| Objective:To study the positive inotropic effect of PF-04449613,a phosphodiesterase 9 inhibitor,in normal rat heart and its underlying mechanism.To explore whether phosphodiesterase 9 inhibition can be used as a new target for developing the positive inotropic drugs for the treatment of heart failure in the clinical settings.Chemicals:PF-04449613,a PDE9 inhibitor:purchased from Tocris Bioscience with a specification of 10 mg,catalog no:5915,molecular formula C21H25N5O3,molecular weight 395.45,purity>99.6%.Methods:1.In vivo recording of left ventricular and arterial hemodynamic parameters in ratsAfter anesthesia with 20%urethane(5 ml/kg,ip),the Millar double pressure volume catheter was inserted into the rat left ventricle through the carotid artery(close chest)approach.The two pressure sensors in a catheter were placed in the aorta and the left ventricle respectively.The arterial,left ventricular pressure and left blood volume were measured at the same time.The P-V loop was derived from the left ventricular pressure and left blood volume.The relative parameters of cardiac and arterial hemodynamics were analyzed by Labchart8 software,and the overall hemodynamic characteristics of rats in vivo were analyzed by PF-04449613 dissolved in DMSO less than 0.3 mL was administered intraperitoneal.2.In vitro intraventricular pressure recording from isolated rat heartsThe isolated heart of Langendorff was perfused via retrograde aorta,and the baroreceptor was inserted into the left ventricle.In the conditions of pacing and nonpacing(4Hz,5 V),the RM6240 multi-channel physiological signal acquisition and processing system were used to record the changes of left ventricular developed pressure,heart rate and ±dp/dtmax of left ventricular before and after administration of PF-04449613.3.Ca2+ transient recording of rat left ventricular myocytes triggered by field stimulationThe rat ventricular myocytes were acutely isolated with collagenase type II(CollagenaseⅡ).The intracellular Ca2+ level and dynamic changes induced by field stimulation were recorded by laser scanning confocal microscope(CLSM)and single wavelength fluorescence excitation of Fluo-4 acetyl hydroxymethyl ester(AM).The data was collected by high sensitive image acquisition equipment CCD(Cool Charge-Coupled Device).The decay rate constants of rapid decline phase and slow decline phase were analyzed.4.The combined activity analysis of NCX and PMCA from rat left ventricular myocytes triggered by caffeineHigh concentration of caffeine completely opened the sarcoplasmic reticulum type 2 ryanodine receptor(RyR2)and induced Ca2+ transient,so that there was no Ca2+ concentration gradient between cytoplasm and SR,which indirectly eliminated the activity of SERCA2a.The combined activity of NCX and PMCA(rNCX+PMCA)was obtained by fitting the decay rate constant of the decline phase curve.5.Ca2+leak recordings of rat left ventricular myocytesWhen the cells were perfused with tyrode solution with without Na+and Ca2+in presence of 1 mM of Tetracaine,Na+-Ca2+ exchanger(NCX)activity was stopped and Ca2+leak was blocked from SR RyR2(blocked by Tetracaine).The fluorescence value of F0(0Ca/0Na/Tetracaine)was deemed as the level of diastolic calcium in cytoplasm.In the condition of tyrode solutionwithout Tetracaine,Ca2+and Na+,the Ca2+leak was released from RyR2.The fluorescence value of F0(0Ca/0Na)was measured as the diastolic calcium level in the state of Ca2+leak.RyR2 Ca2+leak=F0(0Ca/0Na)-FO(0Ca/0Na/Tretracation).The PMCA activity alone was separated from caffeine-evoked Ca2+ transient in the condition of Na+,Ca2+-free solution.The single exponential curve fittings were used to estimate the activities of PMCA(rpMCA).6.Western blotting for analysis of PLB and RyR2 phosphorylationThe protein was extracted from acute isolated primary cardiomyocytes and divided into normal control group and PF-04449613 group.The levels of phosphorylation of phospholamban 16 serine(p-PLB Ser-16)and 17 threonine(p-PLB Thr-17)in cardiomyocytes were analyzed by Western blotting.Also,the levels of phosphorylation of RyR2 serine-2808(p-RyR2 Ser-2808)and RyR2 serine-2814(p-RyR2 Ser-2814)in cardiomyocytes were analyzed.Result:1.PF-04449613 increased left ventricular inotropy of rat heart in vivoPF-04449613(5.5 mg/kg)significantly increased stroke work,cardiac output,stroke volume,end-systolic pressure and ejection fraction in rats.However,PF-04449613(5.5 mg/kg)significantly reduced end-systolic volume,end-diastolic volume and end-diastolic pressure without affecting the heart rate.In addition,the effect on vascular hemodynamics showed that PF-04449613 significantly increased systolic blood pressure(SBP)and decreased diastolic blood pressure(DBP),and arterial elasticity(EA)which was proportional to peripheral vascular resistance.2.PF-04449613 increased left ventricular inotropy of isolated rat heartsIn the conditions of pacing and nonpacing,PF-04449613(0.001,0.01,0.1,1 μM)significantly increased the left ventricular developed pressure and the ±dp/dtmax of left ventricles in a concentration-dependent manner.3.PF-04449613 enhanced the Ca2+uptaking function by promoting SERCA2a activityPF-04449613 significantly enhanced the transient amplitude of Ca2+triggered by field stimulation.Compared to the normal group,PF-04449613(5 μM)significantly increased the SERCA2a-mediated rapid decay phase rate constant(α)and decreased the slow component rate constant(β)mediated by the combined action of NCX and PMCA.4.PF-04449613 had no significant effect on the combined activity of NCX and PMCAThe rate constant of the slow decay phase of Ca2+transient induced by caffeine reflects the combined activity of NCX and PMCA without the participation of SERCA2a(rNCX+FMCA).The result of the slow component rate constant(β)showed that PF-04449613(5 μM)had no significant function change of the combined activity of NCX and PMCA.5.PF-04449613 reduced the Ca2+leak rate without affecting the activity of PMCACompared to the normal group,PF-04449613(5 μM)had no effect on PMCA activity.However,PF-04449613(5 μM)reduced RyR2-mediated calcium leak rate from SR.6.PF-04449613 enhanced PLB phosphorylationPF-04449613(5 μM)significantly increased the phosphorylation level of p-PLB Thr-17 but did not change the phosphorylation level of p-PLB Ser-16.However,PF-04449613(5 μM)did not significantly affect the phosphorylation of 2808 serine ryanodine receptor(p-RyR2 Ser-2808)and 2814 serine ryanodine receptor(p-RyR2 Ser-2814).Conclusion:In this study,double pressure-volume catheter experiment(P-V loop)showed that PDE9 inhibitor PF-04449613 had positive inotropic effect based on its increasing systolic blood pressure,decreased diastolic blood pressure and peripheral vascular resistance.The isolated heart experiments also indicated that PF-04449613 had positive inotropic effect,and this effect was independent of neurohumoral regulation.PF-04449613(5 μM)increases the phosphorylation of PLB through the CaMKII pathway,which enhances the activity of SERCA2a for the re-uptaking of Ca2+and the calcium capacity of SR.During myocardial contraction phase,the Ca2+transient amplitude was increased resulting in increased heart contractile ability. |
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