The Role Of CCAAT/enhancer-binding Protein β In Atorvastatin Attenuating Myocardial Hypertrophy

Objective:Myocardial hypertrophy is an important independent risk factor for many cardiovascular diseases,including heart failure,myocardial infarction,arrhythmia and sudden death.And this transition is a very complex pathophysiological process,whose key feature is cardiomyocyte death,especially apoptosis.Possibly,the apoptosis in pathological hypertrophy results to reduced contractile tissue,heart dilate and stiffen eventually.Thus,some researchers proposed that inhibiting modes of cardiomyocyte apoptosis might be a promising therapeutic approach to prevent the transition from compensated hypertrophy to decompensated hypertrophy and heart failure.The competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA coenzyme A reductase(HMG-CoA,statins)are the commonly used lipid lowering drugs in the clinic.And independent of reducing blood lipids,statins also could improve endothelial cells function,inhibit oxidative stress,anti-inflammatory,enhance the stability of atherosclerotic plaques and reduce myocardial hypertrophy and remodeling.But the pharmacological action of statins on myocardial hypertrophy is so complex,which has not been explained satisfactorily.Even there are some controversy about the effect of statin on mitochondrion.And the effect of statins on myocardial cell metabolism and mitochondrial related regulatory proteins is also one of the research hotspots recently.The CCAAT/enhancer-binding protein β is a member of C/EBP transcription factors family,the main function of which is to regulate the transcription of cell proliferation and differentiation genes.Previous study also reports that C/EBPβ is significantly downregulated with exercise stimulation,and it plays an important role in the physiologic hypertrophy and proliferation.Although recently it is found that C/EBPβ has significant regulation function in pathological cardiac hypertrophy,and knockdown of C/EBPβ can attenuate PE-induced hypertrophic responses.Relatively,studies about the function of C/EBPβ in pathological cardiac hypertrophy are scarce,and the specific regulation mechanism need more studies to elucidate.In addition,as an organ with high oxygen consumption and high energy consumption,the normal energy metabolism is very important for its normal physiological function.While the myocardial hypertrophy or heart failure happens,the mitochondrial energy metabolism function is also beginning to change or be damaged.But whether atorvastatin prevents myocardial hypertrophy and reverses mitochondrial metabolic function via C/EBPβ in cardiomyocyte has not been reported before.The present study aimed to investigate the anti-hypertrophic and anti-apoptosis effect of atorvastatin using cultured H9c2 cells and spontaneously hypertensive rats(SHR),and tried to explore whether or not the underlying mechanism was due to the inhibition of C/EBPβ activation.Possibly our results provided a new perspective on understanding of the cardioprotective effect of atorvastatin.Methods: Part I: The H9c2 rat ventricular cells were cultured with fresh medium containing various concentrations(0,0.01,0.1,1 or 10 μmol/L)of Angiotensin II and various concentrations(1,5,10 or 50 μmol/L)of atorvastatin for 24 hours.Cell viability was evaluated with the MTS assay to determine the optimum concentration of intervene.Rhodamine-phalloidin was employed to visualize actin fragment and show the cell morphologic changes in this assay.For C/EBPβ overexpression,H9c2 cardiomyocytes were transiently transfected with flag-C/EBPβ plasmid.The early and late apoptosis rate of H9c2 was evaluated by flow cytometry using a commercial apoptosis detection kit.Western blot analysis was used to detect the protein expression of marker protein of pathological cardiac hypertrophy(ANP and BNP),C/EBPβ、PGC-1α、apoptosis related protein(Bcl-2 and Bax).Immunofluorescence analysis was used to show the expression of PGC-1α in cardiomyocytes cytoplasm.Rhodamine 123 was used in present study to detect changes in mitochondrial membrane potential,and the fluorescence intensity was analyzed with microplate reader.Part II: Effect of atorvastatin on myocardial hypertrophy in spontaneously hypertensive rats(SHR).Animal experiment: The male SHR rats and age-matched male Wistar-Kyoto(WKY)rats were used in this study at 11 weeks of age with body weight 254.5 ± 5.6 g and 248.8 ± 5.4 g respectively.The male and age-matched animals were randomly divided into three groups: control group(8 WKY rats),SHR(8 rats)and intervention group(8 SHR).The SHR in intervention group were administered by oral gavage with atorvastatin(suspension in distilled water,10 mg/Kg once a day)for 6 weeks,and the other two groups were administered by gavage with equal quantity distilled water.The body weight of each rat was weighed once a week.Systolic blood pressure,diastolic blood pressure and heart rate of rats were measured every weeks using a standard tail cuff sphygmomanometer.Left ventricular(LV)dimensions and heart function were measured under M-mode tracings using Doppler echocardiograph at the age of 16 weeks.After observation of the general appearance of the isolated heart,atrium and right ventricular free wall were cut along the atrioventricular ring.Then the remaining septal and left ventricular free wall were dried with filter paper which were weighed as left ventricular