| Objective:To study the inotropic effect of Rolipram,a phosphodiesterase Ⅳ inhibitor,and its underlying mechanism in normal rats,and to provide the theory for the development of positive inotropic drugs to treat heart failure with phosphodiesterase Ⅳ as a new target.Chemicals:Rolipram:purchased from MedChem Express(MCE)with a specification of 10mg,the purity>99.56%and the catalogue number HY-16900.Methods:1.In vivo recording of left ventricular and arterial hemodynamic parameters in ratsAfter anesthesia with 20%urethane(5 ml/kg,ip),MILLAR double pressure catheter was inserted into the right carotid artery.With two baroreceptors at the tip of the catheter placed in the aorta and left ventricle respectively,the pressures of the artery and left ventricle were measured simultaneously.With left ventricular pressure as Y axis and volume as X axis,P-V loop are calculated.Based on the P-V loop and arterial pressure data,the hemodynamic parameters before and after Rolipram in normal rat were analyzed.2.In vitro heart left ventricular systolic force measurement in ratsLangendorff setup was used to analyze the contractility of the isolated heart.The baroreceptor probe was inserted into the left ventricle through the left atrium by intubation.The pressure transducer with RM6240 multi-channel physiological signal acquisition processing system are linked together,the left ventricular systolic pressure curve before and after dosing were recorded.There are two types of pressure recording modes:non-fixed frequency stimulation and fixed frequency stimulation(4Hz,5v).3.Analysis of Ca2+ transient triggered by field stimulation of single cardiac myocytes in normal ratsRat ventricular myocytes were isolated by enzymatic method and loaded with calcium fluorescent indicator Fluo-4 AM.Ca2+transient images of single cardiac myocytes were obtained by calcium fluorescence imaging system.SERCA2a activity and combined activity of Na+/Ca2+Exchanger(NCX)and plasma membrane Ca2+-ATPase(PMCA)were separated by curve fitting of Ca2+transient decaying phase.By using Caffeine combined with Na+ and Ca2+free extracellular fluid,the activities of combined NCX and PMCA,PMCA only,Ca2+leakage and Ca2+content of sarcoplasmic reticulum were measured.4.Measurement of Ca2+ transient triggered by action potential of single cardiac myocytes in normal ratsRat ventricular myocytes were isolated by enzyme and loaded with Fluo-4 AM as a Ca2+fluorescence indicator.Patch clamp combined with Ca2+ fluorescence imaging system was used.Action potential and Ca2+ transient were recorded simultaneously.Meanwhile,durations at 50%and 90%full repolarization levels(APD50 and APD90)of action potential and the amplitude of Ca2+transient of single cardiac myocyte were measured.Results:1.In vivo effects of Rolipram on left ventricular systolic force in rats Rolipram(3 mg/kg)significantly increased the work output,cardiac output,stroke output,end-systolic pressure,heart rate,ejection fraction,maximal rate of left ventricular pressure increase and decreased end-systolic volume and end-diastolic volume decrease in normal rats,but did not significantly change end-diastolic pressure.2.In vivo effects of Rolipram on left ventricular systolic and diastolic properties in ratsRolipram(3 mg/kg)increased the slope of end-systolic pressure-volume relationship(ESPVR)and decreased the slope of end-diastolic pressure-volume relationship(EDPVR),which indicated that Rolipram(3 mg/kg)could improve ventricular systolic performance,ventricular diastolic performance and cardiac compliance in normal rats.3.In vivo effects of Rolipram on arterial pressure and vascular elasticity in ratsRolipram(3 mg/kg)can increase the intraventricular pressure,systolic pressure and diastolic pressure of normal rats,shorten the aortic closure time,and reduce the ventricular-arterial coupling index.The results show that Rolipram exerted inotropic effect in normal rats without obvious changes in arterial elasticity.4.In vitro effects of Rolipram on left ventricular systolic force in isolated rat heartsRolipram(0.1,1,10 μM can increase the contractility of isolated rat heart,and increase the left ventricular development pressure,heart rate,and the maximum rate of increase of left ventricular pressure in a concentration-dependent manner.5.Effects of Rolipram on Ca2+ transients and Sarcoplasmicreticulum calcium adenodinetrip hosphatase 2a(SERCA2a)activity in single rat cardiac myocyteRolipram(10 μM)significantly enhanced the field stimulation induced Ca2+ transient amplitude of rat left ventricular myocytes,but did not change the diastolic baseline fluorescence intensity.By using two-exponential and four-parameter curve fitting of the Ca2+ transient decaying phase,Rolipram(10 μM)significantly enhanced the Ca2+ transient fast component rate constant(α)mediated by SERCA2a and reduced slow component rate constant(β)mediated by combined NCX and PMCA.6.Effects of Rolipram on caffeine-induced NCX and PMCA activities in single rat cardiac myocyteCaffeine induced Ca2+ transient decaying phase fitted by a single exponential equation.Rolipram(10 μM)did not significantly affect combined NCX and PMCA activity rate constant r(NCX+PMCA),nor did affect rNCX or rPMCA,but significantly increased SR Ca2+ content and increased diastolic Ca2+ leakage.7.Effect of Rolipram on action potential-triggered Ca2+ transients in rat left ventricular myocytesIn action potential induced Ca2+ transients,Rolipram(10 μM)prolonged durations at 50%and 90%full repolarization levels(APD50 and APD90)of action potential and increased the amplitude of Ca2+ transient.Conclusion:In this P-V loop study,the results demonstrated that Rolipram,a PDE4 inhibitor,significantly increased the rat heart rate and contractility based on its enhanced end-systolic left ventricular pressure,stroke volume,cardiac output,ejection fraction,slope of ESPVR and reduced end-systolic volume.Meanwhile,Rolipram increased the systolic,diastolic blood pressure and pulse pressure without changing the arterial elastance which is proportional to the peripheral vascular resistance.Rolipram mismatched the ventricular-arterial coupling by reducing the normal value in control to the low level indicating that Rolipram exerted positive inotropy with insignificant vascular effect in normal rats.However,the decreased effect on ventricular-arterial coupling by Rolipram might be beneficial in heart failure of rats which have higher value of ventricular-arterial coupling.Although this study addressed the inotropic effects of Rolipram both in in vivo and in vitro and its underlying mechanism was mediated by enhancing the SERCA2a activity,however,there are still a few limitations in this study.First,PDE4 constitutes a large fraction of the total PDE activity in rodent heart than in human heart(Eschenhagen,2013),suggesting the data in this study should be applied to human cautiously.Secondly,the mechanism of enhanced SERCA2a activity by rolipram is still needed to further explore,such as involvement with p-phospholamban mediated by PKA,CaMK Ⅱ and protein phosphatase,etc.In conclusion,Rolipram,a PDE4 inhibitor,exerted positive inotropic effect both in vivo and in vitro without insignificant peripheral arterial elastance,which resulted in the mismatched ventricular-arterial coupling in normal rats.The inotropic effect was mediated by Ca2+handling in which Rolipram enhanced SERCA2a activity and Ca2+ leak and reduced NCX and PMCA activities.However,Rolipram had no direct effects on rate constants of NCX and PMC A.These results indicated that this PDE4 inhibitor may serve as a potential positive inotropic agent for the treatment of heart failure. |
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