Progesterone Regulates Calcium Handling And Protects Against Bisphenol A-induced Arrhythmogenesis In Cardiac Myocytes From Female Rats

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Progesterone Regulates Calcium Handling through Membrane Progesterone Receptors(mPRs)in the Cardiac Myocytes from Female RatBackgroundCardiovascular disease(CVD)is now one of the most common cause of women health and death worldwide and is the leading cause for women in China.In recent years,sex hormones play an important role in the occurrence,development,and prognosis of CVD in women.It is well-recognized that endogenous estrogens exert a variety of protective effects on cardiovascular(CV)system such as vasodilation.However,the recognition of the role of natural progesterone(P4)and its receptors in CV system is mostly based on studies which mainly aim to elucidate the CV actions of estrogen.Indeed,there have been very few studies investigaing the direct effect of P4 on CV system.Previous studies have shown that P4 exerts a potential protective effect on CV system,including vasodilatation,lowers blood pressure,and protects against myocardial ischemia reperfusion injury.However,the underlying mechanisms of these P4-induced CV actions remains unclear.Therefore,elucidation of the direct effect of P4 on CV system and its underlying mechanism will benefit us to better understanding the role of sex hormone in the genesis and development of CVD and may provide effective approaches and theoretical basis for the prevention and treatment of CVD in menopausal women.Classically,P4 exerts a vast range of biological effects through binding to nuclear progesterone receptorb(nPR)that regulate gene transcription by recruiting transcription factors to progesterone response elements.In addition to the classical mechanism of transcriptional actiation,the nongenomic mechanism of P4 has received growing research attention.Such P4 mechanism is independent of gene transcription and protein synthesis,but is mediated by membrane-initialed signaling.Growing evidence has shown that the nongenomic mechanism plays an important role in the P4-induced biological effects.However,the possible receptors and mechanism mediating such nongenomic action of P4 have not been fully understood.Membrane progesterone receptors(mPRs)is a novel candidate for the intermediary in P4' s rapid actions.Growing evidence has shown that mPRs present in a wide range of vertebrate species including humans.Moreover,mPRs could independently mediate the nongenomic effect of P4.Evidence has been obtained that P4 promoted NO synthesis in human vascular endothelial cells through mPRs.However,the potential role of mPRs in P4 regulation of heart physiology remains unknown.Myocyte calcium handling plays a critical role in the maintainance of normal cardiac physiology.Recently,it has been shown that chronic P4 exposure significantly shorten the duration and decay time of calcium reuptake in rabbit cardiac myocytes,suggesting P4 may play an important role in the regulation of myocyte calcium handling.Thereby,further study on the role of mPRs in myocyte calcium handling will be of great scientific significance.AimsThe aim of this study is to investigate the gene/protein expression and localization of all mPRs isoforms in heart tissues/myocytes and to determine the role of mPRs in the P4 regulation of myocyte calcium handling,and to explore the underlying molecular mechanisms.MethodsAdult female SD rats were used for all experiments.1.The expression of mPRs gene/protein were confirmed in female rat heart using RT-PCR and Western blot.2.The cellular localization of mPRs were examined in female rat myocytes using immunofluorescence method.3.Calcium transient,sarcoplasmic reticulum(SR)calcium load and calcium spark,L-type calcium current,and cyclic adenosine monophosphate(cAMP)level were respectively examined in cardiac myocytes by calcium photometry system,confocal microscopy,patch clamp,and ELISA methods to examine the role of mPRs in the P4 regulation of myocyte calcium handling under control,P4,mPRs agonist Org-OD,P4+mPRs antagonist PTX,Org-OD+PTX,and other intervention groups.4.The phosphorylation/total levels of RyR(ryanodine receptor,RyR)and PLN(phospholamban,PLN)phosphorylated sites,calcium/calmodulin-dependent protein kinase II(CAMKII),Akt,and endothelial nitric oxide synthase(eNOS)were assessed using Western blotting under control,P4,Org-OD,P4+PTX,Org-OD+PTX,and other intervention groups to further explore the underlying molecular mechanism of P4's rapid action.Results1.The mRNAs and proteins of mPRs were expressed in various heart tissues of female rat.Using rat ovary tissue as positive control,we showed that the mRNAs and proteins of mPR??mPR? and mPR? were positively detected in the myocardium of left ventricle.Compared with left ventricle,the mRNA levels of left atrial and right atrial were significantly increased.2.The cellular localization of mPRs proteins in female rat cardiac myocytes.We observed that there was an obvious green fluorescence around plasma membrane in all myocytes incubated by specific mPRs antibody.Blockade mPRs antibody with corresponding blocking peptides eliminated all observed membrane green fluorescences in cardiac myocytes.These results suggest that mPRs are localized in plasma membrane of cardiac myocytes.3.P4 increased