodeling, function, and homodynamic parameters in isoproterenol-induced heart failure was evaluated in the present study. A subcutaneous injection of isoproterenol (5 mg/kg/day) for 10 days was used for the induction of heart failure. Isoproterenol administration produced intensive myocardial necrosis and fibrosis with a significant decrease in the arterial pressure indices, heart rate, contractility (LVdP/dt max) and relaxation (LVdP/dt min), but an increase in the left ventricular end-diastolic pressure. Rats were randomly assigned to control, treatment with only atorvastatin, and treatment with atorvastatin plus coenzyme Q10. Histopathological analysis showed a marked attenuation of myocyte necrosis and interstitial fibrosis in all atorvastatin treated groups (P < 0.001). A low dose of atorvastatin (5 mg/kg/day) significantly improved the left ventricular systolic pressure, contractility and relaxation (P < 0.01). On the contrary, a high dose of atorvastatin (20 mg/kg/day) worsened the isoproterenol-induced left ventricular dysfunction by a further reduction of LV dP/ dt max from + 2780 ± 94 to + 1588 ± 248 (mm Hg/s; P < 0.01) and LV dP/ dt min from − 2007 ± 190 to − 2939 ± 291 (mm Hg/s; P < 0.05). Co-administration of coenzyme Q10 with atorvastatin reversed the hemodynamic depression and the left ventricular dysfunction to a high level (P < 0.001). There was a lower level of LVEDPs in the atorvastatin + coenzyme Q10 treated groups (3 ± 1 and 4 ± 1.4 versus 8 ± 3.5 and 14 ± 3.6 mm Hg, respectively), thereby suggesting improvement in the myocardial stiffness by the combined coenzyme Q10 and atorvastatin treatment. The atorvastatin therapy attenuated myocardial necrosis and fibrosis in isoproterenol-induced heart failure. However, a high dose of the drug considerably worsened the left ventricular dysfunction and hemodynamic depression, which was reversed by coenzyme Q10 co-administration.

CPU228, a derivative of dofetilide, relieves cardiac dysfunction by normalizing FKBP12.6, NADPH oxidase and protein kinase C ε in the myocardium

The aim of this study was to determine if CPU228, a derivative of dofetilide, is more effective than dofetilide in attenuating isoproterenol-induced heart failure by recovering downregulated FK506 binding protein (FKBP12.6), and suppressing oxidative stress, upregulated NADPH oxidase and protein kinase C epsilon (PKC epsilon) hyperphosphorylation in the myocardium. Heart failure was induced by isoproterenol (1 mg/kg s.c. for 5 days) in male Sprague-Dawley rats. Intervention with either CPU228 or dofetilide (2 mg/kg on Days 3-5) was then conducted in vivo and in vitro. Isoproterenol produced compromised left ventricular systolic pressure, left ventricular pressure rise (dp/dt(max)) and fall (dp/dt(min)), and left ventricular end-diastolic pressure, associated with oxidative stress, abnormal FKBP12.6, NADPH oxidase p67phox and PKC epsilon in the myocardium. CPU228 was more effective in attenuating these changes than dofetilide in vivo. Dofetilide produced a prolonged QTc to replace a shortened one. In primary neonatal cardiomyocytes, cultured with isoproterenol and treated with either CPU228 or dofetilide at 10(-8), 10(-7) and 10(-6) mol/l, isoproterenol produced a hyperadrenergic state characterized by downregulated FKBP12.6, upregulated NADPH oxidase p67phox and PKC epsilon in vitro. CPU228 was more effective than dofetilide in recovering these changes in a dose-dependent manner without a prolonged QTc. CPU228 was more effective than dofetilide in attenuating heart failure by normalizing isoproterenol-induced changes, including downregulation of FKBP12.6, upregulation of NADPH oxidase and PKC epsilon hyperphosphorylation in vivo and in vitro.

