Isoprenaline-induced Cardiac Hypertrophy Research Articles

Discovery of 3-trifluoromethyl-substituted pyrazoles as selective phosphodiesterase 10A inhibitors for orally attenuating isoprenaline-induced cardiac hypertrophy

Recently, MP-10, a previous drug candidate with potent inhibition of phosphodiesterase 10A (PDE10A) in clinical phase II trials for schizophrenia or Alzheimer's disease, has shown significant potential in preventing and treating cardiovascular diseases. However, its poor metabolic stability and high permeability across the blood-brain barrier (BBB) make it unsuitable for preventing and treating peripheral cardiovascular diseases. Herein, the hit-to-lead optimization was performed to discover novel 3-trifluoromethyl-substituted pyrazole derivatives as potent and selective PDE10A inhibitors. The structure-activity relationships, biological characterization, molecular mechanism, and drug-like evaluation were discussed to identify compound C7 which showed potent inhibition against PDE10A (half maximal inhibitory concentration, IC50 = 11.9 nmol/L), more than 840-fold selectivity over other PDE subtypes, enhanced liver microsomes stability (T1/2 = 239 min) compared to MP-10 and low BBB permeability. Importantly, oral pretreatment with C7·3HCl at a dose of 5.0 mg/kg significantly attenuated the pathological and functional changes induced by isoprenaline (ISO)-induced pathological cardiac hypertrophy in mice, particularly suppressing increase of cardiac weight, atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) hypertrophic markers along with cardiac fibrosis. These findings further support that targeting PDE10A provides an innovative therapeutic approach for preventing and treating cardiac diseases.

Inhibition of miR-146b-5p alleviates isoprenaline-induced cardiac hypertrophy via regulating DFCP1

Pathological cardiac hypertrophy often precedes heart failure due to various stimuli, yet effective clinical interventions remain limited. Recently, microRNAs (miRNAs) have been identified as critical regulators of cardiovascular development. In this study, we investigated the role of miR-146b-5p and its underlying mechanisms of action in cardiac hypertrophy. Isoprenaline (ISO) treatment induced significant hypertrophy and markedly enhanced the expression of miR-146b-5p in cultured neonatal rat cardiomyocytes and hearts of C57BL/6 mice. Transfection with the miR-146b-5p mimic led to cardiomyocyte hypertrophy accompanied by autophagy inhibition. Conversely, miR-146b-5p inhibition significantly alleviated ISO-induced autophagy depression, thereby mitigating cardiac hypertrophy both in vitro and in vivo. Our results showed that the autophagy-related mediator double FYVE domain-containing protein 1 (DFCP1) is a target of miR-146b-5p. MiR-146b-5p blocked autophagic flux in cardiomyocytes by suppressing DFCP1, thus contributing to hypertrophy. These findings revealed that miR-146b-5p is a potential regulator of autophagy associated with the onset of cardiac hypertrophy, suggesting a possible therapeutic strategy involving the inhibition of miR-146b-5p.

Bellidifolin ameliorates isoprenaline-induced cardiac hypertrophy by the Nox4/ROS signalling pathway through inhibiting BRD4

To date, there is no effective therapy for pathological cardiac hypertrophy, which can ultimately lead to heart failure. Bellidifolin (BEL) is an active xanthone component of Gentianella acuta (G. acuta) with a protective function for the heart. However, the role and mechanism of BEL action in cardiac hypertrophy remain unknown. In this study, the mouse model of cardiac hypertrophy was established by isoprenaline (ISO) induction with or without BEL treatment. The results showed that BEL alleviated cardiac dysfunction and pathological changes induced by ISO in the mice. The expression of cardiac hypertrophy marker genes, including ANP, BNP, and β-MHC, were inhibited by BEL both in mice and in H9C2 cells. Furthermore, BEL repressed the epigenetic regulator bromodomain-containing protein 4 (BRD4) to reduce the ISO-induced acetylation of H3K122 and phosphorylation of RNA Pol II. The Nox4/ROS/ADAM17 signalling pathway was also inhibited by BEL in a BRD4 dependent manner. Thus, BEL alleviated cardiac hypertrophy and cardiac dysfunction via the BRD4/Nox4/ROS axes during ISO-induced cardiac hypertrophy. These findings clarify the function and molecular mechanism of BEL action in the therapeutic intervention of cardiac hypertrophy.

