The Effect And The Possible Mechanism Of A Soluble Guanylate Cyclase Stimulator(s GC003) On Cardiac Hypertrophy

OBJECT Cardiac hypertrophy is a common pathological process of many cardiovascular diseases including hypertension, myocardial infarction, ischemic heart disease and heart valve disorders, which is also thought as an independent risk factor to induce cardiovascular disease. Thus, amelioration of cardiac hypertrophy is of great significance for treating cardiovascular diseases. Current treatments on cardiac hypertrophy are largely based on inhibition of the renin-angiotensin system, yet these therapies tend to slow progression of the hypertrophy, rather than reversing it. Consequently the development of new drug targets is needed. Soluble guanylate cyclase(s GC) plays a key role in nitric oxide(NO) signaling pathway. NO activates s GC, and then the activated s GC catalyzes the synthesis of the second messenger cyclic guanosine monophosphate(c GMP) from guanosine triphosphate(GTP), regulating several physiological processes including vasodilation, platelet aggregation, vascular remodeling, apoptosis, inflammation and neurotransmission. s GC is abundantly expressed in cardiovascular system. Many studies have also demonstrated that the expression and activity of s GC is significantly changed in the nosogenesis and progression of cardiovascular diseases. These provide theory basis for s GC regarding as a drug target. Riociguat, the first s GC-targeted drug to treat pulmonary hypertension(PH) which is developed by Bayer Healthcare AG(Germany), is approved by the US, European Union, Japan Food and Drug Administration to treat pulmonary arterial hypertension(PAH) in adults and chronic thromboembolic pulmonary hypertension(CTEPH). s GC003, a compound with independent intellectual property and novel chemical structure, is synthesized by Beijing institute of pharmacology and toxicology. The early pharmacodynamics evaluation showed that s GC003 has good protective effect in amelirating PH induced by hypoxia and monocrotaline(MCT). Pretreatment with s GC003 could reduce the pulmonary pressure, decrease the right ventricular hypertrophy index and inhibit the hypertrophy of right ventricle. However, the antihypertrophic effect appeared secondary to the attenuation of hypertension via vasodilatation. The direct effect of s GC003 on heart so as to attenuate cardiac hypertrophy has yet been unknown. This study aims to investigate the direct impact of s GC003 on heart and the effect of s GC003 on cardiomyocyte hypertrophy including the possible mechanism, hope to provide experimental evidence for the clinical use of s GC003.METHODS 1. The Langendorff technique was used to measure changes on coronary artery flow, heart rate(HR), pressure of left ventricle(Pmax), left ventricular developed pressure(LVDP), rate pressure products(RPP), +d P/dtmax and-d P/dtmax of isolated hearts from Sprague-Dawley(SD) rats in the absence or presence of s GC003(10-4, 10-6, 10-8 mol/L) and riociguat(10-6 mol/L). 2. The SD rats were exposed to chronic hypoxia in a hypobaric oxygen chamber for two weeks to induce right ventricular hypertrophy. Then the Langendorff technique was used to measure changes on coronary artery flow, HR, Pmax, LVDP, RPP, +d P/dtmax and-d P/dtmax in isolated hearts from right ventricular hypertrophy rats in the absence or presence of s GC003(10-4, 10-6, 10-8 mol/L) and riociguat(10-6 mol/L). After the Langendorff experiments, the right ventricular hypertrophy index was detected to determine the extent of hypertrophy. 3. Cardiomyocytes were isolated from neonatal Sprague-Dawley rats using serial enzymatic digestion and then incubated with endothelin-1(ET-1) 10-8 mol/L in the absence or presence of s GC003(10-8、10-7、10-6 mol/L). Hypertrophic responses including cardiomyocyte area(Image-Pro Plus6.0 software), the expression of ANP m RNA(RT-PCR mehtod) and total protein content(BCA method) were detected. 4. The c GMP concentration of neonatal rats cardiomyocytes after s GC003 stimulation was detected with enzyme-linked immunosorbant assay(ELISA) to determine the effect of s GC003 on s GC activity. 5. The influence of s GC003 on translocation to nuclear of nuclear factor of activated T-cell(NFATc4) was measured by immunofluorescence, and then the ratio of fluorescence of nucleus and cytoplasm was analyzed by an image manipulation software(Image-Pro Plus6.0).RESULTS 1. s GC003(10-8、10-6、10-4 mol/L) increased coronary artery flow, HR, Pmax, LVDP, RPP, +d P/dtmax and-d P/dtmax of isolated hearts from normal SD rats in a concentration-dependent manner. 2. s GC003(10-6、10-4 mol/L) increased coronary artery flow of isolated hearts from right ventricular hypertrophy rats while had no significant effect on HR, Pmax, LVDP, RPP, +d P/dtmax and-d P/dtmax. The right ventricular hypertrophy index of rats exposed to chronic hypoxia is notably raised to 0.3 from 0.2(P<0.001). 3. After 48 h stimulation with ET-1 10-8 mol/L, the cardiomyocyte area increased 80%(P<0.01),the total protein content increased 120%(P<0.01) and the expression of ANP m RNA up-regulated 140%(P<0.01). s GC003(10-8、10-7、10-6 mol/L) elicited antihypertrophic actions, including inhibition of ET-1-mediated increase in cardiomyocyte area(P<0.01), raise of total protein content(P<0.05) and upregulation of ANP m RNA(P<0.05). 4. s GC003(10-7 mol/L) increased c GMP concentration of cardiomyocytes from 3.61 pmol/m L to 14.66 pmol/m L(P<0.001). Pretreatment with ODQ(10-5 mol/L), an s GC inhibitor, significantly inhibited the activation of s GC, with the concentration of c GMP reduced to 7.57 pmol/m L(P<0.01 vs s GC003 group, P>0.05 vs con group). 5. ET-1(10-8 mol/L) provoked the translocation of NFATc4 into nuclear, the ratio of fluorescence in nucleus and cytoplasm was increased from 1.11 to 1.47 compared with con group(P<0.001). s GC003(10-7 mol/L) inhibited ET-1-induced translocation of NFATc4 into nuclear, the ratio was decreased to 1.19(P<0.001). Pretreatment with ODQ and KT5823, a selective inhibitor of PKG, inhibited the effect of s GC003, the ratios are 1.49(P<0.001) and 1.39(P<0.001), respectively.CONCLUSIONS 1. s GC003 increases the coronary artery flow of isolated hearts from normal SD rats to facilitating to provide more oxygen and nutrition, enhances the contractile and diastolic function of heart. 2. s GC003 increases the coronary artery flow in isolated hearts from rats with right ventricle hypertrophy, has no significantly effect on contractile and diastolic function. 3. The cardiomyocyte-selective antihypertrophic effects of s GC003 may be related with the regulation of s GC-c GMP-PKG-NFAT pathway.