The study of ISO induced heart failure rat model

ObjectiveTo find a more excellent large-dose isoproterenol (ISO) induced heart failure (HF) rat model. Methods166 male Wistar rats were divided into four groups, normal group (n=10), 85/85 mg/kg group (n=50), 85/340 mg/kg group (n=50) and 340/340 mg/kg group (n=56). HF was induced by two subcutaneous injections of homologous ISO on 2 consecutive days. We calculated death rate of each ISO group on different time points. And all the survival ISO rats were examined by echocardiography separately on 2 weeks, 3 weeks and 4 weeks. We defined the EF<45% rats as heart failure, and calculated EF<45% rate of three ISO groups. 4 weeks after the last injection, all of the rats were sacrificed. H&E and Masson staining were used to evaluate inflammatory infiltration and myocardial fibrosis, and immunohistochemistry was used to measure the levels of IL-1, IL-6 and IL-17. We also treated adult rat cardiac fibroblasts with IL-1, IL-6, IL-17, or PBS for 24 h, and real-time PCR was used to measure the expressions of MMP-2 and MMP-9 in treated fibroblasts. ResultsThrough 4-week observation, we found that the period within 2 days after the last injection was the most dangerous one for all the ISO rats, regardless of injection dose. Then, the death rate increased slowly. 4 weeks later, the death rates were respectively 30% in 85/85 mg/kg group, 60% in 85/340 mg/kg group, and 68% in 340/340 mg/kg group, and there were statistical differences among three experimental groups. Moreover, the EF<45% rate and cardiac fibrosis rate were 100% in 85/340 mg/kg group as same as 340/340 mg/kg group, and much higher than 85/85 mg/kg group. Furthermore, we also found that pro-inflammatory cytokines (IL-1, IL-6 and IL-17) increased in 85/340 mg/kg group compared with normal group, and above cytokines could induce MMP-2 and MMP-9 expression in cardiac fibroblasts. Conclusion85/340 mg/kg ISO induced HF rat model was superior to the other two dose ISO induced HF rat models. Pro-inflammatory cytokines might contribute to HF and myocardial fibrosis through promotion of MMPs expression in cardiac fibroblasts.

IL-17 induces myocardial fibrosis and enhances RANKL/OPG and MMP/TIMP signaling in isoproterenol-induced heart failure

Objective This study was designed to investigate whether IL-17 can regulate the expression of the MMP/TIMP system, the OPG/RANK/RANKL system, or type-I and type-III collagen fibers in a rat model of isoproterenol-induced heart failure (HF). We also investigated the effect of IL-17 on myocardial fibrosis in this model. Methods HF was induced in Wistar–Kyoto rats by hypodermic injection of isoproterenol (ISO) twice every 24 h. After 2 months, the surviving rats were divided into three groups: monoclonal Anti-IL-17 Ab (100 μg/day), IgG (100 μg/day) or PBS were injected five times every 48 h (i.p.). One day after the last injection, all of the rats were sacrificed. H&E and Masson staining were used to evaluate myocardial fibrosis, and immunohistochemistry was used to measure the levels of MMP-1, TIMP-1, TIMP-4, OPG, RANKL, type-I and type-III collagen fibers. We also treated adult rat cardiac fibroblasts with IL-17 (10 ng/ml), IL-17 (10 ng/ml) + OPG (10 ng/ml), IL-17 (10 ng/ml) + anti-RANKL Ab (100 ng/ml), or PBS for 24 h, realtime RT-PCR was used to measure the expressions of MMP-1. Results The expressions of MMP-1, RANKL, and type-I and -III collagen fibers decreased, and the expressions of TIMP-1, TIMP-4, and OPG increased in the Anti-IL-17 group compared to controls. H&E and Masson staining revealed that blockade of IL-17 can improve myocardial fibrosis in HF. IL-17 increased the expression of MMP-1 in cardiac fibroblasts, and OPG and anti-IL-17 Ab could inhibit this function partly. Thus, IL-17 was dependent on the RANKL/OPG system to induce MMP-1 partly. Conclusion Our study demonstrates the contribution of IL-17 to myocardial fibrosis in isoproterenol-induced HF. IL-17 can regulate the RANKL/OPG and MMP/TIMP systems in this model. The RANKL/OPG system is one of intermediaries between IL-17 and MMP-1 in cardiac fibroblasts. As a harmful cytokine, anti-IL-17 treatment is a potential therapeutic strategy in HF.

