Comparative Studies On The Cardiac Functions Of Protein Phosphatases And Na~+-k~+-ATPease In Normal Rats

Objective:To investigate the differential cardiac hemodynamic functions and underlying mechanisms of potential and existent positive inotropic targets of protein phosphatase(Calyculin A,an inhibitor of protein phosphatase)and Na+-K+-ATPase(deslanoside,a inhibitor of Na+-K+-ATPase)in normal rats,and to analyze the perspective of protein phosphatase as a potential target of positive inotropic drug development.Chemicals:1.Calyculin A(CA),batch number:101932-71-2,purchased from Genne Operation company.2.Deslanoside Injection:Prescription drug with registerd number of H32021538,purchased from Chengdu Beite Pharmaceutical Co.Ltd.Methods1.In vivo left ventricular hemodynamic parameters(pressure-volume loop)recordingThe rats were anaesthetized by 20%urethane(5 mL/kg,ip).The rat left ventricular hemodynamic parameters were assessed by a pressure and volume microtip catheter(MILLAR,SPR-901,840-8188,Houston,USA)connected to a PowerLab 4/30 data acquisition system(AD Instruments,PowerLab 4/30,Australia).This catheter was inserted into the left ventricle to measure left ventricular pressure-volume relationship and aortic pressure from right carotid artery.All data were analyzed with Labchart 8 from AD Instruments.2.Ex vivo intraventricular pressure recording from isolated rat heartsRats were anaesthetized by 20%urethane(5 ml/kg,ip)and their hearts were rapidly excised and cannulated through the aorta above the coronary ostia.The hearts were then perfused in the Langendorff non-recirculating mode,at a perfusion pressure of 80 mmHg,with perfusion solution(37 ?)containing(in mM):NaCl 117,KCI 5.7,CaCl2 1.8,MgCl2 1.7,NaHCO3 4.4,NaH2P04 1.5,HEPES 20,Glucose 11 gassed with 95%02 plus 5%CO2(pH 7.4 with NaOH).Drugs were added to the perfusion solution and infused via retrograde perfusion of the coronary artery.Intraventricular pressure under non-isovolumic conditions of contraction was measured by a 4 channel amplifier(Chengdu instruments company,Chengdu,China)conneted to polyethylene tube inserted into the left ventricle via the left atrium.The hearts were beating spontaneously(non-paced).3.Ca2+ transient recordings of adult rat cardiomyocytes by fluorescence imagingAdult rat cardiomyocytes were loaded with the membrane permeable acetoxymethyl(AM)ester form of the fluorescent Ca2+ indicator Fluo-3(5 ?M)for 30 min at room temperature.Fluo-3 was excited at 488 nm(Lambda DG-4,Sutter instruments,USA)and emitted fluorescence measured with a 515 nm long pass filter.The region of interest was restricted to a single cell with the aid of an adjustable window.The transient amplitudes were calculated as a difference of the peak and diastolic fluorescent values.Background fluorescence levels were used to correct raw fluorescence data.Ca2+ spark fluorescent intensity of Fluo-3 loaded myocytes was recorded during electrical pacing(0.2 Hz,10 ms duration,15 V,alternating polarities)with two platinum electrodes at imaging frequencies of 100 Hz.Images were recorded with an ANDOR ZYLA-5.5-CL3 CCD camera(Andor Technologe,EU)connected to an inverted microscope(Olympus ?53,Olympus,Tokyo,Japan)that was synchronized by a real-time analog-digital processor unit and Meta fluor acquisition software(64-bit,version 7.8.10.0,Molecular Device,USA).Results1.Effects of Calyculin A and deslanoside on rat heart contractility.Both Calyculin A and deslanoside enhanced the cardiac output,stroke work,systolic pressure and decreased systolic volume significantly.Moreover,Calyculin A also increased stroke work and ejection fraction significantly in normal rats.These results show the powerful effect of Calyculin A compared to deslanoside on rat left ventricular hemodynamic parameters.2.Effects of Calyculin A and deslanoside on rat heart systolic and diastolic functionsComparing to deslanoside which can only enhance contractile properties of left ventricle,Calyculin A could not only improve the contractility but also improve the diastolic function indicating the potential for treating heart failure with decreased diastolic function.3.Effects of Calyculin A and deslanoside on arterial pressure,arterial impedance and ventricular-vascular coupling.Both Calyculin A and deslanoside significantly enhanced arterial systolic and diastolic pressure.However,Calyculin A showed the superior effects on lowering the arterial impedance and improving ventricular-vascular coupling compared with deslanoside.4.Effects of Calyculin A and deslanoside on isolated rat heart positive inotropyBoth Calyculin A and deslanoside significantly increased the contractility of isolated rat hearts and decreased the heart rat independent on neuro-humoral mediation.Moreover,Calyculin A increased peak rate of rise of left ventricular pressure(+dp/dtmax).5.Effects of Calyculin A and deslanoside on rat left ventricular myocyte Ca2+ transientCalyculin A enhanced the systolic period fluorescence intensity and decreased the end-diastolic period fluorescence intensity.These effects of Calyculin A were mediated by reducing dephosphorylation which increases the SERCA2a activity and enhances the Ca2+recovery rate.Deslanoside increased both systolic and diastolic period fluorescence intensity without affecting the SERCA2a activity.These effects of deslanoside were mediated by inhibiting Na+-K+-ATPase.These results indicated that Calyculin A had more powerful effect on intracellular Ca2+ recovery into sarcoplasmic reticulum by SERCA2a compare with deslanoside.Conclusion:The results demonstrated that both Calyculin A and deslanoside increased positive inotropy in P-V loop and isolated rat heart experiments and enhanced the rat left ventricular myocyte Ca2+ transients.Moreover,Calyculin A increased stroke work,ejection fraction,improved the diastolic function and ventricular-vascular coupling,decreased the arterial impedance.Also,Calyculin A enhanced the Ca2+ recovery rate and decreased the end-diastolic period fluorescence intensity.These effects of Calyculin A and deslanoside were mediated by inhibiting the protein protein phosphatase and Na+-K+-ATPase,respectively.Protein phosphatase which was mediated the superior hemodynamic effects by its inhibitor of Calyculin A may serve as potential target of developing positive inotropic drug for treating heart failure.