NHE-1 participates in isoproterenol-induced downregulation of SERCA2a and development of cardiac remodeling in rat hearts

Impaired Ca(2+) handling is one of the main characteristics in heart failure patients. Recently, we reported abnormal expressions of Ca(2+)-handling proteins in isoproterenol (ISO)-induced hypertrophied rat hearts. On the other hand, Na(+)/H(+) exchanger (NHE)-1 inhibitor has been demonstrated to exert beneficial effects in ischemic-reperfusion injury and in the development of cardiac remodeling. The aims of the present study are to investigate the role of NHE-1 on Ca(2+) handling and development of cardiac hypertrophy in ISO-infused rats. Male Wistar rats were randomly divided into vehicle [control (CTL)] and ISO groups without or with pretreatment with a selective NHE-1 inhibitor, BIIB-723. ISO infusion for 1 wk significantly increased the ratios of heart to body weight and left ventricle (LV) to body weight and collagen accumulation. All of these increases were antagonized by coadministration with BIIB-723. The ISO-induced significant increase in LV wall thickness was suppressed significantly (P < 0.05) by BIIB-723. ISO-induced decreases in cardiac stroke volume and a total mechanical energy per beat index, systolic pressure-volume area at midrange LV volume, were normalized by BIIB-723. The markedly higher expression of NHE-1 protein in the ISO group than that in CTL group was suppressed (P < 0.05) by BIIB-723. Surprisingly, ISO induced downregulation of the important Ca(2+)-handling protein sarcoplasmic reticulum Ca(2+)-ATPase 2a, the expression of which was also normalized by BIIB-723 without changes in phosphorylated phospholamban (PLB)/PLB expression. We conclude that NHE-1 contributes to ISO-induced abnormal Ca(2+) handling associated with cardiac hypertrophy. Inhibition of NHE-1 ameliorates cardiac Ca(2+)-handling impairment and prevents the development of cardiac dysfunction in ISO-infused rats.     

LKB1IP promotes pathological cardiac hypertrophy by targeting PTEN/Akt signalling pathway

Pathological cardiac hypertrophy represents a leading cause of morbidity and mortality worldwide. Liver kinase B1 interacting protein 1 (LKB1IP) was identified as the binding protein of tumour suppressor LKB1. However, the role of LKB1IP in the development of pathological cardiac hypertrophy has not been explored. The aim of this study was to investigate the function of LKB1IP in cardiac hypertrophy in response to hypertrophic stimuli. We investigated the cardiac level of LKB1IP in samples from patients with heart failure and mice with cardiac hypertrophy induced by isoproterenol (ISO) or transverse aortic constriction (TAC). LKB1IP knockout mice were generated and challenged with ISO injection or TAC surgery. Cardiac function, hypertrophy and fibrosis were then examined. LKB1IP expression was significantly up-regulated on hypertrophic stimuli in both human and mouse cardiac samples. LKB1IP knockout markedly protected mouse hearts against ISO- or TAC-induced cardiac hypertrophy and fibrosis. LKB1IP overexpression aggravated ISO-induced cardiomyocyte hypertrophy, and its inhibition attenuated hypertrophy in vitro. Mechanistically, LKB1IP activated Akt signalling by directly targeting PTEN and then inhibiting its phosphatase activity. In conclusion, LKB1IP may be a potential target for pathological cardiac hypertrophy.     