Naringenin Attenuates Isoprenaline-Induced Cardiac Hypertrophy by Suppressing Oxidative Stress through the AMPK/NOX2/MAPK Signaling Pathway

Cardiac hypertrophy is accompanied by increased myocardial oxidative stress, and whether naringenin, a natural antioxidant, is effective in the therapy of cardiac hypertrophy remains unknown. In the present study, different dosage regimens (25, 50, and 100 mg/kg/d for three weeks) of naringenin (NAR) were orally gavaged in an isoprenaline (ISO) (7.5mg/kg)-induced cardiac hypertrophic C57BL/6J mouse model. The administration of ISO led to significant cardiac hypertrophy, which was alleviated by pretreatment with naringenin in both in vivo and in vitro experiments. Naringenin inhibited ISO-induced oxidative stress, as demonstrated by the increased SOD activity, decreased MDA level and NOX2 expression, and inhibited MAPK signaling. Meanwhile, after the pretreatment with compound C (a selective AMPK inhibitor), the anti-hypertrophic and anti-oxidative stress effects of naringenin were blocked, suggesting the protective effect of naringenin on cardiac hypertrophy. Our present study indicated that naringenin attenuated ISO-induced cardiac hypertrophy by regulating the AMPK/NOX2/MAPK signaling pathway.

Activation of FMS-like tyrosine kinase 3 protects against isoprenaline-induced cardiac hypertrophy by improving autophagy and mitochondrial dynamics.

FMS-like receptor tyrosine kinase 3 (Flt3) expression was reported to increase in the heart in response to pathological stress, but the role of Flt3 activation and its underlying mechanisms remain poorly elucidated. This study was designed to investigate the role of Flt3 activation in sympathetic hyperactivity-induced cardiac hypertrophy and its mechanisms through autophagy and mitochondrial dynamics. In vivo, cardiac hypertrophy was established by subcutaneous injection of isoprenaline (6mg/kg·day) in C57BL/6 mice for 7 consecutive days. The Flt3-ligand intervention was launched 2 h prior to isoprenaline each day. In vitro, experiments of cardiomyocyte hypertrophy, autophagy, and mitochondrial dynamics were performed in neonatal rat cardiomyocytes (NRCMs). Our results revealed that the expression level of Flt3 protein was significantly increased in the hypertrophic myocardium provoked by isoprenaline administration. Flt3-ligand intervention alleviated isoprenaline-induced cardiac oxidative stress, hypertrophy, fibrosis, and contractile dysfunction. Isoprenaline stimulation impaired autophagic flux in hypertrophic mouse hearts, supported by the accumulation of LC3II and P62 proteins, while Flt3-ligand restored the impairment of autophagic flux. Flt3 activation normalized the imbalance of mitochondrial fission and fusion in the hearts of mice evoked by isoprenaline as evidenced by the neutralization of elevated mitochondrial fission markers and reduced mitochondrial fusion markers. In NRCMs, Flt3-ligand treatment attenuated isoprenaline-stimulated hypertrophy, which was abolished by a Flt3-specific blocker AC220. Activating Flt3 reversed isoprenaline-induced autophagosome accumulation and impairment of autophagic flux probably by enhancing SIRT1 expression and consequently TFEB nuclear translocation. Flt3 activation improved the imbalance of mitochondrial dynamics induced by isoprenaline in NRCMs through the SIRT1/P53 pathway. Activation of Flt3 mitigated ISO-stimulated hypertrophy probably involves the restoration of autophagic flux and balance of mitochondrial dynamics. Therefore, activation of Flt3 attenuates isoprenaline-induced cardiac hypertrophy in vivo and in vitro, the potential mechanism probably attributes to SIRT1/TFEB-mediated autophagy promotion and SIRT1/P53-mediated mitochondrial dynamics balance. These findings suggest that activation of Flt3 may be a novel target for protection against cardiac remodeling and heart failure during sympathetic hyperactivity.

GDH promotes isoprenaline-induced cardiac hypertrophy by activating mTOR signaling via elevation of α-ketoglutarate level.

