Loss Of MD1 Exacerbates Pressure Overload-induced Left Ventricular Structural And Electrical Remodelling

BackgroundCardiac remodelling plays an important role in the development and progression of heart failure(HF).To date,the factors and mechanisms that modulate pathological cardiac remodelling have not yet been fully elucidated.Evidence gathered over the past two decades has shown that activation of Toll-like receptor 4(TLR4)signalling can lead to pathological cardiac remodelling(both structural and electrical),whereas therapies targeting against TLR4 have shown effectiveness in attenuating murine cardiac remodelling caused by pressure overload.Therefore,regulation of TLR4 signalling may be a new strategy for the prevention and treatment of pathological cardiac remodelling.Myeloid differentiation protein 1(MD1),an endogenous negative modulator of TLR4 signalling,is not only expressed predominantly in immune cells,but also widely expressed in heart tissues.To the best of our knowledge,no previous study has reported whether MD1 regulates cardiac structural and electrical remodelling in HF.AimsTo determine the functional significance of MD1 in the regulation of aortic banding(AB)-induced left ventricular(LV)structural and electrical remodelling and its underlying mechanisms.MethodsPart one:Samples of human failing hearts were collected from the left ventricles of DCM patients undergoing heart transplants.Control samples were obtained from the left ventricles of normal heart donors who had died as a result of accidents,and whose hearts were not used for transplantation due to non-cardiac reasons.Quantitative real-time PCR(qRT-PCR)was used to detect the mRNA levels of BNP and β-MHC,and Western blot analysis was used to determine MD1 expression in heart samples of DCM patients and normal donors.Male and wild-type C57BL/6(WT)mice with body weight ranged from 23.5-27.5g and aged 8-10 weeks were selected as experimental subjects,AB surgery was performed to establish experimental HF model,and the mice undergone sham operation was used as control group.To ensure the induction of adequate constriction of the aorta,Doppler echocardiography was performed to evaluate the LV internal diameter and wall thickness at various times after surgery.At 4 weeks after surgery,Doppler echocardiography was performed to obtain LV ejection fraction(EF)and LV fractional shortening(FS)values of the mice,and Western blot analysis was used to detect MD1 expression in the mouse hearts samples.Part two:Male WT and MD1 knockout(MD1-KO)mice with body weight ranged from 23.5-27.5g and aged 8-10 weeks were selected as experimental subjects,the mice undergone AB surgery and sham operation were used as the experimental HF group and control group,respectively.To ensure the induction of adequate constriction of the aorta,echocardiography was performed to evaluate the LV internal diameter and wall thickness of mice at various times after surgery.At 4 weeks after surgery,we performed pressure-volume relationship measurements and echocardiography to obtain these values:LVEF,LVFS,LVEDD,LVESD,LVESP,IVSd,IVSs,LVPWd,LVPWs,dp/dt max and dp/dt min.Ratios of heart weight(HW)/body weight(BW),HW/tibia length(TL),and lung weight(LW)/TL were used to assess weight changes of the mouse hearts and lungs.H&E and PSR staining of the heart tissue slices were used to evaluate the cross-sectional area(CSA)of LV cardiomyocytes and the collagen volume of LVs,respectively.QRT-PCR was used to detect the mRNA levels of cardiac hypertrophy markers(BNP and β-MHC)in LV tissues of the mice.The LV myocytes of the mice were isolated using collagenase Ⅱ digestion and Langendorff perfusion technique,then the isolated cells were incubated with Fluo-4 Ca2+ indicator.Systolic Ca2+ transients and sarcoplasmic reticulum(SR)Ca2+ content of LV myocytes were recorded and analyzed by fluorescence microscopy system.The diastolic Ca2+ sparks of LV myocytes were recorded by confocal microscopy system and were analysed by professional softwares.To investigate possible molecular mechanisms,Western blot analysis was used to detect the expression and phosphorylation of associated proteins in left-ventricle samples of the groups.Part three:Male WT and MD1-KO mice with body weight ranged from 23.5-27.5g and aged 8-10 weeks were selected as experimental subjects,experimental animal models were developed using the methods described in part two.At 4 weeks after surgery,the