Positive Inotropic Effect Mediated By Acting Sarcoplasmic Reticulum Calcium Release In Isolated Rat Hearts And Cardiac Safety Evaluation Of Liguzinediol

Objectives:The purpose in the present work was to investigate the mechanism underlying positive inotropic effect in rat isolated hearts and evaluate cardiac safety of Liguzinediol.Methods:1. Intraventricular pressure recording from isolated rat hearts:Left ventricle contractile function was measured using the Langendorff non-recirculating mode of isolated rat hearts. Effects of LZDO and LZDO in presences of ai receptor, β receptor, dopamine D1, Hi receptor antagonist, Phosphodiesterase, Na+-K+ATPase, Na+-Ca2+exchange inhibitor, L-type calcium channel antagonist, Ryanodine receptor, or SR Ca2+ATPase inhibitor. Left ventricular systolic pressure (LVSP) and left ventricular end diastolic pressure (LVEDP) as major cardiac contractile function were calculated. Also, heart rate (HR), peak rate of rise of left ventricular pressure (+dp/dtmax), and peak rate of fall of left ventricular pressure (-dp/dtmax) of isolated rat hearts were measured.2. The stimulatory activity assay on31GPCRs:The stable cell lines expressing31GPCRs respectively were applied for the screening. LZDO was diluted to100μM and screened in duplication. The cells were validated with the reference compounds. Their EC50/IC50values were similar to the reported values and can be applied in the screening. During screening, the reference agonist for each GPCR was assayed as positive control and at concentration stimulating maximal activity. The activation activity of LZDO to each GPCR was normalized with the positive control and shown as%activation.3. Recording of action potential from rat heart papillary muscle:The action potential (AP) duration at50%level (APD50) and at90%level (APD90) from rat heart papillary muscle were recorded using conventional intracellular recording technique. Effects of LZDO on above parameters were analyzed. 4. Whole-cell current clamp recording:The action potential (AP) duration at50%level (APD50) and at90%level (APD90) of rat left myocyte were recorded using patch clamp current clamp configuration. Effects of LZDO on above paremeters were analyzed.5. Whole-cell voltage clamp recording:Whole-cell voltage clamp technique was used to analyze the effects of LZDO on L-type Ca2+current which underlies mechanism of LZDO’s positive inotropic effect and hNav1.5、 hERG currents which serve for cardiac safety evaluation of LZDO.6. Intracellular Ca2+imaging:Intracellular Ca2+imaging was used to investigate the effect of LZDO on Ca2+transient.7. In vivo and in vitro electrocardiogram (ECG) recording:ECG was recorded by conventional guinea pig in vivo and in vitro ECG techniques, we analyzed the effects of LZDO on P-R and QTc intervals.Results:1. LZDO increased left ventricular contractility in the isolated rat heart in a dose-dependent manner. L-type calcium channel antagonist, Ryanodine receptor inhibitor or SR Ca2+-ATPase inhibitor completely blocked the positive inotropic effect of LZDO:LZDO significantly enhanced LVSP, LVEDP,+dp/dtmax, and-dp/dtmax, but not heart rate. α1adrenergic receptor antagonist (Prazosin1μM), β adrenergic receptor antagonist (Propranolol1μM), Dopamine D1receptor antagonist (SCH233901μM), H1receptor antagonist (Fexofenadine1μM), Phosphodiesterase inhibitor (IBMX5μM), Na+-K+-ATPase inhibitor (Ouabain1μM) or Na+-Ca2+exchange inhibitor (KB-R79431μM) had no effect on blocking contractile function of LZDO100μM (P<0.05). Meanwhile, L-type calcium channel antagonist (Nimodipine1μM), L-type calcium channel antagonist (Verapamil1μM), Ryanodine receptor inhibitor (Ruthenium red5μM) or SR Ca2+-ATPase inhibitor (Thapsigargin2μM) completely blocked the contractile function of LZDO (P>0.05). It is suggested that the positive inotropic effect of LZDO was mediated by L-type calcium channel, Ryanodine receptor and SR Ca2+-ATPase.2. The31GPCRs were not activated by LZDO:Results showed that LZDO100μM did not activate31GPCRs which related to cardiac contractility.3. LZDO did not significantly change APD50and APD90in rat heart papillary muscle:LZDO100μM did not significantly change APD50and APD90in rat heart papillary muscle (P>0.05).4. LZDO did not significantly change APD50and APD90in rat left ventricular myocyte:LZDO100μM did not significantly change APD50and APD90in rat left ventricular myocyte (P>0.05).5. LZDO failed to increase the L-type calcium current in rat left ventricular myocyte, LZDO did not significantly suppress the hNavl.5and hERG current:LZDO100μM did not significantly change L-type calcium current in rat left ventricular myocyte (P>0.05), it is suggested that LZDO’s target is not L-type calcium channel. LZDO (1,10,100,300μM) did not significantly suppress the hNavl.5and hERG current.6. LZDO significantly enhanced intracellular Ca2+transient in rat left ventricular myocyte:LZDO100μM significantly enhanced intracellular Ca2+transient in rat left ventricular myocyte (P<0.05), Moreover, LZDO100μM restored the sarcoplasmic reticulum depletion effect of caffeine on Ca2+transient.7. LZDO did not significantly change P-R and QTc intervals of in vivo and in vitro guinea pig ECG:LZDO1.7g-kg-1in vivo and LZDO300μM in vitro could not significantly prolong P-R and QTc intervals (P>0.05).Conclusions:The positive inotropic effect of LZDO in isolated rat hearts was mediated through an elevation of SR Ca2+transient. LZDO had no direct effect on L-type calcium channel and may act on SR Ca2+ATPase. LZDO did not significantly change P-R and QTc intervals of in vivo and in vitro guinea pig ECG Moreover, LZDO did not significantly suppress the hNav1.5and the hERG current. These data suggested LZDO had no prearrythmia effect. LZDO has a unique biological mechanism without proarrythmia effect that may prove effective in treating heart failure in clinic.