Numerous studies reveal that metabolism dysfunction contributes to the development of pathological cardiac hypertrophy. While the abnormal lipid and glucose utilization in cardiomyocytes responding to hypertrophic stimuli have been extensively studied, the alteration and implication of glutaminolysis are rarely discussed. In the present work, we provide the first evidence that glutamate dehydrogenase (GDH), an enzyme that catalyzes conversion of glutamate into ɑ-ketoglutarate (AKG), participates in isoprenaline (ISO)-induced cardiac hypertrophy through activating mammalian target of rapamycin (mTOR) signaling. The expression and activity of GDH were enhanced in cultured cardiomyocytes and rat hearts following ISO treatment. Overexpression of GDH, but not its enzymatically inactive mutant, provoked cardiac hypertrophy. In contrast, GDH knockdown could relieve ISO-triggered hypertrophic responses. The intracellular AKG level was elevated by ISO or GDH overexpression, which led to increased phosphorylation of mTOR and downstream effector ribosomal protein S6 kinase (S6K). Exogenous supplement of AKG also resulted in mTOR activation and cardiomyocyte hypertrophy. However, incubation with rapamycin, an mTOR inhibitor, attenuated hypertrophic responses in cardiomyocytes. Furthermore, GDH silencing protected rats from ISO-induced cardiac hypertrophy. These findings give a further insight into the role of GDH in cardiac hypertrophy and suggest it as a potential target for hypertrophy-related cardiomyopathy.

Cardioprotective effect of ethanol extracts of Sugemule-3 decoction on isoproterenol-induced heart failure in Wistar rats through regulation of mitochondrial dynamics

Sugemule-3 decoction (SD-3) is a commonly used prescription in Mongolian medicine which composed of the herbs Baidoukou (the fruit of Amomum compactum Sol. ex Maton), Baijusheng (the fruit of Lactuca sativa L.) and Biba (Piper longum L.). SD-3 has remarkable effect on the cardiovascular diseases, but its pharmacological mechanism has not been elucidated. To evaluate the cardioprotective effects and the potential mechanisms of the ethanol extracts of SD-3 against isoproterenol (ISO)-induced heart failure (HF) in rats. The ethanol extracts of SD-3 were prepared and analyzed by LC-ESI-MS/MS. One hundred male Wistar rats were randomly divided into five groups: control, ISO (HF) and different doses of SD-3 (0.4, 0.2, 0.1g/kg/d) groups. HF model rats were established by intraperitoneal injecting of ISO. The left ventricular function was evaluated by echocardiography. Myocardial injury and fibrosis were examined by hematoxylin-eosin (HE) and Masson staining. Western-blot analysis was performed to determine the protein expression of apoptosis and mitochondrial dynamics in all the groups. Moreover, the structural changes in the mitochondria of cardiomyocytes were also observed by transmission electron microscopy. Fifteen compounds were detected in the ethanol extracts of SD-3, include piperine, piperanine, etc. Rats administered with ISO showed a significant decline in the left ventricular function. The cardiac histopathological changes such as local necrosis, interstitial edema, and cardiac fibrosis were also observed in the ISO group. The treatment with SD-3 significantly inhibited these effects of ISO. ISO was found to increase the protein expression of Bax, cleaved-PARP and cleaved-caspase-3, -7 -9, destroy the balance between mitochondrial fusion and fission, and alter the mitochondrial morphology. The ethanol extracts of SD-3 could rebalance mitochondrial fusion and fission, and ameliorates the morphological abnormalities induced by ISO in mitochondria. The current study demonstrated that ethanol extracts of SD-3 improved isoprenaline-induced cardiac hypertrophy and fibrosis through inhibiting cardiomyocyte apoptosis and regulating the mitochondrial dynamics.