Isoproterenol‐induced heart failure in the rat is associated with nitric oxide‐dependent functional alterations of cardiac function

The role of nitric oxide (NO) in heart failure (HF) is complex and remains controversial. We tested the hypothesis that the role of NO in isolated atria and cardiomyocytes is altered in isoproterenol-induced HF. Rats received isoproterenol (ISO, 5 mg/kg/day, intraperitoneally) or vehicle for 1 week. Haemodynamic parameters were obtained by left ventricular catheterization. Effects of NOS inhibition on isolated atria and on electrically paced left ventricular myocytes were determined. Additionally, expressions of nitric oxide synthases and their allosteric modulators hsp90, caveolin-1, and caveolin-3 proteins in the left ventricles were measured. ISO increased left ventricular mass by 33% and decreased indices of left ventricular systolic and diastolic function dp/dtmin and dp/dtmax (both P<0.05). Isolated atria from HF rats had a lower spontaneous beating rate (P<0.05). NOS inhibition by L-NAME increased basal frequency and attenuated the positive chronotropic effect of beta-adrenergic stimulation in the HF group (P<0.05). Ventricular myocytes from failing hearts had impaired cell shortening. L-NAME decreased contractility of control, but not failing myocytes. Left ventricular expressions of eNOS, hsp90, iNOS, but not nNOS or caveolins, were increased. Despite the increased capacity for NO synthesis in isoproterenol-induced HF, NO does not sustain contractility of failing myocytes. NO may contribute to the decreased basal heart rate and it may accelerate beta-adrenergic stimulation of chronotropy.

Combined Deficiency of Dystrophin and β1 Integrin in the Cardiac Myocyte Causes Myocardial Dysfunction, Fibrosis and Calcification

The dystrophin-glycoprotein complex is a large complex of membrane-associated proteins linking the cytoskeleton to the extracellular matrix in muscle. Transmembrane heterodimeric (alphabeta) integrins serve also as cellular adhesion molecules and mechanotransducers. In the animal model for Duchenne muscular dystrophy, the mdx mouse, loss of dystrophin causes more severe abnormalities in skeletal than in cardiac muscle. We hypothesized that ablation of cardiac myocyte integrins in the mdx background would lead to a severe cardiomyopathic phenotype. Mdx mice were crossed to ones with cardiac myocyte-specific deletion of beta1 integrin (beta1KO) to generate beta1KOmdx. Unstressed beta1KOmdx mice were viable and had normal cardiac function; however, high mortality was seen in peri- and postpartum females by 6 months of age, when severe myocardial necrosis and fibrosis and extensive dystrophic calcification was seen. Decreased ventricular function and blunted adrenergic responsiveness was found in the beta1KOmdx mice compared with control (Lox/Lox, no Cre), beta1KO, and mdx. Similarly, adult beta1KOmdx males were more prone to isoproterenol-induced heart failure and death compared with control groups. Given the extensive calcification, we analyzed transcript levels of genes linked to fibrosis and calcification and found matrix gamma-carboxyglutamic acid protein, decorin, periostin, and the osteoblast transcription factor Runx2/Cbfa1 significantly increased in beta1KOmdx cardiac muscle. Our data show that combined deficiency of dystrophin and integrins in murine cardiac myocytes results in more severe cardiomyopathic changes in the stressed myocardium than reduction of either dystrophin or integrins alone and predisposes to myocardial calcification.

Cardioprotective effect of fluvastatin on isoproterenol-induced myocardial infarction in rat

The present study was designed to investigate whether fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, would attenuate the acute myocardial infarction in isoproterenol-treated rat model via maintaining activities of endogenous antioxidant enzymes. Hemodynamic and electrocardiograph parameters were monitored and recorded continuously, cardiac marker enzymes and antioxidative parameters of plasma and heart tissues were measured, and histopathological examination of heart tissues was performed. Isoproterenol-treated rats showed lower of left-ventricular systolic pressure (LVSP), maximum (LVd P/d t max) and minimum rate of developed left ventricular pressure (LVd P/d t min), and higher of left ventricular end-diastolic pressure (LVEDP), in addition, a significant rise in ST-segment and increase in content of lactate dehydrogenase, glutamic oxalacetic transaminase, creatine kinase and malondialdehyde, as well as fall in activities of glutathione peroxidase, superoxide dismutase and catalase were observed. Oral administration of fluvastatin (5, 10 and 20 mg/kg, respectively) significantly prevented almost all the parameters of isoproterenol-induced heart failure and myocardial injury that mentioned above. The protective role of fluvastatin on isoproterenol-induced myocardial damage was further confirmed by histopathological examination. There was no significant change in heart rate in all experimental groups. Compared with control group, any indexes in sham rats treated with fluvastatin (20 mg/kg) alone were unaltered (all P > 0.05). Our results suggest that fluvastatin has a significant effect on the protection of heart against isoproterenol-induced myocardial infarction through maintaining endogenous antioxidant enzyme activities.