surface-lead ECG(lead Ⅱ)was recorded and ECG parameters were analysed in WT and MD1-KO mice;Langendorff-perfused hearts were prepared and epicardial monophasic action potential(MAP)was recorded from the LV anterior free wall,S1-S1 pacing was used for determining 90%action potential duration(APD90)and APD alternans threshold and burst pacing was used to test for ventricular tachycardia(VT)inducibility;the LV myocytes of the mice were isolated using collagenase II digestion and Langendorff perfusion technique,then the isolated cells were used to record Ica,L activation and inactivation applying the whole-cell patch-clamp technique;diastolic spontaneous Ca2+ waves of the LV myocytes incubated with Fluo-4 Ca2+indicator were recorded and analyzed by using fluorescence microscopy system;diastolic Ca2+ sparks of the LV myocytes were recorded using confocal microscopy system and were analysed by applying professional softwares.ResultsPart one:Western blot analysis revealed that MD1 protein levels were markedly lower in the heart samples of DCM patients than in those of normal donors.Moreover,the diminished MD1 protein levels were accompanied with increased mRNA levels of cardiac hypertrophy markers(BNP and β-MHC)and decreased EF values.Similarly,MD1 protein levels,EF values,and FS values in WT mice at 4 weeks after AB were significantly reduced as compared with the corresponding values in the sham-operated group.Part two:Under basal conditions,MD1-KO mice showed no alterations in cardiac phenotype.However,at 4 weeks after AB,MD1-KO mice exhibited a marked deterioration of LV hypertrophy as compared with their WT littermates,which was confirmed by the measurement in the KO mice of a relatively larger CSA,revealed by H&E staining,and higher ratios of HW/BW,HW/TL,and LW/TL.Consistently,the mRNA levels of foetal genes(BNP,β-MHC)were markedly higher in the LV tissues of MD1-KO mice than in those of WT mice.Furthermore,MD1-KO mice exhibited exaggerated LV dilation and dysfunction,as shown by measurements of the following echocardiographic and haemodynamic parameters:LVEDD,LVESD,LVEF,LVFS,LVESP,dp/dt max and dp/dt min,but this wasn’t the case with the increase of IVSd,IVSs,LVPWd and LVPWs.Cardiac fibrosis,a major feature of maladaptive cardiac remodelling,was more prominent in MD1-KO-AB mice than in WT-AB mice.Under baseline conditions,the expression of TLR4 signalling proteins(radio-protective protein 105(RP105),MD2,and TLR4)and CaMKII signaling proteins(oxidised-CaMKII(ox-CaMKII),CaMKII,and phosphorylation of CaMKIIβ+γ+δ),the expression and phosphorylation of major Ca2+ handling proteins,and the expression of Cav1.2,calsequestrin 2(CASQ2),and calcineurin A were similar in WT and MD1-KO hearts.After 4 weeks of AB,MD1-KO mice exhibited a hyperactivation of TLR4 signalling and CaMKII signaling as compared with WT mice,which were confirmed by the increased levels of MD2,TLR4,ox-CaMKII,and CaMKII expression,and phosphorylation of CaMKHβ+γ+δ,as well as by the decreased levels of RP105 expression in the KO mice;SR Cat+ATPase 2(SERCA2)expression and SERCA2 phosphorylation were drastically decreased,whereas the expression of Na+/Ca2+exchanger 1(NCX1)and phospholamban(PLN)were significantly increased in both MD1-KO and WT hearts,although these changes were considerably greater in MD1－KO hearts than in WT hearts;PLN phosphorylation was increased,markedly and to a similar extent,in both MD1-KO and WT hearts;phosphorylation of cardiac ryanodine receptor(RyR2)and the expression of CASQ2 and calcineurin A were markedly increased,whereas Cavl.2 expression was significantly decreased in both WT and MD1-KO hearts,although these changes were significantly greater in MD1-KO hearts;total RyR2 expression was not altered in both WT and MD1-KO hearts.At baseline,the sham-operated WT and MD1-KO mice displayed similar Ca2+transients in LV myocytes.However,the peak amplitude of caffeine-induced Ca2+transients was lower in MD1-KO-Sham mice than WT-Sham mice.After 4 weeks of AB,the LV myocytes of MD1-KO mice showed reduced peak amplitude of Ca2+ transients and percentage of calculated rate constants of SERCA2 to rate constants of systolic Ca2+transient decay,increased percentage of calculated rate constants of NCX1 to rate constants of systolic Ca2+ transient decay,further reduced SR Ca2+ content,as well as prolonged time to peak[Ca2+]i transient amplitude and duration of[Cal+]i transient decay(P<0.05 vs.WT-AB).As for the