Vasonatrin peptide, a synthetic natriuretic peptide, attenuates myocardial injury and oxidative stress in isoprenaline-induced cardiomyocyte hypertrophy

Isoprenaline-induced cardiac hypertrophy can deteriorate to heart failure, which is a leading cause of mortality. Endogenous vasonatrin peptide (VNP) has been reported to be cardioprotective against myocardial ischemia/reperfusion injury in diabetic rats. However, little is known about the effect of exogenous VNP on cardiac hypertrophy. We further explored whether VNP attenuated isoprenaline-induced cardiomyocyte hypertrophy by examining the levels and activities of cGMP and PKG. In this study, we found that VNP significantly attenuated isoprenaline-induced myocardial hypertrophy and cardiac fibroblast activation in vivo. Moreover, VNP effectively halted the activation of apoptosis and oxidative stress in the isoprenaline-treated myocardium. VNP promoted superoxide dismutase (SOD) activity. Further study revealed that the protective effects of VNP might be mediated by the activity of the cGMP-PKG signaling pathway in vivo or in vitro, while the use of agonists and antagonists confirmed these results. Therefore, we demonstrated that the antiapoptosis and antioxidative stress effects of VNP depends on elevated cGMP-PKG signaling activity both in vivo and in vitro. These results suggest that VNP may be used in the treatment of myocardial hypertrophy.

Xanthohumol attenuates isoprenaline-induced cardiac hypertrophy and fibrosis through regulating PTEN/AKT/mTOR pathway

Emerging evidence suggests the cardiovascular protective effects of Xanthohumol (Xn), a prenylated flavonoid isolated from the hops (Humulus lupulus L.). However, the cardioprotective effect of Xn remains unclear. Present study aimed to investigate the protective role of Xn against isoprenaline (ISO)-induced cardiac hypertrophy and fibrosis, and elucidate the underlying mechanism. The cardiac hypertrophy and fibrosis model were established via subcutaneously administration of ISO. ISO reduced the left ventricular contractile function and elevated myocardial enzyme levels, suggesting cardiac dysfunction. Moreover, the increased cardiac myocyte area, heart weight/body weight (HW/BW) ratio and ANP/BNP expressions indicated the ISO-induced hypertrophy, while the excessive collagen-deposition and up-regulation of fibrosis marker protein (α-SMA, Collagen-I/III) expression indicated the ISO-induced fibrosis. The ISO-induced cardiac dysfunction, hypertrophy and fibrosis were significantly attenuated by oral administrated with Xn. PTEN/AKT/mTOR pathway has been reported to involve in pathogenesis of cardiac hypertrophy and fibrosis. We found that Xn administration up-regulated PTEN expression and inhibited the phosphorylation of AKT/mTOR in ISO-treated mice. Moreover, treating with VO-ohpic, a specific PTEN inhibitor, abolished the cardioprotective effect of Xn. Collectively, these results suggested that Xn attenuated ISO-induced cardiac hypertrophy and fibrosis through regulating PTEN/AKT/mTOR pathway.

The Expression of microRNA in Adult Rat Heart with Isoproterenol-Induced Cardiac Hypertrophy.

Cardiac hypertrophy is a common pathological condition and an independent risk factor that triggers cardiovascular morbidity. As an important epigenetic regulator, miRNA is widely involved in many biological processes. In this study, miRNAs expressed in rat hearts that underwent isoprenaline-induced cardiac hypertrophy were identified using high-throughput sequencing, and functional verification of typical miRNAs was performed using rat primary cardiomyocytes. A total of 623 miRNAs were identified, of which 33 were specifically expressed in cardiac hypertrophy rats. The enriched pathways of target genes of differentially expressed miRNAs included the FoxO signaling pathway, dopaminergic synapse, Wnt signaling pathway, MAPK (mitogen-activated protein kinase) signaling pathway, and Hippo signaling pathway. Subsequently, miR-144 was the most differentially expressed miRNA and was subsequently selected for in vitro validation. Inhibition of miR-144 expression in primary myocardial cells caused up-regulation of cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The dual luciferase reporter system showed that ANP may be a target gene of miR-144. Long non-coding RNA myocardial infarction associated transcript (LncMIAT) is closely related to heart disease, and here, we were the first to discover that LncMIAT may act as an miR-144 sponge in isoproterenol-induced cardiac hypertrophy. Taken together, these results enriched the understanding of miRNA in regulating cardiac hypertrophy and provided a reference for preventing and treating cardiac hypertrophy.

