| Research backgroundCharacterized with increased cardiomyocyte protein synthesis and increased cell volume, myocardial hypertrophy (MH) is crucial for the transition of cardiac function from adaptive to maladaptive status and progression to irreversible change. Although some degree of cardiac hypertrophy serves to reduce wall stress and helps compensate for increased load on the myocardium, sustained prohypertrophic signaling within cardiomyocytes is detrimental and a major factor contributing to the progression to heart failure (HF).so far, a variety of signaling pathways involved in the occurrence of cardiac hypertrophy. Among them, enhancement of calcium-regulated signaling pathways plays a major role in the development of pathological cardiac hypertrophy. Accordingly, manipulation of cardiac Ca2+ signaling represents a logical approach to treatment of cardiac hypertrophy.Cardiac Ca2+ signaling is a delicate interplay of multiple Ca2+ handling proteins. At systole, the activation of L-type Ca2+ channel (LTCC) allows extracellular Ca2+ influx into cardiomyocytes upon membrane depolarization, triggering sarcoplasmic reticulum (SR) Ca2+ release via Ca2+-induced Ca2+ release (CICR) mechanism. At diastole, the increased intracellular Ca2+ is sequestrated into SR by the SR Ca2+-ATPase (SERCA) and extruded from cytoplasm by the sodium/calcium (Na+/Ca2+) exchanger and sarcolemmal Ca2+-ATPase. The activity of LTCC can be inhibited by Ca2+ channel blockers which have been confirmed to be therapeutically effective for the treatment of cardiac hypertrophy in many animal models. Given the essential role of intracellular Ca2+ in cardiac contraction, blockade of LTCC would decrease cardiac contractile function. Furthermore, blockade of noncardiac Ca2+ channels induces side effects, including systemic hypotension, constipation, edema, etc. Thus, new agents (drugs) that can suppress cardiac Ca2+ signaling without causing the side effects are highly expected.Polydatin (PD), a resveratrol glucoside with a 3,4’,5-trihydroxystilben-3-b-D-mono-D-glucoside molecular structure, is a natural component extracted from the perennial herbage Polygonum Cuspidatum Sieb.et Zucc. So far, there is only one study reporting that PD attenuated ventricular remodeling in vivo by inhibiting the activation of neurohormone. Whether PD has a direct anti-hypertrophic effect and whether it modifies the heart failure process in vivo remain unknown. Recently, we found that PD modulated Ca2+ handling and cardiac contractility. When acutely applied to isolated rat ventricular myocytes, PD slightly increased rather than decreasing cardiac contractility. The findings in normal cardiomyocytes lead us to the hypothesis that PD suppresses hypertrophic stimuli-induced upregulation of Ca2+ signalings and thus exerts direct anti-hypertrophic effect, without compromising cardiac contractile function. If the hypothesis is true, combining with previous findings that PD did not reduce blood pressure and interfere with systemic hemodynamics in normal animals, it’s rational to speculate that PD is a good candidate drug for the treatment of cardiac hypertrophy.Thus, To test this hypothesis, we explored the therapeutic effect of PD on attenuation of hypertrophy in cultured neonatal rat ventricular myocytes(NRVMs) stimulated with phenylephrine (PE) and in pressure overload (PO)-induced hypertrophic mouse model. Furthermore, we investigated the effects of PD on cardiac Ca2+ handling and contractility, as well as Ca2+/calmodulin-activated calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway which has been shown to play a major role in the development of pathological cardiac hypertrophy. Here, we demonstrated that PD prohibited the development of cardiac hypertrophy in vitro as well as in vivo by inhibiting Ca2+ signal and calcineurin-NFAT signaling pathway. Objective:In the current study, we will investigate whethe the PD can attenuate hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs)stimulated with phenylephrine (PE) and in pressure overload (PO)-induced hypertrophic