Verapamil prevents isoproterenol-induced cardiac failure in the rat

Virgin, male Sprague-Dawley rats were used to study the production of cardiac failure by a single subcutaneous injection of 85 mg/kg dl-isoproterenol (ISO) and the possible preservation of cardiac function by a pre-treatment of 50 mg/kg verapamil (VER) 5 min prior to 85 mg/kg ISO. At 24 hrs after drug injections cardiac function was assessed in anesthetized, open-chest rats by the measurement of cardiac output and by a volume loading of the heart with a 2 min, 15.3 ml/min jugular vein infusion of Tyrode's solution. Peak cardiac index and peak stroke index were depressed by ISO. VER completely prevented these signs of ISO-induced cardiac failure. A second group of rats was sacrificed 24 hrs after ISO and VER-ISO and their left ventricular calcium contents were determined. ISO caused a significant increase in left ventricular calcium content. VER attenuated the ISO-induced increase in myocardial calcium content, but did not prevent it. This data raises questions as to whether VER's property as a transarcolemmal calcium flux inhibitor was the mechanism of preservation of cardiac function following ISO administration. It is possible that VER may have preserved cardiac function by altering ISO-induced hemodynamic changes in the rat.     

The Novel Mas agonist,CGEN-856S,Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats

CGEN-856S is a novel Mas agonist. Herein, we examined the effects of this peptide on isoproterenol (ISO)-induced cardiac remodeling and myocardial infarction (MI) injury. We also sought to determine whether CGEN-856S activates the underlying mechanisms related to Mas receptor activation. Heart hypertrophy and fibrosis were induced by ISO (2 mg·kg⁻¹·day⁻¹) in Wistar rats. After a 7-day treatment period with CGEN-856S (90 µg·kg⁻¹·day⁻¹) or vehicle, the cardiomyocyte diameter was evaluated in left ventricular sections stained with hematoxylin and eosin, and immunofluorescence labeling and quantitative confocal microscopy were used to quantify the deposition of type I and III collagen and fibronectin in the left ventricles. MI was induced by coronary artery ligation, and CGEN-856S (90 µg·kg⁻¹·day⁻¹) or saline was administered for 14 days. The Langendorff technique was used to evaluate cardiac function, and left ventricular sections were stained with Masson’s trichrome dye to quantify the infarct area. Using Chinese hamster ovary cells stably transfected with Mas cDNA, we evaluated whether CGEN-856S alters AKT and endothelial nitric oxide synthase (eNOS) phosphorylation. CGEN-856S reduced the degree of ISO-induced hypertrophy (13.91±0.17 µm vs. 12.41±0.16 µm in the ISO+CGEN-856S group). In addition, the Mas agonist attenuated the ISO-induced increase in collagen I, collagen III, and fibronectin deposition. CGEN-856S markedly attenuated the MI-induced decrease in systolic tension, as well as in +dT/dt and -dT/dt. Furthermore, CGEN-856S administration significantly decreased the infarct area (23.68±2.78% vs. 13.95±4.37% in the MI+CGEN-856S group). These effects likely involved the participation of AKT and NO, as CGEN-856S administration increased the levels of p-AKT and p-eNOS. Thus, our results indicate that CGEN-856S exerts cardioprotective effects on ISO-induced cardiac remodeling and MI-mediated heart failure in rats through a mechanism likely involving the eNOS/AKT pathway.     

Speckle Tracking Based Strain Analysis Is Sensitive for Early Detection of Pathological Cardiac Hypertrophy

Cardiac hypertrophy is a key pathological process of many cardiac diseases. However, early detection of cardiac hypertrophy is difficult by the currently used non-invasive method and new approaches are in urgent need for efficient diagnosis of cardiac malfunction. Here we report that speckle tracking-based strain analysis is more sensitive than conventional echocardiography for early detection of pathological cardiac hypertrophy in the isoproterenol (ISO) mouse model. Pathological hypertrophy was induced by a single subcutaneous injection of ISO. Physiological cardiac hypertrophy was established by daily treadmill exercise for six weeks. Strain analysis, including radial strain (RS), radial strain rate (RSR) and longitudinal strain (LS), showed marked decrease as early as 3 days after ISO injection. Moreover, unlike the regional changes in cardiac infarction, strain analysis revealed global cardiac dysfunction that affects the entire heart in ISO-induced hypertrophy. In contrast, conventional echocardiography, only detected altered E/E’, an index reflecting cardiac diastolic function, at 7 days after ISO injection. No change was detected on fractional shortening (FS), E/A and E’/A’ at 3 days or 7 days after ISO injection. Interestingly, strain analysis revealed cardiac dysfunction only in ISO-induced pathological hypertrophy but not the physiological hypertrophy induced by exercise. Taken together, our study indicates that strain analysis offers a more sensitive approach for early detection of cardiac dysfunction than conventional echocardiography. Moreover, multiple strain readouts distinguish pathological cardiac hypertrophy from physiological hypertrophy.     

