| Dissertationâ… :The Relationship between Expression of Protein Kinase D1 and Left Ventricular Remodeling in Spontaneously Hypertensive Rats as well as Atorvastatin Interventive StudyBackgroundHypertension is the most important risk factor for cardiovascular disease.The adult heart undergoes myocardial remodeling when subjected to longstanding hypertension. Hypertensive myocardial remodeling is an independent risk factor for lethal cardiovascular events,and is the key pathological manifestations during the transition of heart function from compensation to decompensation.Hearts respond to such stress stimuli by increasing cell size and extracellular matrix,reorganizing sarcomeres and activating a fetal cardiac gene program.Although these responses may initially normalize wall stress,the prolonged hypertrophy increases the risk for chamber dilation,heart failure and sudden death.Recent years,with the development of molecular biology,cardiac remodeling has been shown to be the basic mechanism of heart failure.Cardiac remodeling is a complex course,and the pathogenesis mechanism still remains unclear.It is well-known that abnormal activation of neurohumoral factors,oxidative stress and cytokines are involved in cardiac remodeling.A complex set of signal-transduction pathways and downstream transcription factors underlie hypertensive myocardial remodeling.Therefore,the researches on the pathogenesis and control of cardiac remodeling have become a worldwide hot topic. Protein Kinase D(PKD) is a recent addition to the calcium/calmodulin-dependent serine/threonine protein kinase.PKD family consists of 3 isoforms,these are the original PKD1 which now is also referred to as PKD, PKD2 and PKD3.Increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system,particularly in the regulation of myocardial contraction,hypertrophy and remodeling.Lots of studies have reported that cardiac specific expression of a constitutively active PKD mutant in transgenic mice leads to cardiac hypertrophy,and a chronic increase in PKD activity is sufficient to induce adverse myocardial remodeling.Studies also show that mice lacking cardiac PKD display an impaired response to stress signals that normally lead to cardiac hypertrophy.As hypertension is often accompanied by dyslipidemia,the treatment frequently involves 3-hydroxy-3-methylglutaryl coenzymeA(HMG-CoA) reductase inhibitors (statins).More and moer evidences have been established that statins not only efectively reduced serum cholesterol level,but also exerted pleiotropic beneficial efects on cardiovascular disease,including improvement of endothelial function, reduction of plaque thrombogenicity,prevention of cardiac hypertrophy or remodeling. However,the exact mechanism remains unclear.This study was therefore designed to observe the temporal profile of the expression of PKD and analyze its relationship with hypertensive cardiac remodeling; to study the role of PKD related signal-transduction pathways and downstream factors; to investigate the effects and the mechanism of atorvastatin in the prevention and treatment of cardiac remodeling.The purpose of the study is to elucidate the cellular and molecular mechanisms of hypertensive cardiac remodeling and the interventional effects of atorvastatin on it,and to provide novel theoretical evidences and strategy for prevention and treatment of hypertensive cardiac remodeling.Objectives1.To investigate the expression of PKD and related molecular pathway at both the mRNA and protein levels in myocardium of spontaneously hypertensive rats (SHR). 2.To investigate the involvement and the signal-transduction pathway of PKD in left ventricular remodeling in SHR.3.To evaluate the effects and the mechanism of atorvastatin in the prevention and treatment of cardiac remodeling.MethodsTwenty-eight 8-week-old WKY,fourty-one SHRs were obtained from company. WKY rats were randomly divided into the following four groups:A Group:8 weeks (n=7);B Group:16 weeks(n=7);C Group:24 weeks(n=7);G Group:WKY placbo (n=7).SHR rats were randomly divided into the following five groups:D Group:8 weeks(n=7);E Group:16 weeks(n=7);F Group:24 weeks(n=7);H Group:SHR placbo(n=10);I Group:SHR atorvastatin((n=10).All animals were feeded by normo-fortage and