The Regulatory Effects Of LSD1 On Pressure Overload-induced Cardiac Hypertrophy And Fibrosis And The Underlying Mechanisms

Heart failure remains a leading cause of morbidity and mortality worldwide,leading to a serious threat to human health.Pathological ventricular remodeling,including cardiac hypertrophy and fibrosis,represents a final common pathway in response to a variety of pathological stimuli.Studies have shown that epigenetic regulation,especially histone lysine methylation modification is closely related to myocardial hypertrophy and fibrosis.Histone lysine specific demethylase 1(LSD1)can specifically remove mono-or dimethylated groups of histone 3(H3)lysine 4(H3K4)and H3 lysine 9(H3K9).LSD1 is reported to be highly expressed in a wide variety of tumors,such as gastric cancer,small cell lung cancer(SCLC)and acute myeloid leukemia(AML).LSD1 has been regarded as a new target for anti-tumor therapy.Interference with the expression of LSD1 or targeted inhibition of LSD1 activity can significantly inhibit tumor growth,invasion and metastasis.Multiple LSD1 specific inhibitors,such as GSK2879552,ORY-1001 and TCP are undergoing phase Ⅰ/Ⅱ clinical trials.However,the underlying side effects of LSD1 inhibition on the cardiovascular system are currently unclear.In view of this,transverse aortic constriction(TAC)induced pressure overload was used to explore the roles of LSD 1 in myocardial hypertrophy and fibrosis,as well as in the progress of heart failure.The main work was listed as below:1.The expression of LSD1 in failing hearts and its in vitro study1.1 The expression of LSD1 in failing heartsWe firstly used immunohistochemical experiments and found that the expression of LSD 1 protein in the hearts of dilated cardiomyopathy(DCM)patients was significantly higher than that in the normal(Donor)hearts.Next,we performed TAC surgery to induce pressure overload model on C57BL/6 mice.The results showed that,compared to the Sham group,the LSD1 protein level in the failing mouse hearts was increased to 1.33-fold and its substrates H3K4me2 and H3K9me2 were reduced to 49.4%and 58.2%,respectively.In addition,the expression of LSD1 in the cardiac fibroblasts(CFs)isolated from the failing mouse hearts,was also significantly upregulated,compared to that in the sham group.These findings suggested that LSD1 may be involved in the progress of human and mouse cardiac failure,especially in the myocardial fibrosis.1.2 The effect of LSD1 knockdown or activity inhibition in vitro on the activation of neonatal rat cardiac fibroblasts induced by angiotensin Ⅱ(Ang Ⅱ)The neonatal rat cardiac fibroblasts(NRCFs)were isolated and induced to myofibroblasts by Ang Ⅱ,which showed increased protein level of collagen Ⅰ.We found that,consistent with the above findings of human failing hearts and the holistic animal research in mouse,the expression of LSD1 protein in activated NRCFS was also significantly increased.Meanwhile,we isolated neonatal rat cardiomyocytes(NRCMs)and found that the statistic difference of LSD1 expression in AngⅡ-induced hypertrophic cardiomyocytes was not observed.Then,we generated LSD1 small interfering RNA(siRNA)and found that siRNA-mediated LSD1 knockdown can significantly downregulate the protein expression of α-SMA,collagen Ⅰ and collagen Ⅲ in NRCFs induced by Ang Ⅱ.On the other hand,ORY-1001,a selective small molecule inhibitor of LSD1,was chosen.We found that,treatment by ORY-1001 significantly reversed Ang Ⅱ-induced the expression of these fibrotic markers.These findings suggested that LSDl was involved in the activated NRCFs and knockdown or activity inhibition of LSD1 in vitro can effectively reverse the activation of NRCFs induced by Ang Ⅱ.2.The regulatory effects of myofibroblast-specific LSD1 conditional knockout on the pressure overload-induced cardiac hypertrophy and fibrosis and the underlying mechanisms2.1 The generation of myofibroblast-specific LSD1 conditional knockout mouseIn vitro experiments have confirmed that LSD1 was upregulated in the activated NRCFs,and LSD1 knockdown or activity inhibition can significantly reverse the activation of NRCFs induced by Ang Ⅱ,suggesting that LSD1 may play pro-fibrotic roles.In order to further evaluate the roles of LSD1 in in vivo myocardial fibrosis,we then generated a myofibroblast-specific LSD1 conditional knockout mouse.Periostin(encoding gene Postn),is rarely expressed in the normal heart tissues,but only expressed in myofibroblasts that are activated after heart injury.Thus,the mice containing a tamoxifen inducible Cre recombinase(MerCreMer)cDNA cassette inserted into exon 1(E1)of Postn genetic locus(PostnMCM)was used in this study.Meanwhile,mice with a single Rosa26-loxp-Stop-loxp-RFP(R26RFP)reporter allele were