Mechanistic Study Of Liguzinediol On The Inotropic Effect Via Inhibition Of Protein Phosphatases Activities

Objective:The study was to investigate the mechanism underlying positive inotropic effects of Liguzinediol (LZDO) in normal rats hearts.Methods1. In vivo left ventricular Pressure-Volume loop recordingTo measure the left ventricular systolic function and analyze the relationship between pressure and volume in presence of LZDO in rat hearts using the pressure-volume catheter. The indexes including:Heart rate, end-systolic Volume, end-diastolic Volume, end-systolic Pressure, Stroke Volume, Ejection Fraction, Cardiac Output, Peak rate of rise of left ventricular pressure, Stroke Work, end-systolic pressure-volume relationship and end-diastolic pressure-volume relationship.2. Left ventricular myocyte intracellular Ca2+ imagingUsing intracellular Ca2+ imaging to observe the effects of LZDO on Ca2+ transient in normal left ventricular myocytes of rats.3. Ex vivo intraventricular pressure recording from isolated rat heartsThe left ventricular function was recorded by the Langendroff non-recirculating mode. Effects of LZDO in presences of Ryanodine receptor agonist, protein phosphatase inhibitor; left ventricular developed pressure (LVDP), Peak rate of rise of left ventricular pressure (+dP/dtmax) and Heart rate (HR) of isolated rat hearts were measured.4. Measurement of SERCA2a activityThe sarcoplasmic reticulum (SR) vesicles were separated for analyzing the activity of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) in presence of LZDO.5. Western immunoblot analysisVentricular myocytes from rats were isolated to investigate the phosphorylations of 16-Ser-PLB and 17-Thr-PLB in presence of LZDO with different concentrations (0,1,10,100 μM).6. Measurements of PP1/PP2 A activitiesUsing ProFluor(?) Ser/Thr PPase Assay kit to measure the activities of PP1/PP2A in myocardial homogenates in presence of LZDO with different concentrations (0,1,10,100 μM).Results:1. LZDO exerts the inotropic effect of rats in vivoLZDO significantly increased slope of end-systolic pressure-volume relationship curve in rat hearts which indicated that LZDO enhanced left ventricular systolic function, intrinsic myocardial contractility. Also, ventricular passive diastolic function was not significantly affected. The value of end-diastolic stiffness with a slightly downward trend reminds that LZDO might have the ability of improving myocardial compliance.2. LZDO presented a significant increase of Ca2+ transient in left ventricular myocytes.LZDO 100 μM significantly inscreased intracellular Ca2+ transient in left ventricular myocytes from the begaining of the perfusion up to 30 min. Thapsigargin, an inhibitor of SERCA2a, significantly reduced the SR Ca2+ transient and the inhibition reached stable level at the time of 30 min of perfusion. LZDO 100 μM failed to restore the decreased Ca2+transient induced by thapsigargin.3. LZDO restored the negative inotropic and chronotropic effects induced by caffeine, and did not altered the increased contracitility induced by Calyculin.Caffeine, known as an agonist of RyR2, can massively release Ca2+ in its early action. As time goes on, the effect of caffeine can cause calcium concentration gradient descent. Therefore, caffeine presents negative inotropic and chronotropic effects. LZDO 100 μM restored the reduced myocardial contractility induced by caffeine 0.5 mM at time of 30 min of perfusion, which indicated that LZDO might reverse the depletion effect of sarcoplasmic reticulum calcium concentration caused by caffeine.Calyculin A, an inhibitor of PP1 and PP2A, enhanced the left ventricular contractivity. However, LZDO did not further increase the positive inotropic effects induced by calyculin A (4 nM). These results showed that the positive inotropic effect of LZDO may be related to intracellular protein phosphatase in rats heart.4. LZDO increased the activity of SERCA2a from rat left ventricular myocyteThe activities of sarcoplasmic reticulum Ca2+-ATPase2a (SERCA2a) were increased in presence of LZDO with different concentrations (1,10,100 μM)in a concentration dependent manner.5. LZDO increased the PLB phosphorylations at both Ser-16 and Thr-17PLB phosphorylations at both Ser-16 and Thr-17 were increased in presence of LZDO (100 μM), which indicated that LZDO’s inotropic effect might be mediated by PLB phosphorylations.6. LZDO inhibited the activities of PP1 and PP2ALZDO (100 μM) significantly decreased the activities of PP1 and PP2A from ventricular myocytes. The results indicated that LZDO might influence the activities of PP1 and PP2A by which myocardial contractility is increased.Conclusions:The positive inotropic effect of LZDO was mediated by the inhibition of PP1 and PP2A activities which reduce the dephosphorylation of PLB. Moreover, dephosphorylation of PLB increases the SERCA2a activity. Based on its novel target on inotropic effect, LZDO might serve as a potential drug for heart failure treatment in clinical setting.