Inhibition Of Isoprenaline-induced Cardiac Remodeling By Calcitonin Gene-related Peptide And The Underlying Mechanisms

Cardiac remodeling is a pathological condition and ultimately progresses into heart failure. Although the initial remodeling seems to be adaptive and shows beneficial effects on maintaining the heart function in a short term, persistent remodeling may contribute to functional decompensation. Recently, it has been shown that the dysfunction of capsaicin-sensitive sensory nerves and the expression abnormality of microRNA-1, microRNA-133a and connective tissue growth factor (CTGF) may be involved in cardiac remodeling. Deletion of transient receptor potential vanilloid (TRPV1, also named capsaicin receptor, VR1) could exacerbate cardiac remodeling after myocardial infarction, however, stimulating the release of neurotransmitter of sensory nerves by activating VR1 could improve the cardiac function. These reports suggested that cardiac remodeling under pathological conditions might be related to the dysfunction of sensory nerve. Rutaecarpine (Rut) is a major quinazolinocarboline alkaloid isolated from Chinese herbal drug Wu-Chu-Yu, which exerts cardiovascular protective effects by stimulating the synthesis and release of calcitonin gene-related peptide (CGRP), the principal neurotransmitter of sensory nerves, by activation of VR1. Since the dysfunction of sensory nerve was involved in cardiac remodeling, the present study was designed to evaluate the role of CGRP in isoprenaline (Iso)-induced cardiac remodeling and the underlying mechanisms. The effects of Rut on cardiac remodeling and the underlying mechanisms were also explored.METHODSAnimal experimentsCardiac remodeling was induced by administration of isoprenaline (5 mg/kg, s.c.) for 10 days, different concentrations of rutaecarpine (10 or 40mg/kg, i.g.) were co-administrated with isoprenaline. At the end of the drugs treatment, the cardiac morphology changes were measured by echocardiogram; the hearts were harvested, and the heart weight (HW), left ventrucular weight (LVW) were recorded and normalized for body weight (BW); the morphology and the size of cardiac cells were evaluated by hematoxylin-eosin staining; the apoptosis of cardiomyocytes was analyzed by TUNEL staining; the collagen deposition was evaluated by Masson's trichrome staining; the plasma level of CGRP were measured by RIA assay; the expression of microRNA-1 and microRNA-133a was determined by real time PCR; the mRNA expression of atrial natriuretic peptide (ANP, a hypertrophy marker) and brain natriuretic peptide (BNP, a hypertrophy marker), Bax and Bcl-2, collagen typeⅠ,Ⅲand CTGF, and CGRP (αandβisoforms) were examined by RT-PCR.Cell experimentsThe cardiomyocytes and cardiac fibroblasts were pretreated with CGRP at different doses for 1 h before incubating with isoprenaline (Iso) for 48 h. At the end of the drugs treatment, the cell viability was measured by MTT; the rate of apoptotic cells was examined by hoechst staining and flow cytometry; the mRNA expression of Bax, Bcl-2, collagen typeⅠandⅢ, and connective tissue growth factor (CTGF) was detected by RT-PCR; the expression of microRNA-1 and microRNA-133a was measured by real time PCR; The intracellular reactive oxygen species (ROS) production was examined by DCFH-DA flurescent probe.RESULTSAnimal experimentsIn comparison with control group, isoprenaline (Iso) treatment significantly increased the left ventricular end-systolic diameter (LVEDs), left ventricular end-systolic posterior wall thickness (LVPWTs) and left ventricular end-diastolic posterior wall thickness (LVPWTd); the ratio of HW/BW and LVW/BW, caridac myocyte cross-sectional area (CSA) and hypertrophic gene expression were also dramatically increased. Administration of Iso significantly increased the percentage of TUNEL-positive cells and Bax mRNA expression, while the Bcl-2 mRNA expression was markedly decreased. Iso treatment could also remarkably increased myocardial collagen deposition and collagen typeⅠ,Ⅲand CTGF mRNA expression. In Iso-treated rats, the expression of microRNA-1 was significantly up-regulated, while the microRNA-133a was markedly down-regulated. In the this study, we also found that the CGRP mRNA expression in dorsal root ganglion and concentration in plasma were significantly decreased. Co-administration of rutaecarpine (Rut) with Iso could remarkably stimulate the synthesis and release of CGRP, and in turn attenuates the cardiac remodeling. However, the beneficial effect of Rut was inhibited by pretreatment of capsaicin, which selectively depletes endogenous CGRP.Cell experimentsIsoprenaline (Iso) treatment significantly decreased the cell viabilty, increased the rate of apoptotic cells in H9c2 cardiomyocytes; the mRNA expression of Bax, a proapoptotic gene, was also remarkably up-regulated after Iso treatment; while the expression of Bcl-2, an antiapoptotic gene, was no significant change. Iso treatment could also increase the expression of microRNA-1, and decrease the expression of microRNA-133a. In addition, the intracellular reactive oxygen species (ROS) level was significantly increased in Iso-treated group. Pretreatment of calcitonin gene-related peptide (CGRP) could markedly decrease the generation of intracellular ROS, correct the abnormal expression of microRNA-1 and microRNA-133a induced by Iso, and in turn decreased the rate of apoptotic cells. However, the protective effect of CGRP was abolished by pretreatment of CGRP receptor antagonist CGRP8-37.In cardiac fibroblasts, Iso treatment markedly increased the proliferation activity; the mRNA expression of collagen typeⅠandⅢand connective tissue growth factor (CTGF) was also significantly up-regulated; In addition, the intracellular reactive oxygen species (ROS) level was significantly increased in Iso-treated group. Pretreatment of CGRP could markedly inhibt these detrimental effects induced by Iso, the beneficial effect of CGRP was abolished by CGRP8-37.CONCLUSIONS1.The dysfunction of capsaicin-sensitive sensory nerves and the abnormal expression of microRNA-1, microRNA-133a and CTGF may be involved in Iso-induced cardiac remodeling.2. Rut could inhibit Iso-induced cardiac remodeling, which was related to regulating the expression of microRNA-1, microRNA-133a and CTGF through stimulating the synthesis and release of CGRP.3.The effect of CGRP on the expression of microRNA-1, microRNA-133a and CTGF may be associated with inhibiting the generation of intracellular ROS.