water.WKY placbo,SHR placbo,SHR atorvastatin receiving distilled water or atorvastatin at 50 mg/kg/day for 16 weeks by intragastric administration.Animals were killed when they were 8 weeks,16 weeks and 24 weeks old by decapitation.The hearts were immediately harvested and weighed.The follwing parameters were measured during the study:(1) All the rats have their body weight,heart rat and tail blood pressure measured once per 2 week;(2) Blood was collected from jugular vein at 8 weeks,16 weeks and 24 weeks respectively.Plasm lipid was determined using routine method;(3) Echocardiography was used to evaluate the function of heart, LVEDd,IVSd and LVPWd were measured,RWT and LVEF were calculated;(4) Histopathological study of heart tissue,for the detection of remodeling,the ventdcular tissue was stained with H-E and Masson's trichrome staining;(5) The left ventricular mass index(LVMI) was used to estimate the degree of cardiac hypertrophy.(6) Hydroxyproline content assay,CVF and PVCA were used to estimate the cardiac fibrosis;(7) Western-blot,RT-PCR for the expression of PKD, ERK5 and MEF2C.Results1.The experimental animals Two rats of SHR group died in the entire experiment,one of WKY group died. A total of 66 rats finished the study,27 rats in WKY group,39 rats in SHR group.2.Comparisons of SBP,BW,HR and Lipids level between WKY and SHR groups.There were no significant differences in terms of body weight,heart rat,tail systolic blood pressure and lipids at the beginning of the experiment.SBPs in SHRs at 16 and 24 weeks are higher than those in WKY rats(P＜0.01).There were no significant differences in the BW,HR and total cholesterol level among the SHR and WKY groups..In SHR group,SBP increased during the course,while the one in WKY-V group remained unchanged.After treatment with atorvastatin,SBP decreased significantly(P＜0.01).3.Eehoeardiographie evaluationEchocardiography was taken at 8,16 and 24 weeks respectly.Interventricular septum (IVS),left ventricular posterior wall(LVPW) and left ventricular diastolic diameter (LVDd) of SHR in 16W and 24W group increased significantly(P＜0.01) compared with the SHR 8W and those in WKY group,and these changes were attenuated by atorvastatin(P＜0.01).4.HE stainingHE staining slides under optical microscopy showed that in WKY group,cell size of cardiomyocytes was smaller,uniform,and array was regular;In SHR 16W and 24W groups,cardiomyocytes size was greater,not uniform,cell form was irregular,and cell arrange was disorder;Cell wall was not clear and cell fusion was observed at intersection of cell.Cardiomyocytes in atorvastatin group lied between WKY and SHR group.5.Left Ventricular Mass Index analysisThe left ventricular mass index(LVMI) is the ratio of left ventricular weight(in milligrams) to body weight(in grams)(LVW/BW),LVMI of SHR in 16W and 24W group increased significantly(P＜0.01) compared with the SHR 8W and those in WKY group,and these changes were attenuated by atorvastatin(P＜0.01).6.Cardiac fibrosis of SHRs As depicted in Masson,marked deposition of collagen was detected in the cardiac interstitial and perivascular areas of the SHRs in 16W and 24W compared with the WKY rats,which was ameliorated by atorvastatin.Consistent with this, hydroxyproline content,CVF and PVCA were elevated in the left ventricle of SHRs in 16W and 24W compared with WKY rats(P＜0.01),and atorvastatin significantly attenuated this elevation(P＜0.05)7.Expression of PKD,p-PKD1 by Western-blotCompared to WKY group,the groups of SHRs in 16W and 24W showed a significant rise in p-PKD protein content in heart tissue(P＜0.01).In SHR group,expression of p-PKD increased during the course(P＜0.01),while the one in WKY-V group remained unchanged.Consistent with this,p-ERK5 and p-MEF2C in SHRs of 16W and 24W increased significantly(P＜0.05),and these changes were attenuated by atorvastatin (P＜0.01).8.Relationship among blood pressure,cardiac hypertrophy,fibrosis and expression of p-PKD1744/748,p-PKD1916The LVMI,IVS,LVPW,LVDd,hydroxyproline content,CVF and PVCA were all positively related to the systolic blood pressure,and reduction of blood pressure by atorvastatin contributes to the attenuation of cardiac hypertrophy and fibrosis.The LVMI was also significantly correlated with the hydroxyproline