Comparative Study On Hemodynamic Differences Between Deslanoside And Milronone In Normal Rats

ObjectiveTo investigate the hemodynamic differences between deslanoside,a positive inotropic glycoside agonist,and milronone,a non-cardiac glycoside positive inotropic agent,and to analyze the possible mechanism why long-term use of Milrinone may leads to the incr,eased mortality in clinical settings.Chemicals1.Deslanoside Injection:Prescription drug with registerd number of H32021538,specifications of 0.4 mg/2 mL,purchased from Chengdu Beite Pharmaceutical Co.Ltd.2.Milrinone Injection:Prescription drug with registerd number of H1097005,specifications of 5 mg/5 mL,purchased from Taizhou People’s Hospital.Methods1.In vivo left ventricular hemodynamic parameters（Pressure-Volume Loop）recordingMale Sprague Dawley rats were used in this study.The rats were anesthetized.After pressure validation,the Millar double pressure-volume microtip catheter connected to a PowerLab 4/30 data acquisition system was inserted into the left ventricle of rats from the right carotid artery in parallel.The date were recorded and the hemodynamic parameters were analyzed with Labchart 8 from AD Instruments.2.Ex vivo intraventricular pressure recording from isolated rat heartsThe rat heart was perfused in the Langendorff non-recirculating mode.Deslanoside and Milrinone were dissolved in the perfusion solution and infused via retrograde perfusion of the coronary artery.Biological acquisition system（Chengdu instruments company,Chengdu,China）was used to record left ventricular systolic index（HR,LVDP,dp/dtmax）.3.Ca²⁺ transient recordings of adult rat cardiomyocytes by fluorescence imagingCardiac Myocyte Isolation:Hearts were removed from adult male SD rat anesthetized by 20%urethane（5mL/kg,ip）.The aorta was cannulated for Langendorff perfusion,and ventricular myocytes were isolated by a standard enzymatic technique（Collagenase II 2 mg/mL,protease 0.1 mg/mL）.The cells were maintained at room temperature in a modified KB solution.Measuring the intracellular calcium transient:Adult rat cardiomyocytes were loaded with the membrane permeable acetoxymethyl（AM）ester form of the fluorescent Ca²⁺ indicator Fluo-4（5 μM）for 30 min at room temperature and then placed on the stage of an inverted fluorescent microscope.Cells were stimulated by a field stimulatorat with a frequency of 1Hz.Fluo-4 was excited at 488 nm and emitted fluorescence measured with a 510 nm long pass filter using an camera obtain value of the fluorescence intensity.Calcium concentration was described as △lntensity%（△Intensity%= I-I0,I0 was the fluorescence intensity value of baseline）.Results1.In vivo effects of deslanoside and milrinone on myocardial contractility in rats.Both Deslanoside and Milrinone can enhance left ventricular myocardial contractility in rats.At the same,both can increase cardiac output,stroke volume,left ventricular end systolic pressure,ejection fraction significantly.However,Deslanoside mainly increased stroke work,the maximum rate of left ventricular pressure rise and reduced left ventricular end-systolic volume without increasing HR.Milrinone primarily enhanced stroke volume,HR,and decreased left ventricular contraction/diastolic volume and left ventricular systolic/diastolic pressure.2.In vivo effects of deslanoside and milrinoneon on rat left ventricular inotropic and lusitropic properties.The slope of the end-systolic pressure-volume（ESPVR）relationship（end-systolic Elastance,Ees）and the slope of the end-diastolic pressure-volume（EDPVR）relationship（end-diastolic Elastance,Eed）were significantly increased by deslanside.Milrinone significantly increased Ees and descreased Eed of rat left ventricle.3.In vivo effects of deslanoside and milrinoneon on arterial pressure,arterial impedance and ventricular-vascular coupling.Deslanside significantly increased arterial systolic and diastolic pressures,and shorten the time of aortic closure and prolonged left ventricular ejection time in rats.However,deslanoside had insignificant effect on arterial impedance and ventricular-vascular coupling.Milrinone reduced the left ventricular and arterial pressures and arterial impedance.Also milrinone improved ventricular-vascular coupling.4.Effects of deslanoside and milrinone on cardiac contractility in isolated rat hearts.Both deslanside and milrinone increased the left ventricular pressure and the maximal rate of left ventricular pressure rise in a concentration-dependent manner,suggesting that both of them could increase the contractility of myocardium in rats.The difference is that deslanside significantly reduced HR,which was different from the previous in vivo study,presumably due to neuro-humoral regulation.However,milrinone increased HR of isolated rat heart,indicating that increased HR is due to its direct effect.5.Effects of deslanoside and milrinone on Ca²⁺ transient from left ventricular myocytes of rats.Deslanoside increased cardiomyocyte systolic and diastolic fluorescence intensity.The effect was mediated by suppressing the slope of slow component（Kother mechanism）and slope of fast component（KsERCA2a）of reuptake phase of Ca²⁺ transient.Milrinone increased the amplutide of Ca²⁺ transient mediated by enhancing the KSERCA2a and suppressing the Kother mechanism respectively.Conclusion:From the overall effect,both could increase left ventricular contractility in rats.Deslanside mainly reduced the ventricular end-systolic volume,didn’t change the diastolic performance and HR;Milrinone principally increased myocardial contractility to reduce vascular resistance,and increased HR,we speculate that increased HR may be the underlying mechanism for the increased mortality caused by long-term medication.From the perspective of the ventricular-vascular coupling,compared with deslanside,milrinone could lower the peripheral vascular resistance,optimize the ventricular-vascular coupling and more efficient performance.From the results of ex vivo experiments,we found that deslanoside decreased the isolated heart HR without changing in vivo rat heart HR,indicating that deslanoside’s effect on HR depends on the neuro-humoral regulation.However,milrinone increased HR both in vivo and in vitro,indicating milrinone had the direct effect on HR in vitro independent of neuro-humoral regulation.In addition,Deslanside could simultaneously increase ventricular systolic and diastolic cytoplasmic Ca²⁺ concentration and enhanced myocardial contractility,but easily caused Ca²⁺ leakage and led to arrhythmia.Milrinone increased cytoplasmic Ca²⁺concentration while reducing diastolic Ca²⁺ concentration.In coclusion,milrinone excerted the stronger inotropic effect with increased HR in vivo compared with deslanside.The effect of milrinone on increasing HR may explain the fact that long-term use of milirinone may increase mortality of patients in clinical setting..