2,5-Dimethylcelecoxib prevents isoprenaline-induced cardiac hypertrophy and fibrosis via the activation of glycogen synthase kinase-3

2,5-Dimethylcelecoxib prevents isoprenaline-induced cardiac hypertrophy and fibrosis via the activation of glycogen synthase kinase-3

Mechanistic insights to the cardioprotective effect of blueberry nutraceutical extract in isoprenaline-induced cardiac hypertrophy

BackgroundLowbush blueberry extract (Vaccinium angustifolium) is abundant with polyphenols (such as chlorogenic acid) with high antioxidant profile. It has received great interest due to its protective role in many disorders such as heart diseases and neurological disorders. HypothesisWe hypothesized that blueberry leaf extract might have a protective effect against cardiac hypertrophy via suppressing oxidative stress, inflammation and fibrosis. MethodBlueberry leaf nutraceutical extract was administered orally to male albino rats at three different doses (25, 50 and 100 mg/kg/day of the extract, equivalent to 3.4, 6.8 and 13.6 mg of chlorogenic acid, respectively) once daily for 28 consecutive days against a dose of isoprenaline (ISO) (5 mg/kg) for 14 days. ResultsThe results indicated that isoprenaline induced significant myocardial damage, characterized by conduction abnormalities, increased heart-to-body weight ratio, increased serum CKMB, AST, c-TnI and LDH. Pretreatment with blueberry extract at a dose of 50 mg/kg/day (equivalent to 6.8 mg chlorogenic acid) protected against ISO-induced ECG changes, leakage of cardiac enzymes and histopathological changes. Also, ISO caused significant glutathione depletion, lipid peroxidation and reduction in activities of antioxidant catalase enzyme. These effects were prevented by pretreatment with blueberry extract. Additionally, ISO elicited inflammatory effects by increasing the expression of NF-κB, COX-2, TNF-α and IL-6 while pretreatment with blueberry extract significantly inhibited these inflammatory responses. Furthermore, ISO induced fibrosis by increasing the level of TGF-β while pretreatment with blueberry extract significantly reduced it. ConclusionThese findings indicate that blueberry leaf extract possessed a potent protective effect against ISO-induced cardiac hypertrophy via suppressing oxidative stress, inflammation and fibrosis.

ClC-3 chloride channel is involved in isoprenaline-induced cardiac hypertrophy

Isoprenaline, an activator of β-adrenergic receptor, has been found to induce cardiac hypertrophy in vivo and in vitro, but the exact mechanism is still unclear. ClC-3 is a member of the chloride channel family and is highly expressed in mammalian myocardium. In the present study, the role of ClC-3 in isopronaline-induced cardiac hypertrophy was investigated. We found that ClC-3 expression was reduced in isoprenaline-induced hypertrophic H9c2 cells, primary rat neonatal cardiomyocytes and myocardium of C57/BL/6 mice, and this reduction was prevented by the pretreatment of propranolol. Adeno-associated virus 9 (AAV9)-mediated ClC-3 expression in myocardium decreased heart mass index, thinned interventricular septum and left ventricular wall and lowered the mRNA expression of natriuretic peptide type A (ANF) and β-myosin heavy chain (β-MHC). Our results showed that ClC-3 played an important role in β-adrenergic cardiac hypertrophy which could be associated with ANF and β-MHC, and all these findings suggested that ClC-3 may be a novel therapeutic target for the prevention or treatment of myocardiac hypertrophy.

The orphan receptor NOR1 participates in isoprenaline-induced cardiac hypertrophy by regulating PARP-1.