content.Cardiac hypertrophy was positively correlated with the expression of p-PKD1744/748 and p-PKD1916,p-PKD1744/748 and p-PKD1916 was positively related to the collagen content.ConclusionsThe aging-related myocardial hypertrophy and fibrosis occured with the development and process of hypertension in SHR model which offers reliable animal mode for the researches on the mechanism of hypertensive LV myocardial remodeling.2.The over-expression of PKD1 at both the mRNA and protein levels has been confirmed in the heart tissue of SHR,and which associated significantly with the myocardial hypertrophic-related parameters.The activation of PKD1 involved in the development and process of myocardial hypertrophy in SHR.3.There was relationship between p-PKD1 and myocardial fibrosis in SHR.4.Atorvastatin could partially reverse the hypertension-induced myocardial remodeling through the down-regulation of PKD activation. Dissertationâ…¡:The Role of Protein kinase D in Cardiomyocyte Hypertrophy And Cardiac Fibroblasts Proliferation Induced by Angiotensinâ…¡Stimulation:Involvement of Signalling PathwayBackgroundThe reason of cardiac remodeling are a series of complex molecular and celluar mechanisms,which contribute to the changes of cardiac structure,function and phenotye.These changes including:myocyte hypertrophy,apoptosis,fibroblast proliferation,reexpression of fetal gene and protein.Among these changes,myocyte hypertrophy and fibroblast proliferation are the key factors of cardiac remoedling. Angiotensinâ…¡(Angâ…¡) is an octapeptide that exerts inotropic,hypertrophic and apoptotic effects on cardiomyocytes[Fabris et al.,2007;Mollmann et al.,2007].Thus, Angâ…¡is central for any process involved in control of hypertrophy and heart failure. The corresponding signal transduction pathways have been demonstrated in fetal, neonatal and adult cardiomyocytes.The renin-Ang system is an important component of the physiological and pathological responses of the cardiovascular system.Through Angâ…¡receptor-1(AT1).Angâ…¡carries out its functions,including hypertrophic remodeling of cardiomyocytes,which involves various downstream signal transduction mechanisms However,the regulatory molecular mechanisms, specifically the signaling cascades,involved in Angâ…¡-induced cardiomyocyte hypertrophy are not fully understood.Mitogen-activated protein kinases(MAPKs) are a family of serine/threonine kinases that play a central role in transducing extracellular cues into a variety of intracellular responses.Activated MAPKs phosphorylate multiple intracellular targets,including numerous transcription factors that induce the reprogramming of gene expression. More recently,some reports have showed that PKD1 is implicated in Angâ…¡-induced proliferation of vascular smooth muscle cells.However,little is known about how extracellular hypertrophic stimulation angtensinâ…¡is perceived and converted into intracellular signals and how these signals change the transcriptional program that eventually leads to cardiac hypertrophy and fibros in vivo.Protein Kinase D(PKD) is a recent addition to the calcium/calmodulin-dependent serine/threonine protein kinase,increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system,particularly in the regulation of myocardial contraction,hypertrophy and remodeling.The laboratories of Olson and McKinsey have reported that cardiac specific expression of a constitutively active PKD mutant in transgenic mice leads to cardiac hypertrophy, and a chronic increase in PKD activity is sufficient to induce adverse myocardial remodeling.Studies also show that mice lacking cardiac PKD display an impaired response to stress signals that normally lead to cardiac hypertrophy.These exciting data,together with the preliminary evidence that PKD expression may be increased in human heart failure,necessitate further investigation of the role of PKD in the development of cardiac remodeling and failure in vivo in response to clinically relevant stresses such as pressure overload and myocardial infarction.Objective1 To validate further the effect of cardiomycyte hypertrophy,fibroblast proliferation and syncesis of collagen induced by Angâ…¡.2 To investigate the mechanism of the signal-transduction pahtway bout