Plantago asiatica L. seeds extract protects against cardiomyocyte injury in isoproterenol- induced cardiac hypertrophy by inhibiting excessive autophagy and apoptosis in mice

BackgroundCardiac hypertrophy is the early stage of many heart diseases, such as coronary heart disease, hypertension, valvular dysfunction and cardiomyopathy. Cardiomyocyte autophagy and apoptosis play an important role in the process of cardiac hypertrophic response. Plantago asiatica L. seeds extract (PASE) is prepared from a traditional herbal medicine in Asia with tremendous pharmacological activities. However, whether PASE could relieve cardiac hypertrophy has not been elucidated. The present study is aimed to investigate the effect of PASE on cardiac hypertrophy and explore its potential underlying mechanism.MethodsCardiac hypertrophy was induced in C57BL/6 mice by subcutaneous injection of isoproterenol (ISO) for two weeks. Meanwhile, the mice were intraperitoneally injected with PASE at dosages of 20, 40 and 80 mg/kg/day. Cardiac hypertrophy was evaluated by echocardiographic examination, haematoxylin and eosin staining and quantitative real-time polymerase chain reaction. Expressions of proteins involved in autophagy and apoptosis such as Beclin1, p62, LC3II, Bax, Bcl-2 and Cleaved-caspase-3 were detected by western blot analysis. Western blot, transient transfection, acridine orange staining, TUNEL staining and autophagy inducer were used to observe the effect and explore the mechanism of PASE on cardiomyocyte and H9c2 cells with excessive autophagy and apoptosis induced by ISO.ResultsISO induction for two weeks disturbed the myocardial contractility and cardiac function of left ventricles of mice. PASE treated mice showed significantly improved cardiac function indexes, including EF, FS, SV and CO, compared with the ISO group. Treatment with PASE also decreased the heart weight/body weight ratio and cardiomyocyte size, and downregulated the mRNA and protein expressions of hypertrophic markers ANP, BNP, and β-MHC. Furthermore, the changes of autophagy and apoptosis markers, such as LC3II, Beclin1, p62, Bcl-2, Bax and Cleaved-caspase-3 induced by ISO were resumed by PASE treatment. Consistently, PASE demonstrated similar effects on ISO-induced H9c2 cells as it did in vivo. In addition, PASE could counteract the increased autophagy induced by the autophagy inducer, rapamycin.ConclusionPASE attenuated ISO-induced cardiac hypertrophy in mice by inhibiting excessive autophagy and apoptosis in cardiomyocytes. The novel findings may pave the way for the clinical usage of PASE for the prevention of heart diseases related with cardiac hypertrophy.     

Overexpression of ornithine decarboxylase decreases ventricular systolic function during induction of cardiac hypertrophy