calcium transient in female rat cardiac myocytes through mPRs.Acute P4 treatment 15min not only significantly increased the amplitude of calcium transients,but also shorten the decay rate of calcium transients.Such effects of P4 were completely mimicked by P4-BSA,a membrane-impermeable BSA conjugated P4,and Org-OD,mPRs special agonist.Moreover,both of P4 and Org-OD induced effects were abolished by the preincubation of inhibitory G(Gi)protein inhibitor PTX,but not nuclear progesterone receptor antagonist RU486.4.P4 increased SR load in female rat cardiac myocytes through mPRs.Compared with control group,the amplitude of caffeine-induced calcium transients of P4 and Org-OD groups were increased significantly.However,such effects of P4 and Org-OD were completely abolished by PTX.Moreover,there was no noticeable change in the decay rate of caffeine-induced calcium transients among various groups.5.P4 increased SR calcium release in female rat cardiac myocytes through mPRs.Compared with control group,the calcium spark frequency of P4 and Org-OD groups were increased significantly.However,such effects of P4 and Org-OD were abolished by PTX.These was no noticeable change in the amplitude of calcium spark frequency among various groups.6.P4 treatment did not affect the L-type calcium current in female rat cardiac myocytes.Compared with control group,the current density of L-type calcium channel was not altered following P4 application.7.P4 increased the phosphorylation levels of two key SR calcium transport proteins RyR and PLN in female rat cardiac myocytes through mPRs.Compared with control group,both P4 and Org-OD greatly increased the phosphorylation levels of CAMKII sites RyR2814 and PLN17,but not PKA sites RyR2808 and PLN16.Also,both P4 and Org-OD induced RyR2814 and PLN17 phosphorylations were abolished by PTX.We further showed that there was a consistent change in the phosphorylation levels of RyR2814 and PLN17 over Org-OD exposure time,with peak at 15minutes.8.The effects of P4 and Org-OD on intracellular cAMP level in female rat cardiac myocytes:Using isoproterenol as positive control,Org-OD,but not P4,treatment rapidly decreased the intracellular cAMP level in cardac myocytes compared with control.However,such effect of Org-OD was abolished by PTX.9.CAMKII was indispensable for P4-induced alterations of calcium handling in female rat cardiac myocytes.CAMKII inhibitor AIP markedly inhibited either P4 or Org-OD induced increases in RyR2814 and PLN 17 phosphorylations,amplitude and decay rate of calcium transients,and calcium spark frequency effects in female rat myocytes.10.IP3/IP3R signaling was not involved in mPRs-induced CAMKII activation.IP3R specific antagonist Xestospongin C did not affect P4-indued increases in the phosphorylation levels of RyR2814 and PLN17 in female rat mycytes.11.PI3K/Akt/eNOS signaling pathway was involved in mPRs-mediated signal transduction.Both P4 and Org-OD induced RyR2814 and PLN 17 phosphorylations were abolished by eNOS inhibitor L-NAME or PI3K antagonist wortmannin.The activities of CAMK? and eNOS were further examined using western blotting.We found that either P4 or Org-OD treatment increased the phosphorylation levels of CAMK? and eNOS.Preincubation with L-NAME or wortmannin abolished both P4 and Org-OD's effects on CAMK? phosphorylation.Moreover,preincubation with wormannin abolished both P4 and Org-OD's effects on eNOS phosphorylation.Furthermore,we showed that both P4 and Org-OD increased the phosphorylation level of Akt in female rat myocytes.However,such effects of P4 and Org-OD were abolished by PTX.12.mPRs-induced alterations of calcium handling in female rat cardiac myocytes were mediated by PI3K/Akt/eNOS signaling pathway.Compared to control group,blockade with wortmannin or L-NAME completely abolished both P4 and Org-OD-induced increases in the amplitude of calcium transients and calcium spark frequency in female rat myocytes.Conclusions1.We fistly show that three mPRs isoforms are widely expressed in various heart tissues in female rat,and are localized on the myocyte membrane.2.Our study,for the first time,shows that P4 exerts a rapid effect on the regulation of calcium handling in female rat cardiac myocytes through mPRs.3.P4 activates PI3K/Akt/eNOS signaling pathway,which causes CAMK?phosphorylation of RyR and PLN and then leading to the increases of SR loand and SR release.These actions underlie the stimulatory effect of P4 on calcium transients in the female rat myocytes.Section ? The Effect and Mechanism of Progesterone onBisphenol A-Induced Arrhythmogenesis in Female RatCardiac MyocytesBackgroundBisphenol A(bisphenol a,BPA)is one of common estrogenic endocrine-disrupting chemicals(EDCs),which is widely used in the production of epoxy resins and polycarbonate plastics.With the extensive use of BPA products in daily production and living,the potential harmful effects of BPA on human environment/health have raised deep concerns from experts and scholars worldwide.It has been shown that BPA exerts a wide range of adverse human health effects through its estrogenic disrupting function including abnormity of brain development and behavior,retarded growth of fetus,and disorders of reproductive and immune systems.Interesting,there is increasingly evidence that BPA exposure exerts a potential toxic effect on cardiovascular(CV)system.Recently,studies in our laboratory have demonstrated that rapid BPA exposure has arrhythmogenic effects in female rat hearts.Thereby,we need to be alert to the potential hazard for heart associated with BPA exposure.To better understand the biological and toxic impact of EDCs and the responsiveness of biological systems to such an impact,it is increasingly recognized that the effects of EDCs should be assessed not in isolation but in the context of native steroid hormones.Native steroid hormones can influence the actions of exogenous EDCs including BPA.For example,17?