AT1/PKC/PKD1/ERK5/MEF2C in hypertrophic cardiomyocytes induced by Angâ…¡.3 To probe the investigate the involvement and the signal-transduction pathway of PKD1 in fibroblast proliferation and syncesis of collagen.MethodsTake 1-3days SD neonatal rats,primary culture in vitro.Study changes of cadiomyocyte after stmulation.Cardiomyocyts were divided into different groups: Control Group:no stmulation factor;Angâ…¡stmulation Group:different dosges Angâ…¡; Angâ…¡stmulation +Losartan Group;Angâ…¡stmulation + PD123319 Group;Angâ…¡stmulation+ PKC inhibitor G(o|¨)6983 Group;Angâ…¡stmulation + PKCεsiRNA Group; Angâ…¡stmulation+ PKD1 siRNA Group;Angâ…¡stmulation+ ERK5 siRNA Group. Dtecting different time group myocyte below:(1) Observing changes of cardiomyocyte form by electron microscope;(2) 3H-leu incorporation rate,evaluated the level of cell hypertrophy;(3) Western blot dectected expression of p-PKCα/β,δ,ζ,εand PKD,p-PKD,ERK1/2,p-ERK1/2,p38,p-p38,ERK5,p-ERK5,MEF2C,p-MEF2C;(4) Added special siRNA to study the relationship among pathway molecule;(5) Determine expression of pathway rnolecul by Immunofluorescence method and EKR5 translocation between cytoplasm and nucelus.The neonate rat CFs as a experimental model were cultured primarily by the different time.To detcet effect of Angâ…¡on CFs hyperplasy and collogen synthesis and expression of PKD1,p-PKD1.Results1.AT1/PKC/PKD/ERK5/MEF2C signal-transduction pathway was involved in hypertrophic cardiomyocytes induced by Angâ…¡.(1) Morphological changes of cardiomyocytes stimulated by Angâ…¡Angâ…¡group:the final concentration is 100nM:Control group:cardiomyocytes were incubated in serum-free medium.Morphological changes were observed by electron microscope after treatment at different times(0min,5min,15min,30min,60min,120min).Hypertrophy phenomenon of cardiomyocytes was significantly greater in Angâ…¡treated cells than in controls,which was in a time-dependent way.Moreover, Angâ…¡treatment increased the cell surface area in a dose-dependent way.The cell surface area of 10nM,100nM and 1000 nM groups were 1422.31±139.26μm2,1931.79±142.661μm2,2032.71+195.73μm2 repectively,which were significantly higher than those of the control group(816.39±92.11μm2)(P＜0.01).(2) Measurement of[3H]-Leu incorporation Cardiomyocytes(3×105 cells/ml) which were incubated in 24 orifice plates were made quiescent by incubation in serum-free DMEM medium for 24 h.Cells were stimulated by different concentrations of Angâ…¡(0,1nM,10nM,100 nM and 1000nM).[3H]-Leu incorporation were determined by using a scintillation counter. 100nM Angâ…¡rapidly increased[3H]-Leu incorporation with peak incorporation at 15 and 30 min.[3H]-Leu incorporation was concentration dependent,beginning at 10 nmol/1 Angâ…¡and with maximum effect at 1000 nmol/1 Angâ…¡.[3H]-Leu incorporation of 10nM,100nM and 1000 nM groups were 1588.71±144.52,2008.14±151.25,2177.38±165.76cpm/well repectively,which were significantly higher than those of the control group(1141.27±134.95cpm/well)(P＜0.01).(3) Western blot for AT1/PKC/PKD1/ERK5/MEF2C signal-transduction pathwayâ—Angâ…¡stimulates PKD activation through a AT1-dependent pathway Angâ…¡receptor has two subtypes:AT1 and AT2,cells were pretreated for 1 hr with with losartan(0.3,1.0,3.0μmol/L,a specific antagonist for AT1,or PD123319(20μmol/L),an antagonist for AT2,then stimulated with Angâ…¡(100 nmol/L) for 0.5 hr. Losartan at 3.0μmol/L completely blocking PKD phosphorylation at Ser744/748 and Ser916,whereas PD123319 had no effect on Angâ…¡activation of PKD.â—Activation of PKD by Angâ…¡is PKC-dependent Cells were pretreated with the general PKC inhibitor G(o|¨)6983(0.3,1,3μmol/L) for 1 hr before exposure to Angâ…¡(100 nmol/L) for 1 hr.G(o|¨)6983 blocked PKD phosphorylation in a dose-dependent manner,which suggests that PKC is involved in Angâ…¡-stimulated PKD phosphorylation in cardiomyocytes.â—PKCεspecifically mediated Angâ…¡-induced PKD phosphorylation Several members of PKC isoforms,includingα,β,δ,εandζ,are expressed in cardiomyocytes,Angâ…¡treatment induced marked phosphorylation of PKCεwithin 15 min but did not induce phosphorylation of PKCα/β,PKCδ,or PKCζ.Expression of p-PKCεis significantly correlated with phosphorylation of PKD(P＜0.05).