Ornithine decarboxylase (ODC), the first enzyme of polyamine metabolism, is rapidly upregulated in response to agents that induce a pathological cardiac hypertrophy. Transgenic mice overexpressing ODC in the heart (MHC-ODC mice) experience a much more dramatic left ventricular hypertrophy in response to β-adrenergic stimulation with isoproterenol (ISO) compared to wild-type (WT) controls. ISO also induced arginase activity in transgenic hearts but not in controls. The current work studies the cooperation between the cardiac polyamines and L-arginine (L-Arg) availability in MHC-ODC mice. Although ISO-induced hypertrophy is well-compensated, MHC-ODC mice administered L-Arg along with ISO showed a rapid onset of systolic dysfunction and died within 48 h. Myocytes isolated from MHC-ODC mice administered L-Arg/ISO exhibited reduced contractility and altered calcium transients, suggesting an alteration in [Ca(2+)] homeostasis, and abbreviated action potential duration, which may contribute to arrhythmogenesis. The already elevated levels of spermidine and spermine were not further altered in MHC-ODC hearts by L-Arg/ISO treatment, suggesting alternative L-Arg utilization pathways lead to dysregulation of intracellular calcium. MHC-ODC mice administered an arginase inhibitor (Nor-NOHA) along with ISO died almost as rapidly as L-Arg/ISO-treated mice, while the iNOS inhibitor S-methyl-isothiourea (SMT) was strongly protective against L-Arg/ISO. These results point to the induction of arginase as a protective response to β-adrenergic stimulation in the setting of high polyamines. Further, NO generated by exogenously supplied L-Arg may contribute to the lethal consequences of L-Arg/ISO treatment. Since considerable variations in human cardiac polyamine and L-Arg content are likely, it is possible that alterations in these factors may influence myocyte contractility.     

Effect of U50,488H, a κ-opioid receptor agonist on myocardial α-and β-myosin heavy chain expression and oxidative stress associated with isoproterenol-induced cardiac hypertrophy in rat

Both oxidative stress and β-MHC expression are associated with pathological cardiac hypertrophy. β-adrenergic receptor stimulation plays an important role in cardiac hypertrophy. Recent studies have reported a negative interplay between opioid receptors and adrenoceptors in heart. This study investigated the effect of U50,488H (a selective κ-opioid receptor agonist) on myocardial oxidative stress and α- and β-MHC expression in isoproterenol-induced cardiac hypertrophy. Male Wistar rats were administered normal saline (control), isoproterenol (ISO) (5 mg/kg BW s.c. OD), and isoproterenol with U50,488H (0.4 and 0.6 mg/kg BW, i.p. OD) for 14 days. In a separate group, nor-binaltorphimine (nor-BNI) (0.5 mg/kg, BW, i.p.) (κ-receptor antagonist) was administered along with ISO and U50,488H. ISO administration caused significant increase in left ventricular (LV) wall thicknesses, LV mass in echocardiography, heart weight to body weight ratio, and myocyte size as compared to control. Both the doses of U50,488H offered significant protection against these changes. The higher dose of U50,488H significantly prevented ISO-induced increase in myocardial lipid peroxidation and depletion of myocardial antioxidants (glutathione, superoxide dismutase, and catalase), while a similar trend (although not significant) was observed with the lower dose also. ISO-induced myocardial fibrosis was also significantly attenuated by both the doses of U50,488H. Isoproterenol-induced β-MHC expression in the hypertrophied heart was not altered by either doses of U50,488H, however, the latter prevented the loss of myocardial α-MHC expression. All these effects of U50,488H were blocked by nor-BNI. This study provides the evidence that U50,488H reduced oxidative stress and preserved expression of α-MHC in isoproterenol-induced cardiac hypertrophy.     

Prolonged administration of a dithiol antioxidant protects against ventricular remodeling due to ischemia-reperfusion in mice

