Studies Of (-)-Stepholidine On Cardiac Contractile Function And Its Mechanism In Isolated Rat Hearts

Objective:(-)-Stepholidine (SPD) has dual roles of dopamine D1 agonistic and D2 antagonistic action. The effects of SPD on the dopamine receptor have been mainly focused on the central nervous system. Recent molecular biology studies confirmed that the heart has D1 receptors, but its physiological role is unclear. The pharmacological effects of SPD in the cardiovascular function confirmed SPD has the protective role of myocardial infarction and anti-arrhythmia. Meanwhile, the function of SPD on cardiovascular function has a close relationship with cardiac ion channels and intracellular Ca2+ concentration, which suggests SPD may regulate the heart function directly and the mechanism needs to be explored. Therefore we attempted to investigate the effects of SPD on heart contractile function and explore the possible underlying mechanism.Methods:1.Isolated Langendorff perfused rat heart:Left ventricle isometric contractile function was recorded using conventional isolated Langendorff perfused rat hearts. Effects of SPD and SPD under the conditions ofα1 receptor,βreceptor, H1 receptor, dopamine D1 antagonist and L-type calcium channel antagonist on left ventricular developed pressure (LVDP), maximum rate of change in left ventricular pressure (±dp/dt)max, heart rate(HR) were analyzed.2.Recording of action potential from rat heart papillary muscle:The action potential (AP) amplitude (APA), AP duration at 50% level (APD50) and at 90% level (APD90) from rat heart papillary muscle were recorded using conventional intracellular recording technique. Effects of SPD and SPD under the conditions of dopamine D1 antagonist and L-type calcium channel antagonist on above parameters were analyzed.3.Whole-cell current clamp recording:The action potential (AP) resting potential (RP), AP amplitude (APA), AP duration at 30% level (APD30), at 50% level (APD50) and at 90% level (APD90) of rat left myocyte were recorded using patch clamp current clamp configuration. Effects of SPD and SPD under the conditions of dopamine D1 antagonist and L-type calcium channel antagonist on above paremeters were analyzed.4. Whole-cell voltage clamp recording:Effects of SPD on L-type Calcium(Ica) and transient outward K+ current (Ito) were analyzed using patch clamp voltage clamp technique.5.Molecular biology techniques:RT-PCR and Western blot were used to analyze the expressions of dapomine D1 and D2 of normal isolated rat left ventricular myocytes at mRNA and protein levels.Results:1.SPD increased the cardiac contractility and dopamine D1 receptor and L-type calcium channel antagonist significantly blocked the above SPD function:SPD(10μM and 100μM) significantly enhanced LVDP,+dp/dtmax,-dp/dtmax in a dose dependent manner (P<0.05).α1 receptor antagonist (Prazosin 1μM) and H1 receptor antagonist (Fexofenadine 1μM) had no effect on blocking contractile function of SPD. Dopamine D1 receptor antagonist (SCH233901μM) and L-type calcium channel antagonist (Nimodipine 1μM) significantly blocked the contractile function of SPD (P<0.05). (βreceptor antagonist (Propranolol 1μM) partially blocked the contractile function of SPD.2.SPD significantly prolonged APD50 and APD90 in rat heart papillary muscle and this effect could be blocked by D1 receptor antagonist and L-type calcium channel antagonist:SPD 100μM significantly prolonged APD50 and APD90 in rat heart papillary muscle (P<0.05)and this effect could be blocked by dopamine D1 receptor antagonist(SCH23390 1μM) and L-type calcium channel antagonist (Nimodipine 1μM). The effect of SPD was independent of stimulation frequency.3.SPD significantly prolonged APD50 and APD90 of rat left ventricular myocyte and this effect could be blocked by D1 receptor antagonist and L-type calcium channel antagonist:SPD 100μM significantly prolonged APD50 and APD90 of rat left ventricular myocyte (P<0.05)and this effect could be blocked by dopamine D1 receptor antagonist(SCH23390 1μM) and L-type calcium channel antagonist (Nimodipine 1μM). SPD 100μM had no effect on RP,APA and APD30.4. SPD had no effect on transient outward K+ current (Ito) of rat left ventricular myocyte:SPD 1μM,10μM and 100μM had no effect on transient outward K+ current (Ito) of rat left ventricular myocyte (P>0.05). The enhanced rat cardiac contractility and prolonged APD50 and APD90 were independent of transient outward K+ current (Ito).5.SPD enhanced L-type calcium current of rat left ventricular myocyte in a dose dependent manner:SPD 10μM and 100μM enhanced L-type calcium current of rat left ventricular myocyte in a dose dependent manner(P<0.05).The result demonstrated that enhanced rat cardiac contractility and prolonged APD50 and APD90 of SPD were mediated by L-type calcium channel.6.Expressions of dopamine D1 and D2 receptor in rat left ventricular myocyte:The expressions of dopamine D1 and D2 at mRNA and protein levels by Reverse transcription PCR and Western blot techniques supported that dopamine D1 and D2 receptor do exist in rat heart and SPD exerted its effect through dopamine receptor.Conclusion:SPD significantly enhanced rat isolated heart contractility and prolonged the APD50 and APD90 of rat left ventricular myocyte and papillary muscle. These effects of SPD were mediated by D1 receptor receptor and L-type calcium channel and independent ofα1, H1 receptor and transient outward K+ current (Ito).βadrenergic receptor may have also mediated the contractile function of high dose SPD.