introduced to track the myofibroblasts in the heart.The PostnMCM R26RFP mice were crossed with LSD1fl/fl mice.Offspring mice were obtained and their tail genomes were extracted.The genotying of PostnMCM R26RFP LSD1fl/fl(MF KO)mice and the control PostnMCM R26RFP(Control)mice was determined by PCR.8-week-old Control mice and MF KO mice were subjected to TAC.These mice were fed tamoxifen diet 48 h before TAC surgery or sham procedure and then maintained on tamoxifen food until harvesting.At 6 weeks after TAC,myocardial interstitial cells of Control mice and MF KO mice were isolated and part of them showed red fluorescence.Then these RFP-labeled cells(myofibroblasts)were separated through Fluorescence-activated Cell Sorting(FACS).The results of western blotting showed that the level of LSD1 in myofibroblasts of MF KO mice was significantly lower than that of Control mice,indicating that myofibroblast-specific LSD1 conditional knockout(MF KO)mice were successfully generated.2.2 The regulatory effects of myofibroblast-specific LSD1 conditional knockout on the pressure overload-induced cardiac hypertrophy and fibrosis and the underlying mechamismsThe cardiac function of Control and MF KO mice was measured by a Vevo2100 system.At 6 weeks after TAC,we found that the LV mass in Control mice was significantly increased,compared to that at baseline,whereas LV mass in MF KO mice was not significantly changed.At 20 weeks after TAC,Control mice exhibited decreased cardiac function(EF%and FS%),compared to those at baseline.MF KO mice displayed significantly improved cardiac function with EF%and FS%,compared to those in Control mice.At 6 and 20 weeks after TAC,obvious hypertrophy of the hearts from Control mice was noted.The hearts of MF KO mice were significantly smaller than those of Control mice.In addition,compared to sham,the HW/BW and HW/TL in Control mice were significantly increased,whereas MF KO mice had lower HW/BW and HW/TL than those in Control mice.20 weeks of TAC resulted in elevated LW/BW and pulmonary edema in Control mice,while the LW/BW and pulmonary edema in MF KO mice was significantly lower and lighter than those in Control mice.Meanwhile,at 6 and 20 weeks after TAC,we found that the mRNA levels of fibrotic markers(α-SMA,collagen Ⅰ and collagen Ⅲ)were significantly elevated in the Control mouse hearts,whereas MF KO mice displayed markedly decreased levels of these three markers.Masson and Sirius red staining showed that Control mice exhibited obvious evidence of fibrosis,compared to those of sham mice.However,MF KO mice presented with diminished fibrosis,compared to that in Control mice.HE staining revealed that Control mice displayed focal cytoplasmic vacuolization,a hallmark of cell injury,whereas MF KO mice exhibited relatively regular and arranged cardiomyocytes.The fluorescence intensity of fibrotic markers(α-SMA and Vimentin)in MF KO mouse hearts was distinctly lower than that in the Control mice.These data revealed that LSD1 deletion in cardiac myofibroblasts significantly reduced TAC-induced hypertrophic and fibrotic response and played protective roles in cardiac function.Next,we explored the mechanisms underlying the protective roles of myofibroblast-specific LSD1 deficiency.The analysis of RNA-seq revealed that LSD1 knockdown was closely associated with the inhibition of transforming growth factor β(TGFβ)-mediated fibrotic signaling pathways.To further verify the findings above,the results of western blotting showed that Ang Ⅱ-induced upregulation of TGFβ1 and its downstream effectors Smad2/3 phosphorylation,as well as the phosphorylation of p38,ERK1/2 and JNK was significantly reversed by LSD1 silence.Conversely,adenoviruses-induced LSD1 overexpression in isolated NRCFs upregulated the fibrotic markers(collagens Ⅰ and Ⅲ),which was associated with the increase of TGFβ1.Moreover,ELISA analysis demonstrated that siRNA-mediated LSD1 knockdown significantly blocked the secretion of TGFβ1 induced by Ang Ⅱ.Meanwhile,the indirect co-culture model revealed that LSD1 deletion in myofibroblasts can reduce the adverse effect of TGFβ1 induced by Ang Ⅱ on cardiomyocytes.Taken together,these findings indicated that the protective effects of LSD1 deficiency in myofibroblasts were mainly related to the inhibition of TGFβ and its related signaling pathway.3.The effects of cardiomyocyte-specific LSD1 conditional knockout on the cardiac function under pressure overload-induced stressed condition and the underlying mechanisms3.1 The generation of cardiomyocyte-specific LSD1 conditional knockout mouseThe findings above revealed that LSD1 participated in the progress of heart failure and played important roles in the myocardial fibrosis.To further explore the roles of LSD1 in the