The prolonged production of reactive oxygen species due to ischemia-reperfusion (I/R) is a potential cause of the pathological remodeling that frequently precedes heart failure. We tested the ability of a potent dithiol antioxidant, bucillamine, to protect against the long-term consequences of I/R injury in a murine model of myocardial infarction. After transiently occluding the left anterior descending coronary artery for 30 min, saline or bucillamine (10 microg/g body wt) was injected intravenously as a bolus within the first 5 min of reperfusion. The antioxidant treatment continued with daily subcutaneous injections for 4 wk. There were no differences in infarct sizes between bucillamine- and saline-treated animals. After 4 wk of reperfusion, cardiac hypertrophy was decreased by bucillamine treatment (ventricular weight-to-body weight ratios: I/R + saline, 4.5 +/- 0.2 mg/g vs. I/R + bucillamine, 4.2 +/- 0.1 mg/g; means +/- SE; P < 0.05). Additionally, the hearts of bucillamine-treated mice had improved contractile function (echocardiographic measurement of fractional shortening) relative to saline controls: I/R + saline, 32 +/- 3%, versus I/R + bucillamine, 41 +/- 4% (P < 0.05). Finally, I/R-induced injury in the saline-treated mice was accompanied by a fetal pattern of gene expression determined by ribonuclease protection assay that was consistent with pathological cardiac hypertrophy and remodeling [increased atrial natriuretic peptide, beta-myosin heavy chain (MHC), skeletal alpha-actin; decreased sarco(endo)plasmic reticulum Ca2+ ATPase 2a, and alpha-MHC-to-beta-MHC ratio]. These changes in gene expression were significantly attenuated by bucillamine. Therefore, treatment with a dithiol antioxidant for 4 wk after I/R preserved ventricular function and prevented the abnormal pattern of gene expression associated with pathological cardiac remodeling.     

Limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation prevents excess mitochondrial Ca2+ accumulation and attenuates myocardial injury.

BACKGROUND: intracellular Na+ accumulation during ischemia and reperfusion leads to cytosolic Ca2+ overload through reverse-mode operation of the sarcolemmal Na+ -Ca2+ exchanger. Cytosolic Ca2+ accumulation promotes mitochondrial Ca2+ (Ca2+ m) overload, leading to mitochondrial injury. We investigated whether limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation (VF) attenuates Ca2+ m overload and lessens myocardial dysfunction in a rat model of VF and closed-chest resuscitation. METHODS: hearts were harvested from 10 groups of 6 rats each representing baseline, 15 min of untreated VF, 15 min of VF with chest compression given for the last 5 min (VF/CC), and 60 min postresuscitation (PR). VF/CC and PR included four groups each randomized to receive before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+ channel blocker lidocaine (5.0 mg/kg), their combination, or vehicle control. The left ventricle was processed for intracellular Na+ and Ca2+ m measurements. RESULTS: limiting sarcolemmal Na+ entry attenuated cytosolic Na+ increase during VF/CC and the PR phase and prevented Ca2+ m overload yielding levels that corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific Na+ -limiting interventions. Limiting sarcolemmal Na+ entry attenuated reductions in left ventricular compliance during VF and prompted higher mean aortic pressure (110 +/- 7 vs. 95 +/- 11 mmHg, P < 0.001) and higher cardiac work index (159 +/- 34 vs. 126 +/- 29 g x m x min(-1) x kg(-1), P < 0.05) with lesser increases in circulating cardiac troponin I at 60 min PR. CONCLUSIONS: Na+ -limiting interventions prevented excess Ca2+ m accumulation induced by ischemia and reperfusion and ameliorated myocardial injury and dysfunction.     

Regression of isoproterenol-induced cardiac hypertrophy

Cardiac hypertrophy was induced in adult female Wistar rats after 8 days of daily subcutaneous injections of isoproterenol (ISO). Regression from hypertrophy was studied following 1, 2, 4, 8, 12, and 20 days of ISO withdrawal. After 8 days of treatment cardiac mass increased 40%. Following ISO withdrawal, ventricular regression occurred during the first 8 days. After 12-20 days of recovery, a new steady-state heart weight to body weight ratio was established that was 12-13% above the controls. The half-time recovery for heart weight was 3.8 days. Ventricular RNA content was stimulated 76% after 8 days of ISO-induced hypertrophy. During regression RNA content decreased rapidly during the first 8 days with a half-time of 3.4 days. Following 20 days of recovery ventricular RNA was still 31% above the controls. However, myocyte RNA was stimulated 86% following 8 days of ISO treatment and returned to control level after 12 days of regression. Myocardial DNA was increased 23% in the hypertrophied hearts and did not change during the recovery period. Hydroxyproline was increased in the ISO-treated hearts and decreased only slightly during the recovery interval. These data indicate that ISO-induced hypertrophy was reversible while ventricular RNA content only partially recovered. Nevertheless, myocyte RNA showed a large stimulation that was completely reversible at least after 12 days of recovery.     