-estradiol(E2)has been shown to either augment or alleviate the impact of BPA in a variety of cell types or systems.We previously showed that in rat female hearts the arrhythmogenic effect of BPA was particularly pronounced in the presence of physiological level of E2.As we known,progesterone(P4)and native estrogens interact either synergistically or antagonistically to regulate diverse reproductive events.Similarity,Interactions between estrogens and P4 are also common in other physiological systems including CV system.However,the interactions between P4 and estrogenic BPA are remain unknown.It is not currently clear the possible influence of P4 on BPA-induced cardiotoxicity,especially its cardiac arrhythmogenesis.AimsOur previous study has shown that rapid BPA exposure exerts an arrhythmogenic effect on female rat hearts.The aims of current study are to examine the acute influence of P4 on BPA-induced arrhythmogenesis in female rat cardiac myocytes,and to elucidate the underlying mechanisms.MethodsIsolated cardiac myocytes from adult female SD rats were used for all experiments.To define the possible effect of P4 on environmental-relevant concentration of BPA-induced triggered activities in female rat cardiac myocytes,myocyte after-contraction and after-transient were respectively measured by video edge detection system and confocal microscopy methods.Then,myocyte calcium spark and caffeine-induced calcium transient were respectively examined by calcium photometry system and confocal microscopy and the phosphorylation/total levels of RyR and PLN phosphorylation sites and Akt were assessed using Western blotting to further explore the underlying cellular and molecular mechanisms of P4 protection against BPA-induced arrhythmogenesis in female rat cardiac myocytes.Results1.P4 treatment rapidly inhibited BPA-induced triggered activities in female rat cardiac myocytes:Preincubation with InM P4 acutely abolished BPA-induced triggered activities in female rat cardiac myocytes,measured by incidences of myocyte after-contraction and after-transient.RU486,an nPR antagonist,blocked the inhibitory action of P4 on BPA-induced triggered activities in cardiac myocytes.2.P4 treatment rapidly inhibited BPA-induced alterations of calcium handling in female rat cardiac myocytes:1)P4 treatment rapidly inhibited BPA-induced increase in SR load:P4 rapidly inhibited BPA-induced increase in the amplitude of caffeine-induced calcium transients,an indication of SR load.However,RU486 eliminated such inhibitory effect of P4.2)P4 treatment rapidly inhibited BPA-induced increase in SR leak:P4 rapidly inhibited BPA-induced increase in the calcium spark frequency,an indication of SR leak.However,RU486 abolished such inhibitory effect of P4.3.P4 treatment inhibited BPA-induced increase in the phosphorylation of PLN17 in female rat cardiac myocytes:P4 treatment blocked BPA induced increase in PLN17 phosphorylation.By contrast,P4 did not affect BPA-induced increase in RyR2808 phosphorylation.RU486 abolished P4's inhibitory effect on BPA-induced PLN17 phosphorylation.4.P4's inhibitory effect is mediated by membrane-initiated signaling:P4-BSA,a membrane-impermeable protein conjugated P4 abolished BPA-induced increases in calcium spark frequency,percentage of myocytes with triggered activities,and PLN17 phosphorylation.RU486 abolished all the observed inhibitory effects of P4-BSA.5.P4 signaling was independent of cSrc activation:the cSrc inhibitor PP2 did not alter the inhibitory effects of P4 on BPA-induced PLN17 phosphorylation and triggered activities in female rat cardiac mycytes.6.P4 signaling involved activation of the inhibitory G protein(Gi):the Gi protein inhibitor PTX significantly suppressed the inhibitory effects of P4 on BPA-induced PLN17 phosphorylation and triggered activities in female rat cardiac myocytes.7.The inhibitory effect of P4 was mediated by nPR-Gi-PI3K signaling cascade:Blockade of PI3K with wortmannin abolished the inhibitory effects of P4 on BPA-induced PLN17 phosphorylation and triggered activities in female rat cardiac myocytes In addition,P4 treatment rapidly increased the phosphorylation level of Akt,however,such effect of P4 was abolished by PTX or RU486.Conclusions1.In the present study,with isolated cardiac myocytes as the experimental model,we firstly show that P4,at the physiological relevant level,exerts a protective effect against BPA-induced arrhythmogenesis in female rat cardiac myocytes.2.The underlying mechanism of P4' s rapid inhibitory effect involves membrane-initiated signaling.P4 inhibits BPA-induced PLN17 phosphorylation and alterations of SR calcium handling through activation of nPR/Gi/PI3K pathway,thus abolishs BPA-induced triggered activities in female rat cardiac myocytes.