â—Activation of MAPKs by Angâ…¡in neonatal rat cardiomyocytes Cardiomyocytes were stimulated by Angâ…¡at 100nM for different times(0,5,15,30, 60min),we examined the potential role of ERK1/2,P38,ERK5.Angâ…¡(100 nmol/L) induced phosphorylation of ERK5 after 5 min,with peak phosphorylation between 15 and 30 min(P＜0.01),which returned to base line after 1 hr.However,the phosphorylation of ERK1/2 and p38 were earlier than that of ERK5,with peak phosphorylation at 5 min(P＜0.01). Total protein level of ERK5,ERK1/2 and P38 did not change.â—PKCεand ERK5 are involved in Angâ…¡-induced activation of MEF2C Cardiomyocytes were stimulated by Angâ…¡at 100nM for different times(0,5,15,30, 60min),then examined the activation of MEF2C.Angâ…¡significantly stimulated the phosphorylation of MEF2C by 5min treatment,with peak phosphorylation at 15min (P＜0.01),which returned to basal levels by 60 min.Activation of MEF2C is positively related to phosphorylation of PKCε,PKD and ERK5.â—Further research in the signal-transduction pathway by specific siRNA PKCεsiRNACardiomyocytes were infected with PKCεsiRNA before stimulated by Angâ…¡at 100nM,then examined the activation of PKD,ERK5 and MEF2C.PKCεsiRNA inhibited the phosphorylation ofPKD,ERK5 and MEF2C(P＜0.01).PKD siRNACardiomyocytes were infected with PKD siRNA before stimulated by Angâ…¡at 100nM,then examined the activation of ERK5 and MEF2C.PKD siRNA inhibited the phosphorylation of ERK5 and MEF2C(P＜0.01).ERK5 siRNACardiomyocytes were infected with ERK5 siRNA before stimulated by Angâ…¡at 100nM,then examined the activation of MEF2C.ERK5 siRNA significantly inhibited the phosphorylation of MEF2C(P＜0.01).(4) Immunofluorescence staining for expression of all moleculars and translocation of ERK5 The expression of p-PKD 1744/748,p-PKD 1916,p-ERK5,p-MEF2C was observed by immunofluorescence staining.After stimulation by Angâ…¡for 15 min, p-PKD744/748 and p-PKD1916 were expressed in cytoplasm while p-ERK5 and p-MEF2C were in the nucleus.Before stimulation with Angâ…¡,ERK5 was located primarily in the cytoplasm of cardiomyocytes;ERK5 nuclear entry was seen at 15 min after Angâ…¡stimulation,with a striking translocation from the cytoplasm to the nucleus.At 60 min of Angâ…¡treatment,ERK5 was gradually shuttled back to the cytoplasm.2.PKD was involved in the proliferation of cardiac fibroblasts(CFs) and collagen synthesis(1) Angâ…¡induced the proliferation of CFs and collagen synthesis in concentration and time dependent mannerâ—Angâ…¡concentration-dependently stimulate the proliferation of CFs and collagen synthesisCompared with the control and FBS groups,A490 of Angâ…¡10,1O0,1000nM groups were significant higher(P＜0.05～0.01),Consistent with this,hydroxyproline content was also elevated(P＜0.05).A490 and hydroxyproline content were concentration dependent,beginning at 10 nmol/1 Angâ…¡and with maximum effect at 1000 nmol/1 Angâ…¡.â—Angâ…¡time-dependently stimulate the proliferation of CFs and collagen synthesisCFs were stimulated by Angâ…¡at 100nM,absorbance value and hydroxyproline content were time-dependently elevated within 24 hr.(2) Activation of PKD in CFs stimulated by Angâ…¡CFs were stimulated by Angâ…¡at 100nM for different times(12,24,48 hr),we examined the phosphorylation of PKD.Angâ…¡(100 nmol/L) induced phosphorylation of PKD at Ser744/748 and Ser916 after 12 hr with peak phosphorylation at 24 hr. Compared with the control group,the p-PKD content in 12,24 and 48hr increased 52%,153%and 110%respectively(P＜0.05～0.01).(3) PKD specific siRNA inhibited the proliferation of CFs and collagen synthesis CFs were infected with PKD siRNA before stimulated by Angâ…¡,then measured the absorbance value and hydroxyproline content.Compared with the Angâ…¡group, p-PKD expression in the PKD siRNA+ Angâ…¡group was significantly inhibited (P＜0.01),and was similar with the control group(P＞0.05).PKD siRNA decreased both the absorbance value and the hydroxyproline content.(P＜0.01). Conclusions1.Angâ…¡induced hypertrophy of cardiomyocytes,proliferation of cardiac fibroblasts and synthesis of collagen.2.AT1/PKC/PKD1/ERK5/MEF2C signal pathway was involved in hypertrophy of cardiomyocytes induced by Angâ…¡.3.PKD1 plays an important role in Angâ…¡induced proliferation of cardiac fibroblasts and synthesis of collagen. |
PDF Full Text Request