The nonpeptide angiotensin-(1-7) receptor Mas agonist AVE-0991 attenuates heart failure induced by myocardial infarction

The nonpeptide AVE-0991, which has been reported as a selective ligand for the angiotensin-(1-7) [ANG-(1-7)] receptor Mas, has actions similar to those attributed to the cardioprotective product of the renin-angiotensin system, ANG-(1-7). In this study, we evaluated the cardiac effects of AVE-0991 in normal and infarcted male Wistar rats. Myocardial infarction was induced by left coronary artery ligation. At the end of the treatment, the Langendorff technique was used to analyze cardiac function. Left ventricle serial sections were dyed with Gomori trichrome stain to quantify the infarcted area. In normal hearts, AVE-0991 produced a significant decrease in perfusion pressure and an increase in systolic tension, rate of tension rise and fall (+/-dT/dt), and heart rate. These effects were completely blocked by the perfusion of the hearts with a solution containing the selective ANG-(1-7) antagonist A-779. N(G)-nitro-l-arginine methyl ester treatment abolished the AVE-0991-induced vasodilation in isolated hearts. AVE-0991 significantly attenuated the decrease in systolic tension (sham operated, 13.00 +/- 1.02 g; infarction, 7.18 +/- 0.66 g; AVE treated, 9.23 +/- 1.05 g, n = 5), +dT/dt, -dT/dt, and heart rate induced by myocardial infarction. Infarction-induced vasoconstriction was completely prevented by AVE-0991 treatment. Furthermore, AVE-0991 significantly decreased the infarcted area (6.98 +/- 1.01 vs. 3.94 +/- 1.04 mm(2) in AVE-treated rats). These data indicate that the compound AVE-0991 produces beneficial effects in isolated perfused rat hearts involving the ANG-(1-7) receptor Mas and the release of nitric oxide. In addition, our results indicate that AVE-0991 attenuates postischemic heart failure.     

Antifibrotic activity of an inhibitor of group IIA secretory phospholipase A2 in young spontaneously hypertensive rats

The development of fibrosis in the chronically hypertensive heart is associated with infiltration of inflammatory cells and cardiac hypertrophy. In this study, an inhibitor of the proinflammatory enzyme, group IIA human secretory phospholipase A2 (sPLA2-IIA), has been found to prevent collagen deposition as an important component of cardiovascular remodeling in a rat model of developing chronic hypertension. Daily treatment of young male spontaneously hypertensive rats (SHR) with an sPLA2-IIA inhibitor (KH064, 5-(4-benzyloxyphenyl)-4S-(phenyl-heptanoylamino)-pentanoic acid, 5 mg/kg/day p.o.) prevented increases in the content of perivascular (SHR 20.6 +/- 0.9%, n = 5; SHR+KH064 14.0 +/- 1.2%, n = 5) and interstitial (SHR 7.9 +/- 0.3%, n = 6; SHR+KH064 5.4 +/- 0.7%, n = 6) collagen in the left ventricle of rat hearts, but did not affect numbers of infiltrating monocytes/macrophages, left ventricular hypertrophy (SHR 2.88 +/- 0.08, n = 12; SHR+KH064 3.09 +/- 0.08 mg/g body weight, n = 9), increased systolic blood pressure, or thoracic aortic responses. This selective antifibrotic activity suggests that sPLA2-IIA may have an important but specific role in cardiac fibrosis, and that its inhibitors could be useful in dissecting molecular pathways leading to fibrotic conditions.     