average fractional release of Ca2+ transient(ratio of twitch Ca2+ transient/caffeine-induced Ca2+ transient),there was no remarkable difference among the four groups tested here.The mean Ca2+ spark frequency(CaSpF)values revealed that diastolic SR Ca2+ leak was markedly higher in MD1-KO-Sham myocytes than in WT-Sham myocytes(3.21± 0.18 vs.2.21 ± 0.19 sparks/100 μm/s,P<0.05).Moreover,at 4 weeks after AB,the CaSpF increase was greater in MD1-KO mice than in WT mice(7.26 土 0.31 vs.4.43 ± 0.22 sparks/100 μm/s,P<0.001).Part three:Mice from the indicated groups showed similar electrocardiographic PR intervals,but MD1-KO-AB mice showed shorter RR intervals and longer QRS intervals than in WT-Sham mice.The QRS intervals of MD1-KO-AB mice were significantly prolonged when compared with those of WT-AB mice.As compared with WT-Sham mice,both WT-AB and MD1-KO-AB mice exhibited prolonged QTc(corrected QT)intervals,but this prolongation was significantly higher in the KO than in the WT mice.At baseline,WT and MD1-KO hearts exhibited similar APD90,threshold interval for APD alternans,and VT inducibility rate.After 4 weeks of AB,APD90 was markedly prolonged in both WT and MD1-KO mice,but the prolongation was considerably greater in the KO mice than in the WT mice.Among the four groups tested here,the changes in the threshold interval for APD alternans were similar to the changes in APD90.The VT inducibility rate was potentially higher in MD1-KO-AB mice than in WT-AB mice(90.9%vs.50%),although there is no significant difference(P＝0.069).Statistical analysis of mean CaSpF values revealed that diastolic SR Ca2+ leak was markedly higher in MD1-KO-Sham myocytes than in WT-Sham myocytes.Moreover,at 4 weeks after AB,the CaSpF increase was greater in MD1-KO mice than in WT mice.However,there was no remarkable difference in Ca2+ spark amplitude among the four indicated groups.At baseline,WT and MD1-KO LV myocytes showed similar spontaneous Ca2+ wave incidences.The incidence of spontaneous Ca2+ waves in both WT and MD1-KO hearts was increased by a 4-week course of AB,although this increase was significantly greater in MD1-KO-AB hearts(P<0.05 vs.WT-AB).Under basal conditions,the peak Ica,L density was significantly decreased in MD1-KO mice vs.WT mice.After 4 weeks of AB,the peak Ica.L density was increased,markedly and to a similar extent,in both MD1-KO and WT mice.As compared with WT-Sham hearts,the other three groups all exhibited notable reduced half-maximal potential(V1/2)for lca,L inactivation,whereas there was no remarkable difference among the latter three groups.Under baseline conditions,the expression and phosphorylation of major Ca2+handling proteins,and the expression of major K+,Na+,and Ca2+ channels in WT and MD1-KO mouse left-ventricle samples.After 4 weeks of AB,the expression and phosphorylation of SERCA2 were drastically decreased,whereas the expression of NCX1 and PLN,and phosphorylation of RyR2 were significantly increased in both MD1-KO and WT hearts,although these changes were considerably greater in MD1-KO hearts than in WT hearts;PLN phosphorylation was increased,markedly and to a similar extent,in both MD1-KO and WT hearts;total RyR2 expression was not altered in both WT and MD1-KO hearts.After 4 weeks of AB,both WT and MD1-KO mice showed a significant reduction in the protein levels of KCNH2,KCNE1 and KCNQ1,Kv4.2 and Kv4.3,and Nav1.5,which generate IKr,IKs,Ito,and INa currents,respectively.However,this reduction was more pronounced in MD1-KO-AB mice than in WT-AB mice.Conclusions1.The expression of MD1 in LV tissues of HF patients and mice was markedly reduced.2.MD1 ameliorates pressure overload-induced LV structural remodelling by inhibiting TLR4signalling,then by inhibiting CaMKII/HDAC,calcineurin/NFAT and AKT signalling pathway.In addition,MD1 improves pressure overload-induced cardiac dysfunction in mice by alleviating the impairment of intracellular Ca2+ homeostasis.3.MD1 plays a protective role in pressure overload-induced LV electrical remodelling in mice by inhibiting the TLR4/CaMKII signalling,and further by decreasing thechanges of expression and function of Ca2+handling proteins,as well as K+ and Na+channel proteins.MD1 ameliorates pressure overload-induced electrophpysiological instability in mouse LVs,at least partly,is associated with reduction of cardiac repolarisation,increase of action potential duration alternans threshold,and decrease of diastolic SR Ca2+ leak.