The density of myocardial and pericardial rat mast cells in isoproterenol-induced heart failure

Myocardial mast cells (MC) respond to cardiovascular pathology. The behavior of MC population in myocardium and pericardium of rats has been studied 24 h, 14, 28 and 60 days after two isoproterenol injections (at 24 h intervals). The extent of heart failure has been estimated by supersonic inspection 28 and 60 days after isoproterenol injections. The density of MCs of different degrees of maturity was estimated on paraffin sections stained with Alcian blue--Safranin. The MC density in myocardium of intact and experimental rats was relatively low: from 4 to 6 cells/mm2. The MC density in pericardium of intact rats was several times higher than in myocardium: 48.6 +/- 13.0 cells/mm2. In 24 h and 14 days after isoproterenol injections the pericardial MC density was 1.5 times higher than in control rats (P < 0.05) at the expense of increase in the number of mature MCs with Safranine-positive granules without the increase in the number of immature cells with Alcian blue-positive granules. In 28 days the pericardial MC density was 2 times higher than in intact rats (P < 0.05) at the expense of increase in number of immature and mature cells. In 60 days after isoproterenol injections the pericardial MC density and the ratio of immature and mature cells compared with control did not reach statistical significance. The changes in pericardial MC population corresponded to severity of heart failure according to functional indices. The findings show active reaction of pericardial MCs on myocardium dysfunction that stimulates the maturation of resident immature MCs in pericardium and migration of immature cells to pericardium of damage heart.

Chronic blocking of β3-adrenoceptor ameliorates cardiac function in rat model of heart failure

Stimulation of the heart beta 3-adrenoceptor (AR) may result in a negative inotropic effect. Being up-regulated, beta 3-AR plays a more important role in the regulation of cardiac function during heart failure. However, the effect of chronic blocking of beta 3-AR on heart failure has not been fully elucidated. In this study, we used a selective beta 3-AR antagonist SR59230A to treat a well defined heart failure rat model chronically, then evaluated its effect on cardiac function and investigated the mechanism. Male Wistar rats were chosen randomly as controls (n = 8). Isoproterenol induced heart failure rats were randomly divided into ISO group (n = 10) and SR group (n = 10). The ISO group received intraperitoneal injection of 1 ml saline twice a day; the SR group received intraperitoneal injection of SR59230A 85 nmol in 1 ml saline twice a day; and the control group received no treatment. The treatment was started 24 hours after the last isoproterenol injection and continued for 7 weeks. Then we measured the following indexes: the ratio of heart weight to body weight (HW/BW) and the ratio of left ventricular weight to body weight (LVW/BW), collagen volume fraction (CVF), left ventricular end diastolic dimension (LVEDd), left ventricular end systolic dimension (LVESd), ejection fraction (EF), fractional shortening (FS) and the ratio of E wave to A wave (E/A), the mRNA and protein expression of beta 3-AR and eNOS, and cGMP level in the heart. The ratios HW/BW and LVW/BW were significantly increased in the ISO group compared with the control group (P < 0.01), but they were limited in the SR group (P < 0.05 compared with the ISO group). CVF increased in the ISO group and the SR group (P < 0.01), but it was significantly attenuated in the SR group (P < 0.01). LVEDd, LVESd and E/A ratio were significantly increased in the ISO group compared with the control group (P < 0.01), while EF and FS were significantly decreased (P < 0.01). Compared with the ISO group, the SR group showed that LVEDd, LVESd and E/A ratio were significantly decreased (P < 0.01), whereas EF and FS were significantly increased (P < 0.01). beta(3)-AR and eNOS mRNA and protein in the ISO group were significantly increased when compared with the control group (P < 0.01). These increases were all attenuated in the SR group compared with the ISO group (P < 0.01). The level of cGMP in myocardial tissue was significantly increased in the ISO group compared with the control group (P < 0.01), whereas SR59230A treatment normalized this increment (P < 0.01). Chronic blocking of beta 3-AR could ameliorate cardiac function in heart failure rats and its mechanism involves inhibition of the negative inotropic effect and attenuation of cardiac remodeling.