The orphan nuclear receptor NOR1 belongs to the NR4A subfamily of the nuclear hormone receptor superfamily, and is involved in glucose and fat metabolism. However, its potential contribution to cardiovascular diseases remains to be assessed. Here, the roles of NOR1 in cardiac hypertrophy induced by isoprenaline and the underlying molecular mechanisms were investigated. NOR1 was expressed in cardiomyocytes treated with isoprenaline. After NOR1 overexpression or knockdown in neonatal rat cardiomyocytes, cellular hypertrophy was monitored by measuring cell surface area and the mRNA of hypertrophic biomarkers. Interactions between NOR1 and PARP-1 were investigated by co-immunoprecipitation. NOR1 expression and PARP-1 activity were measured in rats with cardiac hypertrophy induced by isoprenaline. Treatment with isoprenaline significantly up-regulated NOR1 expression and PARP-1 activity both in vivo and in vitro. Specific gene silencing of NOR1 attenuated isoprenaline-induced cardiomyocyte hypertrophy, whereas NOR1 overexpression exacerbated cardiac hypertrophy. We identified a physical interaction between NOR1 and PARP-1, which was enhanced by NOR1 transfection and thereby led to PARP-1 activation. Overexpression of NOR1, but not C293Y, a NOR1 mutant lacking the PARP-1 binding activity, increased cellular surface area and the mRNA levels of atrial natriuretic factor and brain natriuretic polypeptide, effects blocked by the PARP-1 inhibitor 3-aminobenzamide or siRNA for PARP-1. This is the first evidence that NOR1 was involved in isoprenaline-induced cardiac hypertrophy. The pro-hypertrophic effect of NOR1 can be partly attributed to its regulation of PARP-1 enzymic activity.

Soluble epoxide hydrolase inhibitor, TUPS, protects against isoprenaline‐induced cardiac hypertrophy

We have previously shown that isoprenaline-induced cardiac hypertrophy causes significant changes in the expression of cytochromes P450 (CYP) and soluble epoxide hydrolase (sEH) genes. Therefore, it is important to examine whether the inhibition of sEH by 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) will protect against isoprenaline-induced cardiac hypertrophy. Male Sprague-Dawley rats were treated with TUPS (0.65 mg kg(-1) day(-1), p.o.), isoprenaline (5 mg kg(-1) day(-1), i.p.) or the combination of both. In vitro H9c2 cells were treated with isoprenaline (100 μM) in the presence and absence of either TUPS (1 μM) or 11,12 EET (1 μM). The expression of hypertrophic, fibrotic markers and different CYP genes were determined by real-time PCR. Isoprenaline significantly induced the hypertrophic, fibrotic markers as well as the heart to body weight ratio, which was significantly reversed by TUPS. Isoprenaline also caused an induction of CYP1A1, CYP1B1, CYP2B1, CYP2B2, CYP4A3 and CYP4F4 gene expression and TUPS significantly inhibited this isoprenaline-mediated effect. Moreover, isoprenaline significantly reduced 5,6-, 8,9-, 11,12- and 14,15-EET and increased their corresponding 8,9-, 11,12- and 14,15-dihydroxyeicosatrienoic acid (DHET) and the 20-HETE metabolites. TUPS abolished these isoprenaline-mediated changes in arachidonic acid (AA) metabolites. In H9c2 cells, isoprenaline caused a significant induction of ANP, BNP and EPHX2 mRNA levels. Both TUPS and 11,12-EET significantly decreased this isoprenaline-mediated induction of ANP, BNP and EPHX2. TUPS partially protects against isoprenaline-induced cardiac hypertrophy, which confirms the role of sEH and CYP enzymes in the development of cardiac hypertrophy.

Peripheral benzodiazepine receptor ligand Ro5-4864 inhibits isoprenaline-induced cardiac hypertrophy in rats

Oxidative stress plays a significant role in the pathogenesis of cardiac hypertrophy. Peripheral benzodiazepine receptors are ubiquitously expressed in various tissues, including the heart. Peripheral benzodiazepine receptors have been reported to be involved in the protection of cells against oxygen radical damage. The present study was designed to determine whether Ro5-4864 (a peripheral benzodiazepine receptor ligand) can inhibit isoprenaline-induced cardiac hypertrophy. Male Wistar rats (body weight 150–200g) were administered, isoprenaline (5mg/kg, body weight, subcutaneously) alone or along with Ro5-4864 (0.1 and 0.5mg/kg, body weight, intraperitoneally) once daily for 14days. Control rats received normal saline subcutaneously (1.0ml/kg). Isoprenaline-induced changes in heart weight to body weight ratio, left ventricular wall thickness (M-mode echocardiography and gross morphometry) and myocyte size were significantly prevented by both the doses of Ro5-4864. Ro5-4864 also attenuated isoprenaline-induced increase in interstitial fibrosis, lipid peroxidation and changes in endogenous antioxidants (glutathione, superoxide dismutase and catalase). Isoprenaline-induced cardiac hypertrophy was associated with increased expression of β myosin heavy chain, which was also prevented by Ro5-4864. This is the first study to demonstrate a salutary effect of Ro5-4864 in experimental cardiac hypertrophy.