CYP2J2 Overexpression Protects against Arrhythmia Susceptibility in Cardiac Hypertrophy

Maladaptive cardiac hypertrophy predisposes one to arrhythmia and sudden death. Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) promote anti-inflammatory and antiapoptotic mechanisms, and are involved in the regulation of cardiac Ca²⁺-, K⁺- and Na⁺-channels. To test the hypothesis that enhanced cardiac EET biosynthesis counteracts hypertrophy-induced electrical remodeling, male transgenic mice with cardiomyocyte-specific overexpression of the human epoxygenase CYP2J2 (CYP2J2-TG) and wildtype littermates (WT) were subjected to chronic pressure overload (transverse aortic constriction, TAC) or β-adrenergic stimulation (isoproterenol infusion, ISO). TAC caused progressive mortality that was higher in WT (42% over 8 weeks after TAC), compared to CYP2J2-TG mice (6%). In vivo electrophysiological studies, 4 weeks after TAC, revealed high ventricular tachyarrhythmia inducibility in WT (47% of the stimulation protocols), but not in CYP2J2-TG mice (0%). CYP2J2 overexpression also enhanced ventricular refractoriness and protected against TAC-induced QRS prolongation and delocalization of left ventricular connexin-43. ISO for 14 days induced high vulnerability for atrial fibrillation in WT mice (54%) that was reduced in CYP-TG mice (17%). CYP2J2 overexpression also protected against ISO-induced reduction of atrial refractoriness and development of atrial fibrosis. In contrast to these profound effects on electrical remodeling, CYP2J2 overexpression only moderately reduced TAC-induced cardiac hypertrophy and did not affect the hypertrophic response to β-adrenergic stimulation. These results demonstrate that enhanced cardiac EET biosynthesis protects against electrical remodeling, ventricular tachyarrhythmia, and atrial fibrillation susceptibility during maladaptive cardiac hypertrophy.     

Identification of ryanodine receptor isoforms in prostate DU-145, LNCaP, and PWR-1E cells

We previously reported that left ventricular (LV) slices from isoproterenol (ISO)-induced hypertrophied rat hearts showed an increase of energy expenditure due to remodeling of Ca(2+) handling in excitation-contraction coupling, i.e., suppressed SERCA2a activity and enhanced Na(+)/Ca(2+)exchanger-1 (NCX-1) activity. Na(+)/H(+) exchanger-1 (NHE-1) inhibitor (NHEI) has been demonstrated to exert beneficial effects in the development of cardiac remodeling. We hypothesized that a novel NHE-1 selective inhibitor, BIIB723 prevents remodeling of Ca(2+) handling in LV slices of ISO-induced hypertrophied rat hearts mediated by inhibiting NCX-1 activity. The significant shortening in duration of multi-cellular Ca(2+) transient in ISO group was normalized in ISO+BIIB723 group. The significant increase in amplitude of multi-cellular Ca(2+) waves (CaW) generated at high [Ca(2+)](o) of LV slices in ISO group was also normalized in ISO+BIIB723 group. However, the enhanced NCX-1 activity was not antagonized by BIIB723. We recently reported that ISO-induced down-regulation of a Ca(2+) handling protein, SERCA2a, was normalized by BIIB723. Therefore, it seems likely that BIIB723 normalized shortened multi-cellular Ca(2+) transient duration and increased CaW amplitude in LV slices mediated via normalization of SERCA2a activity. Furthermore, the results presented here suggest the multi-cellular Ca(2+) transient duration and CaW amplitude in LV slices might be better indices reflecting SERCA2a activity than SERCA2a protein expression level.     

Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy

Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure.     

Electrocardiography in two models of isoproterenol-induced left ventricular remodeling