Probucol attenuates oxidative stress and energy decline in isoproterenol-induced heart failure in rat

In the present study, we examined whether the powerful antioxidant probucol (a clinically used lipid-lowering drug) would attenuate the oxidative stress and energy starvation in experimental model of heart failure (HF) using isoproterenol. Rats were injected subcutaneously with isoproterenol (2.4 mg kg −1) daily for 1 week, and then treated with probucol (61 mg/kg) daily for 2 weeks. Oxidative stress was assessed by measuring myocardial lipid peroxides level and antioxidant enzymes activities, glutathione peroxidase (GPx) and superoxide dismutase. In addition, cardiac metabolic damage was estimated by measuring myocardial ATP, ADP and AMP levels as well as ATP/ADP ratio. It was found that isoproterenol induced a significant increase in heart rate by approximately 30% as compared with the pre-value. These changes were significantly attenuated by post-treatment of rats with probucol. Also, isoproterenol induced several pathological changes including lymphocyte infiltration, myofibrillar hemorrhage and degeneration, and these changes were attenuated by probucol. In addition, animals treated with isoproterenol showed a significant increase in myocardial lipid peroxides level up to 163% and a significant decrease in myocardial GPx activity by 35% as compared with the control group. Probucol not only counteracted significantly the pronounced oxidative stress effect of isoproterenol but also it induced a significant increase in myocardial GPx as compared with the control group. The major new finding of the present study is that treatment with probucol induced a significant increase in myocardial ATP level (the source of energy) and ATP/ADP ratio. Moreover, there is a significant correlation between ATP/ADP ratio and myocardial probucol level. In conclusion, the cardioprotective effect of probucol in treatment of HF is a result of not only its antioxidant properties and an enhancement of endogenous antioxidant reserve (mainly GPx) but also an enhancement of myocardial energy state.

Isoproterenol-induced heart failure results in rapid left ventricular dilation and increased matrix metalloproteinase activity in rats

Isoproterenol-induced heart failure results in rapid left ventricular dilation and increased matrix metalloproteinase activity in rats

1031-38 Beneficial Effects of Ramipril in a Model of Isoproterenol-induced Heart Failure in the Rat

To establish an experimental model of chronic heart failure, we assessed the morphologic, hemodynamic and neurohumoral effects of a single large dose for isoproterenol (ISO: 150 mg/kg BW s.c.). Two weeks after ISO-application 45 ISO- and 30 control-rats were examined. In addition, we investigated the effects of chronic treatment with ramipril (RAM: 10 mg/KG/d, n = 20). Echocardiography of ISO- and control-rats was performed in anesthetized rats to measure left ventricular (LV) mass and LV-enddiastolic dimension. An increase of LV-mass (LV diastolic posterior wall thickness: 2.8 ± 0.2 vs. 1.9 ± 0.3 mm, LV systolic posterior wall thickness: 4.3 ± 0.4 vs. 3.6 ± 0.3 mm; p &lt; 0.01) and LV enddiastolic dimension (6.9 ± 0.6 vs. 4.9 ± 0.3 mm p &lt; 0.01) was displayed. RAM-treated animals showed a highly significant decrease of LV mass and LV-enddiastolic dimension (p &lt; 0.001). Heart rate was increased in the ISO-group (p &lt; 0.005) compared to RAM- and control-rats (p &lt; 0.005). Central venous pressure was significantly increased in the ISO-group (5.8 ± 2.3; vs 2.3 ± 0.6 mmHG; p &lt; 0.01) and decreased to normal ranges in RAMrats. Mean arterial pressure was decreased in ISO- (121.3 ± 14.8 mmHg) and RAM-rats, (95 ± 3 mmHG) vs controls (132.4 ± 19.5 mmHG)(p &lt; 0.05). Mean renal blood flow between ISO-treated and control group was not significantly different, in contrast the mean blood flow in the abdominal aorta was decreased in the ISO-group (p &lt; 0.05). Cardiac ACE-activity (p &lt; 0.01), circulating atrial natriuretic peptide, renin- and aldosterone-levels were significantly higher in the ISO-treated group as compared tothe control group (p &lt; 0.05). ISO-treated hearts displayed morphological alterations such as myocyte necrosis, cellular reaction and fibrosis. RAM-treated hearts revealed the same infarct-like lesions but at a lower grade. In summary, a highly reproducible model of chronic heart failure was established. Hemodynamic measurements displayed a typical characteristic for advanced heart failure in the ISO-group. Blockade of the renin-angiotensinsystem (RAS) attenuated the observed changes indicating that the RAS may be involved in the development of catecholamine-induced congestive heart failure.