Stimulation of κ-opioid receptor reduces isoprenaline-induced cardiac hypertrophy and fibrosis

The aim of the present study was to determine whether U50,488H (a selective κ-opioid receptor agonist) inhibits cardiac hypertrophy and fibrosis induced by β-adrenoceptor stimulation in a rat model. Cardiac hypertrophy and fibrosis were developed by intraperitoneal administration of isoprenaline (ip. 3.0 mg/kg/day,14 days). In the isoprenaline-treated group, heart weight and heart-to-body ratio increased significantly. Hypertrophic alterations were observed in light micrographs of tissue and transmission electron micrographs of myocardial ultra structures. Increases in heart weight, heart-to-body ratio, diameter of cardiomyocytes, and morphological hypertrophic alterations induced by isoprenaline were significantly attenuated by U50,488H(ip. 1.25 mg/kg/day). Growth of cardiomyocytes was induced by incubating with isoprenaline (10 − 6 mol/l), which resulted in an increase in optical density (OD) values. The increased OD value was attenuated by U50,488H(10 − 7 mol/l–10 − 5 mol/l) in a dose dependent manner. Animals receiving administration of isoprenaline displayed significant fibrosis. The extent of isoprenaline induced left ventricular fibrosis was dramatically reduced in U50,488H treated animals. Increased cardiac fibroblast proliferation and collagen synthesis induced by isoprenaline, as evidenced by increased OD value, 3H-thymidine, and 3H-proline incorporation, were significantly reduced in the U50,488H treated group. The specific extracellular matrix proteins, including type I, type III collagen and fibronectin, which increased after administration of isoproterenol, were also attenuated by U50,488H. The abovementioned effects of U50,488H were completely abolished by nor-BNI (nor-binaltorphimine), a selective κ-opioid receptor antagonist. The enhanced intracellular Ca 2+ transient and L-type Ca 2+ current elicited by isoprenaline in cardiomyocytes were significantly inhibited by U50,488H. This study provides the first morphological evidence of the inhibitory effect of U50,488H on isoprenaline-induced cardiac hypertrophy and fibrosis via κ-opioid receptor stimulation.

Down-Regulation of Na+Pump α2Isoform in Isoprenaline-Induced Cardiac Hypertrophy in Rat: Evidence for Increased Receptor Binding Affinity but Reduced Inotropic Potency of Digoxin

Cardiac hypertrophy in rats induces a down-regulation of Na(+),K(+)-ATPase alpha(2) isoform, although its functional consequences are poorly understood. Using a mathematical modeling approach that allows differentiation between effects elicited at the receptor and postreceptor level, we studied uptake, receptor binding kinetics, and positive inotropism of digoxin in single-pass Langendorff-perfused hearts of vehicle- and isoprenaline-pretreated rats (2.4 mg/kg per day over 4 days). Digoxin outflow concentration and left ventricular developed pressure data were measured for three consecutive doses (15, 30, and 45 microg) in the absence and presence of the reverse mode Na(+)/Ca(2+) exchange inhibitor 2-[2-[4-(4-nitrobenzyloxyl-)phenyl]ethyl isothiourea methansulfonate] (KB-R7943) (0.1 microM) in perfusate. In hypertrophied hearts, 1) the amount of alpha(2) receptors was reduced to 52% of control levels; 2) the digoxin binding affinity was increased 12-fold due to a decrease in dissociation rate constants of alpha(1) and alpha(2) receptors, and 3) inotropic responsiveness to digoxin the was attenuated on the stimulus-response level, where the coupling ratio of stimulus to response was reduced to 38% of control values. Only in the lowest dose level (15 microg) was this decrease in inotropic potency counterbalanced by the increase in receptor affinity. The Na(+),K(+)-ATPase isoform shift was not responsible for the diminished inotropic effect of digoxin. Coadministration of KB-R7943 significantly reduced cellular response generation at higher digoxin doses to the same limiting stimulus-response relationship in both the vehicle and isoprenaline group.