mouse model; investigating the effect of PD on the Ca2+ signal and cardiac contractility; Discussing whethe the PD reduce cardiac hypertrophy by inhibiting calcineurin-NFAT signaling pathway.Methods:1 Experimental methods1.1 The design of Experimental groupIn vitro,The Experimental groups were set as follow:The control group:ConPolydatin alone group:PDPhenylephrine group:PEPhenylephrine+Polydatin group:PE+PDResveratrol alone group:RVPhenylephrine+Resveratrol group:PE+ResPhenylephrine+Tacrolimus:PE+FK506Phenylephrine+Polydatin+Tacrolimus:(PE+PD+FK506)Phenylephrine+Nifedipine(PE+Nife)Phenylephrine+Polydatin+Nifedipine(PE+PD+Nife).In vivo, Experimental groups were set as follow:Sham-operated group:ShamTransverse aortic constriction group:TACPolydatin alone group:PDTransverse aortic constriction+Polydatin group:TAC+PD1.2 The detection of gene expression of cardiac hypertrophy and the determination of cardiac functionIn vitro, NRVMs were incubated with PE (20μmol/L), PD(50μmol/1,ethanol dissolved) for 72h after culturing 24h, starved 24h,western blot was used to detection the gene expression of ANP and β-MHC, we also determined the area of each group.in vivo, we estimated cardiac structure and function by VisualSonics Vevo770 system at different time point in TAC mouse, among, PD was dissolved with β-cyclodextrin, oral dose was 50mg/kg/day from 1 week to 8 week after operation. 1.3 The detection of Ca2+ signal in NRVMs and in mouseInvestigating the effect of PD on the Ca2+ signal by the confocal laser scan microscope system (Zeiss LSM-780), including contractility, Ca2+ transient, half full duration of half maximum(Tso) and rise time in groups.1.4 The dection of protein activity and Nuclear transfer of Calcineurin-NFAT signaling pathway in NRVMs and mouseInvestigating the activity of calcineurin by a calcineurin kit, western blot was used to monitor the calcineurin protein and the position of NFAT3 transfer. Results:In the first part:Antihypertrophic effect of PD(一) Antihypertrophic effect of PD in vitro1、PD decreased ANP and β-MHC protein level in NRVMsFirst of all, we observed the effect of PD on the ANP and P-MHC protein level in different groups. The results showed that the ANP ' β-MHC were significantly different among different groups (F= 6.259、13.728;P=0.017、0.002). Compared with Control groups, the expression levels of hyperophic mark genes ANP (ANP/β-actin: 1.00±0.00 vs.1.96±0.42, P=0.017)and p-MHC (β-MHC/β-actin:1.00±0.00 vs. 2.22±0.35, P= 0.002)were significantly up-regulated in PE group, while PD significantly inhibited the PE-induced upregulation of ANP and β-MHC, P< 0.05-0.001). Compared with PE group, the cardiomyocyte area in PE+PD group significantly decreased.2-. PD compared with RV in AntihypertrophicWe observed P-MHC mRNA level ANP and β-MHC protein level in different groups. The results displayed that β-MHC mRNA level, ANP and β-MHC protein level were significantly different among different groups (F=6.206、3.860、 9.319;P=0.005、0.038,0.001).PD alone group (50 μM) and RV(25μM)alone group had no effect on them. compared to the control group, the β-MHC mRNA level, ANP and β-MHC protein level of PE group were significantly increased by 0.99 fold,0.96 folds and 1.22 fold. The results displayed that the application of RV and PD remarkably induced the decrease of β-MHC mRNA level, ANP and β-MHC protein level, there were no differerce between PE+RV and PE+PD,Compared to the control group, PE+RV and PE+PD were still significantly up-regulated.