heart,we also generated a cardiomyocyte-specific LSD1 conditional knockout mouse.The mice containing a tamoxifen inducible Cre recombinase(CreERT2)cDNA cassette inserted into the exon of a-myosin heavy chain(Myh6)genetic locus(Myh6Cre)were crossed with LSD1fl/fl mice,which have two loxp sites.Offspring mice were obtained and their tail genomes were extracted.The genotyping of Myh6Cre LSD1fl/fl(CM KO)mice and the control Myh6Cre(Control)mice was determined by PCR.8-week-old Control mice and CM KO mice were intraperitoneally injected with tamoxifen(30 mg/kg/d)for consecutive 5 days.4 weeks later,we found a dramatical decrease of LSD1 protein in CM KO mouse hearts,compared to that in the Control mouse hearts,indicated that cardiomyocyte-specific LSD1 conditional knockout(CM KO)mice were successfully generated.3.2 The effects of cardiomyocyte-specific LSD1 conditional knockout on the cardiac function under pressure overload-induced stressed condition and the underlying mechanismsAt 4 weeks after tamoxifen treatment,the cardiac echocardiographic results showed that,compared to Control mice,CM KO mice exhibited slight myocardial hypertrophy,evidenced by the increase of left ventricular septum(IVS),left ventricular posterior wall(LVPW)and left ventricular weight(LV mass).Meanwhile,the HW/BW and HW/TL in CM KO mice were higher than those in Control mice.In addition,WGA,HE and Masson staining showed that CM KO mice exhibited mild cardiac hypertrophy and fibrosis,compared to those in Control mice.At 16 weeks after tamoxifen treatment,echocardiography and morphological study were also performed in Control and CM KO mice to further evaluate the long term effects of cardiomyocyte-specific LSD1 deficiency on cardiac function.These results showed that EF%and FS%in CM KO mice were slightly decreased compared to those in Control mice.Furthermore,HW/BW,HW/TL,cardiomyocyte cross-sectional area(CSA)and cardiac fibrosis were elevated in CM KO mice,compared to those in Control mice.These findings indicated that cardiomyocyte-specific LSD1 knockout caused mild cardiac hypertrophy,fibrosis and a slight decrease in cardiac function under basal condition.In order to further explore the effects of cardiomyocyte-specific LSD1 deficiency on the cardiac function under pressure overload-induced stressed condition,at 2 weeks after tamoxifen treatment,Control and CM KO mice were subjected to TAC surgery,respectively.We found that,4 weeks of TAC in CM KO mice resulted in increase in hypertrophic markers(IVSd,LVPWd and LV mass),compared to those in Control mice.Upon 16 weeks of TAC,the cardiac function(EF%and FS%)in CM KO mice was lower than that in Control mice.16 weeks of TAC in CM KO mice also resulted in pulmonary congestion with increase in LW/BW,while in Control mice pulmonary congestion was not observed.Furthermore,WGA,HE and Masson staining also demonstrated that TAC in CM KO mice led to larger cardiomyocytes with increase in CSA and increased cardiac fibrosis,compared to that in Control mice.These findings indicated that cardiomyocyte-specific LSD1 knockout can aggravate TAC induced cardiac hypertrophy and fibrosis.Then,we investigated the underlying mechanisms by which LSD1 deletion in cardiomyocyte induced cardiac remodeling.The data of RNA-seq showed that siRNA-mediated LSD1 knockdown in cardiomyocytes was closely involved in the cardiac muscle contraction,hypertrophic cardiomyopathy(HCM)and DCM.We further found that loss of LSD1 in NRCMs downregulated the expression of its cosuppressors REST and CoREST,concomitant with the re-programming of ANP and BNP,which leading to cardiac hypertrophy under basal and stressed conditions.In summary,we firstly generated myofibroblast-specific LSD1 conditional knockout mice and cardiomyocyte-specific LSD1 conditional knockout mice.We found that myofibroblast-specific LSD1 conditional knockout can alleviate TAC-induced cardiac hypertrophy and fibrosis,and play protective roles in cardiac remodeling.The underlying mechanism was involved in the inhibition of TGFβsignaling and its related pathways.On the other hand,cardiomyocyte-specific LSD1 knockout can induce cardiac remodeling and reduced cardiac function under both basal and stressed conditions,which may be related to the downregulation of CoREST and REST protein levels.These findings firstly suggested that LSD1 played different roles in myofibroblasts and cardiomyocytes,and these roles were worthy of further exploration,suggesting the certain significance of developing LSD1 inhibitors targeting myofibroblasts in the treatment of myocardial fibrosis.Moreover,in clinical studies of LSD 1 inhibitors,great attention should be paid to the occurrence of adverse events associated with cardiac hypertrophy and fibrosis.