Molsidomine Attenuates Cardiac Structural And Electrical Remolding In Rats With Chronic ?-adrenergic Receptor Activation

Background:L-arginine is catalyzed by nitric oxide synthase(NOS)in vivo to produce Nitric oxide(NO).The half-life of NO in a biological system is extremely short,which was calculated in seconds.The extremely fast diffusion speed facilitate NO to reach the target and play its regulatory function.Almost all of the cardiomyocytes can produce NO,which plays an extremely important protective effect for cardiovascular system.NO regulates multiple cardiovascular functions through vascular-dependent and non-vascular-dependent pathways.The former includes the regulation of coronary vasoconstriction,coagulation,vascular smooth muscle proliferation,inflammatory response and angiogenesis;the latter plays a direct regulatory role in cardiac energy metabolism and function from several different aspects,including the regulation of cardiomyocyte Excitation-Contraction Coupling,(ECC),the associated signal pathway(eg cGMP/PKG pathway)and mitochondrial respiration.A variety of cardiovascular diseases associated with ?-receptor activation,although it can maintain heart function and improve symptoms in the early phase,but its long-term effects are harmful.With the progress of the disease,the bioavailability of NO gradually decreased,NOS coupling dysregulation,which accelerate the disorders of Ca2+ handling during ECC,leading to intracellular Ca2+ overload,myocardial energy metabolism dysregulation and a series of adverse changes.The above changes further reduce the bioavailability of NO,result in a vicious cycle,serious deterioration of cardiac fu.nction led to myocardial remodeling,and ultimately cause cardiac hypertrophy,arrhythmia,heart failure and even sudden cardiac death(SCD).Plenty studies demonstrated that increased bioavailability of NO plays an extremely important role in the treatment of cardiovascular system diseases.Howwer,previous studies mostly focused on the acute protective effect of NO,although a small number of reports have shown that endogenous NO has a protective effect on myocardial structural remodeling caused by chronic P-receptor activation,but the effect of exogenous NO is still unknown;And the effect of NO on cardiac electrical remodeling in the ?-receptor chronic activation model has not been reported,and the underline mechanisms are still unclear.Therefore,this study used isoproterenol in rats to construct the ?-receptor chronic activation model,and explore whether molsidomine,a long-acting NO donor,can attenuate myocardial structural remodeling and electrical remodeling,and try to clarify its mechanism.Part I:Molsidomine attenuates Cardiac Structural Remodeling in Rats with Chronic Isoprenaline StimulationObjective:To investigate the effects of molsidomine on cardiac structural remodeling in rats with chronic isoprenaline stimulation.Methods:40 SD rats in this study were randomly divided into four groups.The control group of rats was given an equal volume of 0.9%saline;the ISO group of rats was given ISO alone(5 mg/kg/d,intraperitoneally,ip);the ISO + molsidomine group(ISO + M group)of rats was given ISO(5 mg/kg/d,ip)and molsidomine(10 mg/kg/d,ip);and the ISO + molsidomine + soluble guanylate cyclase(sGC)inhibitor,1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one(ODQ)group(ISO + M + O group)of rats was given the same drugs as the ISO + M group with the addition of ODQ(5 mg/kg/d,ip).The drugs or saline treatments were administered for 14 consecutive days(1/3 total doses every 8 hours).ODQ was dissolved in dimethylsulfoxide to a final concentration of lmg/ml,molsidomine and ISO were dissolved in 0.9%sterile saline to a final concentration of 2mg/ml and 1mg/ml,respectively.ODQ treatment was initiated 0.5 hours prior to molsidomine,and molsidomine treatment was 0.5 hours prior to ISO administration.14 days later,(1)Echocardiographic analysis was performed in to assess the left ventricular posterior wall dimensions(LVPWD),interventricular septum thickness at diastole(IVSD),ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),ejection fraction(EF),and fractional shortening(FS);(2)Haemodynamic measurements to measure cardiac systolic and diastolic funtions and recorde systolic blood pressure(SBP)and diastolic blood pressure(DBP);(3)The ratio of heart weight to body weight was calculated to evaluate cardiac hypertrophy;(4)H-E staining was used to evaluate myocardium hypertrophy;(5)Masson staining was used to evaluate cardiac fibrosis.Results:(1)Compared with control group,ISO stimulation increased IVSD,LVPDW,LVESD,LVEDD,while the EF and FS were decreased(all p<0.01),molsidomine attenuated the changes induced by ISO(all p<0.05),however,ODQ partly attenatuate the protect effect of molsidomine(all p<0.05);(2)Compared with control group,ISO stimulation decreased dp/dtmax and dp/dtmin,while the LVEDP was increased(all p<0.01),molsidomine attenuated the changes induced by ISO(all p<0.01),however,ODQ partly attenatuate the protect effect of molsidomine(all p<0.05);(3)Compared with control group,ISO stimulation increased HW/BW ratio(p<0.01),molsidomine attenuated the changes induced by ISO(p<0.01),however,ODQ partly attenatuate the protect effect of molsidomine(p<0.05);(4)Compared with control group,ISO stimulation increased the diameter of cardiomyocyte(p<0.Ol),molsidomine attenuated the changes induced by ISO(p<0.01),however,ODQ partly attenatuate the protect effect of molsidomine(p<0.05);(5)Compared with control group,ISO stimulation increased collagen volume fraction(CVF)(p<0.01),molsidomine attenuated the changes induced by ISO(p<0.01),however,ODQ partly attenatuate the protect effect of molsidomine(p<0.01).Conclusions:Molsidomine attenuates ?