(二) Antihypertrophic effect of PD in vivo1、Treatment with PD attenuated cardiac remodelingWe observed LVPWs, LVPWd at different time point among different groups, all of them were significantly different.LVPWs (F=12.472,20.797,15.359, 15.915,P=0.000、0.000、0.000、0.000), LVPWd (F=15.626、11.234、12.778、 11.359、P=0.000、0.000、0.000、0.001).In contrast, PD significantly slowed the progression of LV wall thickness increase. Similarly, Compared with Sham groups, treatment with PD, the LVIDs, LVIDd were significantly reduced at 13w, it indicated the delay of LV enlargement in TAC+PD group.2、Treatment with PD improved heart failureWe observed LVFS and EF at different time point among different groups. LVFS, EF at 5w after TAC, TAC group was significantly higher compared with that in Sham group (P<0.001), while LVFS,EF in TAC+PD group were significantly lower that in the TAC group (all P<0.05). LVFS, EF at 13w after TAC.TAC group was significantly lower compared with that in Sham group,but LVFS,EF in TAC+PD group were significantly higher that in the TAC group.3, PD attenuated HW/BW of TAC mouseWe observed HW/BW in different groups at 13w, The results displayed that the HW/BW in TAC+PD group was significantly smaller than in TAC group (F=0.000; P=21.143). but Sham group was significantly lower compared with TAC+PD group (P<0.05).4、PD decreased ANP and P-MHC protein level of TAC mouseWe observed the effect of PD on the ANP and P-MHC protein level in different groups. The results showed that the ANP ' β-MHC were significantly different among different groups (F=11.997、12.134;P=0.002、0.001). Compared with Control groups, the expression levels of hyperophic mark genes ANP were significantly up-regulated in PE group, while PD significantly inhibited the TAC-induced upregulation of ANP and β-MHC. Compared with Sham group, the ANP and β-MHC protein level group significantly increased.In the second part:The effect of PD on cardiac signal1、The effect of PD on cardiac signal in NRVMsWe observed cardiac signal among different groups in NRVMs. The results displayed that AF/Fo (F=13.18;P=0.000), rise time (F=6.898;P=0.000), Tso (F= 12.453;P=0.000)were significantly different among different groups.compared to the control group, the value of △F/Fo, rise time,Tso were 6.32±2.12, 170.13±48.42,74.29±27.9 and in PE group, while PD significantly inhibited the PE-induced upregulation of △F/Fo, rise time, T50.2、The effect of PD on cardiac signal in mouseIsolation of adult mouse ventricular myocytes after TAC 5w, we observed cardiac signal among different groups including △F/Fo, Shortening, T50, The result of variance analysis indicated that △F/Fo(F=6.017;P=0.000),T50 (F=10.985;P=0.000)were significantly different among different groups, while Shortening in TAC+PD group was no difference compared with the TAC group, but,compared to Sham group, was significantly increased.In the third part:The effect of PD on calcineurin/NFAT3 signal pathway1、PD inhibited Calcineurin/NFAT3 in NRVMsWe observed the effect of PD on the Calcineurin/NFAT in NRVMs, Compared with the control group, exposure of NRVMs to PE resulted in an increase in the expression of calcineurin activity by 1.3 fold,which was abrogated by treatment with PD. Subcellular localization of NFAT3 was assessed by Western blot. In the control groups, NFAT3 was primarily localized in the cytoplasm, which was translocated into the nucleus in response to PE stimulation.Treatment with PD inhibited PE-induced NFAT3 nuclear translocation.2、PD inhibited Calcineurin/NFAT3 in mouseSimilarly, extracted tissue proteins, using the same methods,we observed the effect of PD on the Calcineurin/NFAT3 in mouse, The results displayed that Calcineurin protein (F= 10.181;P=0.004), Calcineurin activity (F= 7.084;P=0.012),were significantly different among different groups. which was decreased by treatment with PD. Subcellular localization of NFAT3 was assessed by Western blot. In the Sham groups, NFAT3 was primarily localized in the cytoplasm, which was translocated into the nucleus in response to TAC stimulation.Treatment with PD inhibited TAC-induced NFAT3 nuclear translocation.Conclusions:1. PD renders the heart resistant to subsequent hypertrophic stress.2. PD exerts antihypertrophic effect, inhibits cardiac remodeling, improves cardiac function, delays the progression from myocardial hypertrophy to heart failure.3. PD suppresses hypertrophic stimuli-induced upregulation of Ca2+ signalings and thus exerts direct anti-hypertrophic effect, without compromising cardiac contractile function.4. PD stimulation suppresses the hypertrophy by the calcineurin/NFAT signal pathway. |
PDF Full Text Request