mass.And LVM/BW ratio was calculated.Paraffin-embedded slides were stained with Masson’s trichrome for ventricular remodeling and interstitial fibrosis determination.Western blot analysis was used to detect the protein expression of marker protein of pathological cardiac hypertrophy(ANP and BNP),C/EBPβ、PGC-1α、UCP3、apoptosis related protein(Bcl-2 and Bax).Immunohistochemistry was used to detected the expression of C/EBPβ and UCP3 in myocardial tissue slides of rats.Cardiomyocyte apoptosis in myocardial tissue slides of rats was assessed by the TUNEL assay.Results: Part I: 1.According to the viability by MTS assay and the expression of a marker protein of pathological cardiac hypertrophy(BNP),we selected the 1 μmol/L concentration of Ang II to induce the hypertrophy of H9c2 cardiomyocytes,and 10 μmol/L atorvastatin to intervene in subsequent experiments.2.Rhodamine-phalloidin staining and Western blot showed that the cells surface area,as well as the protein expression levels of ANP,BNP,C/EBPβ were significantly increased by Ang II.Western blot analysis also showed that Ang II could promote the expression of pro-apoptosis proteins(Bax),and inhibit the expression of anti-apoptotic proteins(Bcl-2).However,after treatment with atorvastatin at concentration of 10 μmol/L for 24 hours,the cells surface area,the protein expression levels of ANP,BNP,and C/EBPβ were significantly decreased.And the decreasing Bcl-2/Bax ratio was inhibited.3.Atorvastatin effectively reduce the early and late apoptosis rate of H9c2 cardiomyocytes induced by Ang II,reverse the decreasing mitochondrial membrane potential,and increase the expression of PGC-1α that is an important regulator of mitochondrial biogenesis and energy metabolism.4.For overexpression of C/EBPβ,H9c2 cardiomyocytes were transfected with flag-C/EBPβ plasmid.Compared with the negative control group,H9c2 cardiomyocytes with overexpression of C/EBPβ showed some characteristics of myocardial hypertrophy,such as the increasing protein expression of ANP,BNP,and C/EBPβ,apoptosis rate increasing,Bcl-2/Bax ratio,MMP and protein expression of PGC-1α decreasing.Furthermore,the protective effect of atorvastatin on myocardial hypertrophy was weakened by transfected exogenous C/EBPβ.Part II: 1.At the age of 16 weeks,the mean arterial pressure(MAP)of the rats in the three groups was 103.6 ± 6.1,151.8 ± 12.5,and 159.1 ± 6.2 mmHg,respectively.There was no statistically significant difference between the SHR and intervention groups.2.An echocardiographic examination of each rat was performed at 16 weeks,and the LV wall thickness including the IVSd and PWTd in water-treated SHR were the highest,which exhibited myocardial hypertrophy.In comparison with rats in the control group and atorvastatin-treated SHR,the LVM/BW was significantly higher without difference of heart function in water-treated SHR.Furthermore,staining with Masson’s trichrome demonstrated that the interstitial fibrosis of the LV and ventricular remodeling in the water-treated SHR group were most serious.3.After treatment with atorvastatin for 6 weeks,the fibrosis and remodeling of LV was attenuated.And the IVSd and PWTd were also reduced in atorvastatin-treated SHR.4.Western blot exhibited that the protein expression of ANP,BNP,and C/EBPβ increased in water-treated SHR group.From the immunohistochemical analysis,the expression of C/EBPβ increased in the myocardium of SHR,in which the gray color of not only the cytoplasm but also the nucleus was darker in comparison with WKY rats.After treatment with 10mg/kg atorvastatin for 6 weeks,the protein expression of ANP,BNP,and C/EBPβ were reduced significantly.5.By the TUNEL assay,in comparison with WKY rats the percentage of TUNEL-positive cells was higher in SHR,which was accompanied by a decrease in the ratio of the expression of the apoptosis-related protein Bcl-2 to that of Bax.Treatment with atorvastatin for 6 weeks reversed the decrease in the Bcl-2/Bax ratio in SHR and reduced the percentage of TUNEL-positive cells.6.The immunohistochemical analysis showed that the expression of UCP3 in water-treated SHR was significantly reduced,with a decrease in mean IOD.Equally,the protein expression levels of PGC-1α and UCP3 were also shown by the western blot analysis to be lower in water-treated SHR than in the control group.However,the decreases in the expression of PGC-1α and UCP3 could be inhibited by treatment with atorvastatin in SHR.Conclusions: Through the experiments in vitro and in vivo,we further identified the protective effect of atorvastatin on hypertrophic H9c2 cardiomyocytes induced by Ang II and myocardial hypertrophy of SHR induced by hypertension,including inhibiting myocardial hypertrophy and remodeling,rescuing the MMP,reversing the mitochondrial biogenesis and energy metabolism associated factors,and inhibiting apoptosis.And the high expression of transcription factor C/EBPb in pathological cardiac hypertrophy can increase the apoptosis through impairing mitochondrion metabolism function and regulating mitochondrion associated factors.The beneficial effect of atorvastatin inhibiting cardiac hypertrophy and apoptosis might be at least partly attributed to down-regulation of C/EBPb.Thus,the C/EBPβ/PGC-1α/UCP3 signaling pathway might be a novel interpretation for pleiotropic effects of atorvastatin.And C/EBPb might be a new target to rescue mitochondrion function and apoptosis in pathological cardiac hypertrophy.