We studied the ability of the ECG to detect pathological changes in isoproterenol-induced remodeling of rat heart. Myocardial hypertrophy in rats was induced by repeated injections of isoproterenol (5 mg/kg s.c. 7 days, Iso5, n=7). Single overdose of isoproterenol (150 mg/kg s.c., Iso150, n=7) evoked myocardial infarction followed with ventricular remodeling. The electrocardiograms were recorded in anesthetized animals (thiopenthal 45 mg/kg i.p.) and myocardial contractile performance was analyzed in isolated hearts perfused according to Langendorff. The hypertrophic hearts were characterized by increased heart and left ventricular (LV) weight as well as by thicker LV free wall and interventricular septum. Mean values of LV contraction did not significantly differ from controls. Longer QT interval, QRS complex, negative Q and S waves, higher R amplitude were typical characteristics for Iso5 rats. Iso150 animals showed tendency to decreased systolic blood pressure and heart frequency. Decrease in the thickness of LV compared to Iso5 as well as impaired LV function were related to the dilated left ventricle. Iso150 ECG showed longer QRS and QT, deepened negativity of S wave and mild decrease of R(II) compared to Iso5. Voltage criteria showed that Sokolow-Lyon index is a good predictor of left ventricular hypertrophy in isoproterenol-induced cardiac remodeling without systemic hypertension.     

Blocking RhoA/ROCK inhibits the pathogenesis of pemphigus vulgaris by suppressing oxidative stress and apoptosis through TAK1/NOD2-mediated NF-κB pathway

Numerous experimental studies have demonstrated the role of cytochrome P450 1B1 (CYP1B1) and its associated mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) metabolite in the pathogenesis of cardiac hypertrophy. However, the ability of isoproterenol (ISO) to induce cardiac hypertrophy through mid-chain HETEs has not been investigated yet. Therefore, we hypothesized that ISO induces cardiac hypertrophy through the induction of CYP1B1 and its associated mid-chain HETE metabolites. To test our hypothesis, Sprague–Dawley rats were treated with ISO (5 mg/kg i.p.) for 12 and 72 h whereas, human ventricular cardiomyocytes RL-14 cells were exposed to 100 μM ISO in the presence and absence of 0.5 μM tetramethoxystilbene (TMS) a selective CYP1B1 inhibitor, or 25 nM CYP1B1-siRNA. Moreover, RL-14 cells were transiently transfected with the CRISPR-CYP1B1 plasmid. Thereafter, real-time PCR, western blot analysis, and liquid chromatography–electrospray ionization mass spectroscopy were used to determine the level of gene expression, protein expression, and mid-chain HETEs, respectively. Our results showed that ISO induced CYP1B1 protein expression and the level of cardiac mid-chain HETEs in vivo at pre-hypertrophic and hypertrophic stage. In vitro, inhibition of CYP1B1 using TMS or CYP1B1-siRNA significantly attenuates ISO-induced hypertrophy. Furthermore, overexpression of CYP1B1 significantly induced cellular hypertrophy and mid-chain HETEs metabolite. Mechanistically, the protective effect of TMS against cardiac hypertrophy was mediated through the modulation of superoxide anion, mitogen-activated protein kinases (MAPKs), and nuclear factor-κB (NF-κB). In conclusion, our study provides the first evidence that CYP1B1 and its associated mid-chain HETE metabolites are directly involved in the ISO-induced cardiac hypertrophy.     

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.     

大鼠心肌重塑过程中Axin蛋白质的表达变化

为观察大鼠心肌重塑过程中Axin蛋白质表达水平的变化,实验用颈静脉输注去甲肾上腺素(NE)和动静脉造瘘(AVF)方法复制大鼠心肌重塑病理模型,采用超声心动术检测心脏结构和收缩功能。取病理模型大鼠左心室以及分离培养的成年大鼠心肌成纤维细胞,采用Wester blot技术检测Axin蛋白质的表达水平。结果观察到,在颈静脉输注NE 3d后,大鼠心脏发生向心性心肌肥厚和心肌纤维化,其左心室的Axin蛋白表达水平较对照组显著升高。A-V造瘘术一周后引起大鼠离心性心肌肥厚,心肌无明显纤维化,心肌Axin表达量与对照相比无显著变化。在分离培养的成年大鼠心肌成纤维细胞,NE处理24h能明显升高Axin蛋白的表达水平。上述结果表明,大鼠心脏有Axin蛋白质表达,NE致大鼠心肌重塑过程中Axin蛋白表达显著增加,可能与该过程的心肌纤维化有关。