-recepter activation induced cardiac structural remodeling,improve cardiac systolic and diastolic functions.The protect effects of molsidomine maybe due to the release of NO,which combine with sGC and active the downstream signal pathways.Part ?:Molsidomine attenuates Cardiac Electrical Remolding in Rats with Chronic Isoprenaline StimulationObjective:To investigate the effects of molsidomine on cardiac electrical remodeling in rats with chronic isoprenaline stimulation.Methods:The groups and treatment were same with part I.14 days later,(1)ECG were recored and RR interval,QRS,QT interval were measured,QTc were calculated;(2)Hearts were excised,cannulated into the Langendorff perfusion system and retrogradely perfused with HEPES-buffered Tyrode solution,electrophysiological paraments were examined.The monophasic action potential(MAP)in left anterior and posterior basal ventricles(LAB and LPB),left anterior and posterior apices(LAA and LPA),right ventricular outflow tract(RVOT),left ventricular free wall(LVF),and right ventricular free wall(RVF)were recored and APD90,the spatial dispersions of APD90 were calculated;(3)The dynamic APDR curves were constructed,and calculate the maximal slopes(Smax)of the APDR curves and the dispersions of Smax;(4)Dynamic S1 S1 pacing to test the threshold of APD alternans;(5)Burst pacing induce ventricular tachycardias(VTs),and calculate the incidence of VTs in different groups.Results:(1)Compared with control group,ISO stimulation increased RR interval,QRS,QT interval and QTc(all p<0.01),molsidomine attenuated the changes induced by ISO(all p<0.01),ODQ partly attenatuate the protect effect of molsidomine(all p<0.05);(2)Compared with control group,ISO stimulation increased APD90,the spatial dispersions of APD90(all p<0.01),molsidomine attenuated the changes induced by ISO(all p<0.05),while ODQ partly attenatuate the protect effect of molsidomine(all p<0.05);(3)Compared with control group,ISO stimulation increased Smax.and the dispersions of Smax(p<0.01),molsidomine attenuated the changes induced by ISO(p<0.01),while ODQ partly attenatuate the protect effect of molsidomine(p<0.05);(4)Compared with control group,ISO decreased the threshold of APD alternans(p<0.01),molsidomine attenuated the changes induced by ISO(p<0.01),while ODQ partly attenatuate the protect effect of molsidomine(p<0.05),(5)Compared with control group,ISO stimulation increased the incidence of VTs(p<0.01),molsidomine attenuated the changes induced by ISO(p<0.01),while ODQ partly attenatuate the protect effect of molsidomine(p<0.05).Conclusions:Molsidomine attenuates ?-recepter activation induced cardiac electrical remodeling,improve the threshold of APD alternans and decrease the incidence of VTs,these protect effects were due to the decrease of APD90,decrease of ventricular repolaration dispersion,and stabilization of APDR.Part ?:The mechanism of molsidomine on myocardial structure and electrical remodeling in rats with chronic ?-receptors activationObjective:To investigate the possible mechanism of the effect of molsidomine on myocardial structure and electrical remodeling in rats with chronic ?-receptors activation.Methods:The experimental group and the dosing regimen were the same as the first part.14 days later,(1)L-type Ca2+ current activation curve and current density were recorded by whole-cell patch-clamp technique;(2)Epifluorescence microscope(Leica Microsystems,Germany)was used to detect the Ca2+ transient amplitude(?F/Fo),Ca2+ content(?F/F0,Caffeine),50%recovery time(RT50)and the incidence of Ca2+transient amplitude alternans;(3)The content of nitrate/nitrite in serum and myocardium cGMP levels were measured;(4)Western blot was used to detect the expression of Ca2+,eNOS,iNOS,calcium cycle-related protein,CAMKII,p-CAMKII proteins.Results:(1)Compared with the control group,the maximal current density of L-type Ca2+ currents in ISO rats was decreased(P<0.01),molsidomine attenuated the changes induced by ISO(all p<0.01),ODQ partly attenatuate the protect effect of molsidomine(all p<0.05);(2)Compared with the control group,the levels of AF/F0,AF/F0,caffeine were decreased in the ISO group,the RT50 was prolonged and the induction rate of Ca2+ alternans was increased(p<0.05 or p<0.01),molsidomine attenuated the changes induced by ISO(all p<0.01),ODQ partly attenatuate the protect effect of molsidomine(p<0.05 or p<0.01);(3)Compared with the control group,the NOx levels in the ISO group increased and the content of cGMP in the myocardium decreased(all p<0.01),molsidomine further increased the NOx and cGMP levels(all p<0.01),ODQ decreased the NOx,but the difference was not statistically significant(P>0.05),but ODQ attenuated the increase of cGMP level compare with ISO+M group(P<0.05);(4)Compared with control group,the expression of eNOS,PKG,Cav1.2,SERCA2a and p-PLB/t-PLB were decreased,iNOS expression and p-RyR2/t-RyR2,p-CAMKII/t-CAMKII ratio were increased in ISO group(all p<0.01);molsidomine attenuated the changes induced by ISO(all p<0.01),ODQ partly attenatuate the protect effect of molsidomine(all p<0.05).Conclusions:Molsidomine attenuates ?-recepter activation induced cardiac structural and electrical remodeling by stabilizing intracellular Ca2+ handling.The myocardium protection is at least partially mediated through the NO/sGC/cGMP/PKG signaling pathway.