The Regulatory Role Of Hippo Signalling Pathway On Isoprenaline Induced Cardiac Injury

Background Activation of the sympatho-?-adrenergic system is a hallmark of cardiac injury and heart failure.Stimulation of ?-adrenoceptors(?-AR)leads to inotropic and lusitropic actions to maintain cardiac performance.However,in the setting of heart disease,sustained stimulation of ?-AR,due to enhanced sympathetic nervous activation with elevated catecholamine levels is associated with adverse prognosis.It is well known that sustained stimulation of ?-AR leads to adverse cardiac effects notably cardiomyocyte apoptosis and cardiac fibrosis.However,the mechanisms on stimulation of ?-AR induced cardiac injury is still unclear.Recently,researchers paid more attention to the regulatory role of Hippo signalling pathway on cardiac injury and heart failure,but the mechanism is still unknown.Hippo pathway is one of the key signalling pathways on regulating cell apoptosis and proliferation.Main components of the mammalian Hippo pathway include mammalian sterile 20-like kinase 1 and 2(Mst1/2),large tumor suppressor 1 and 2(Lats1/2),and transcription co-regulator yes-associated protein(YAP)/transcriptional coactivator with PDZ-binding motif(TAZ).Stimulation of the Hippo signaling pathway could activate and phosphorylated Mst1,followed by YAP/TAZ phosphorylation,and regulate expression of a range of target genes thereby inhibiting cell proliferation and promoting apopt osis.Recent study found that inhibition of the Hippo signalling pathway is cardioprotective in myocardial infarction.However,it is not clear whether stimulation of ?-AR could induce cardiomyocyte apoptosis and cardiac fibrosis through the Hippo signalling pathway.According to a series of studies from the group of Professor Xiao-Jun Du,my co-supervisor from the Baker Heart and Diabetes Institute in Australia,the ca rdiac expression of pro-apoptotic Bcl-2 interacting mediator of cell death(BIM)and pro-fibrotic Galectin-3(Gal-3)is elevated in isoprenaline(ISO)-treated mice or in transgenic mice with cardiomyocyte-specific overexpression of ?2-AR,two mice models of cardiac injury.BIM is a BH3-only protein of the Bcl-2 family and an essential initiator of apoptosis in diverse physiological and diseased settings.BIM senses pro-apoptotic signals and activates pro-apoptotic Bcl-2 proteins(such as Bax and Bak)while inhibiting anti-apoptotic Bcl-2 proteins(such as Bcl-2 and Mcl-1).Gal-3 is a ?-galactoside-specific lectin that binds to intracellular and extracellular glycoproteins,and acts as a pro-fibrotic and pro-inflammatory mediator under diseased conditions.Previous study showed that BIM gene deletion is cardioprotective against ISO-induced cardiomyocyte apoptosis,hypertrophy and ventricular dysfunction.But whether Gal-3 is involved in ISO-induced cardiac fibrosis and dysfunction is still unclear.The mechanism responsible for the upregulated expression of both molecules after ?-AR activation in heart disease is also unknown.Professor Xiao-Jun Du's group recently observed in transgenic mice with cardiomyocyte-specific overexpression of Mst1 that the expression of Gal-3 was elevated significantly,which suggests Mst1-Hippo signalling pathway may be involved in regulating of Gal-3.In this study,our hypothesis was that cardiac ?-AR stimulation induced BIM and Gal-3 expression as well as subsequent cardiac injury through the Hippo signalling pathway.Methods 1.C57BL/6J mice and transgenic mice with cardiomyocyte-specific overexpression of ?2-AR(?2-TG)were used to examined the effect of ISO on cardiac expression of Mst1,YAP,phospho-YAP(p-YAP)and BIM by Western-Blot.Cardiac expression of Gal-3 was detected by ELISA.The therapeutic effect of ?-antagonists was also evaluated.2.Gal-3 gene knockout mice were treated with ISO.Echocardiography was performed.Myocardial fibrosis was determined by hydroxyproline assay.Cardiac expression of Gal-3 was detected by ELISA.The expression of fibrotic and inflammatory genes,as well as YAP target genes(CTGF,Ankrd1 and Birc5)was determined by RT–q PCR.3.Transgenic mice with cardiomyocyte-specific overexpression of Mst1(Mst1-TG)or dominant-negative mutant Mst1(dn Mst1-TG)were treated with ISO.Cardiac expression of YAP,p-YAP and BIM was detected by Western-Blot.The expression of cardiac Gal-3 was determined by ELISA.Bioinformatics methods were employed to analyze the expression of YAP target genes in Mst1-TG hearts,and the regulatory effect of Gal-3 on the expression of YAP target genes was also detected by RT–q PCR.4.Small interfering RNA(si RNA)-mediated knockdown of YAP was performed in H9c2 cells(rat cardiomyoblasts),and the expression of YAP,BIM and Gal-3 was detected by Western-Blot.H9c2 cells were also treated with ISO and PKA inhibitor H89 or PKI 14-22,or adenylate cyclase activator Forskolin.The expression of p-YAP,YAP,BIM and Gal-3 was also determined.Results 1.ISO induces cardiac injury through upregulating the expression of BIM and Gal-3 1)ISO treatment increased cardiac expression of BIM and Gal-3 in a time-and dose-dependent manner.2)ISO-induced BIM and Gal-3 upregulation was inhibited by treatment with the non-selective ?-antagonists,propranolol or carvedilol.The selective ?1-AR antagonist atenolol or the ?2-AR antagonist ICI-118551 effectively inhibited ISO-induced Gal-3 expression,and there was a trend towards a further reduction when combined.3)Cardiac levels of Gal-3 and BIM were significantly higher in hearts from ?2-TG mice,and the expression of Gal-3 was increased in an age-dependent manner.4)ISO treatment induced an increase in cardiac Gal-3 expression at m RNA and protein level,a 40% reduction in left ventricular fractional shortening,55% increase in the cardiac collagen content and increased expression of inflammatory or fibrotic genes.Gal-3 gene deletion was cardioprotection against ISO-induced cardiotoxic changes in left ventricular fractional shortening,collagen and expression of inflammatory or fibrotic genes.2.ISO activates cardiac Hippo signalling pathway,induces Mst1 expression and YAP phosphorylation 1)ISO treatment increased cardiac expression of Mst1,YAP and inhibitory Ser127 p-YAP.2)ISO-induced increase in YAP and p-YAP was strongly inhibited by treatment with propranolol or carvedilol.3)In hearts of ISO-treated mice,nuclear YAP(n YAP)and cytoplasmic(c YAP)were all significantly increased with the increment of c YAP more pronounced.n YAP as the ratio of c YAP was significantly lower in ISO-treated hearts.4)The expression of Mst1 and p-YAP was significantly and age-dependently upregulated in ?2-TG hearts.3.?-AR upregulates BIM and Gal-3 expression through activating Hippo signalling pathway 1)The hearts from Mst1-TG mice exhibited enhanced YAP expression and phosphorylation,which was associated with increased expression of BIM and Gal-3.A further increase of Gal-3 was observed in Mst1-TG hearts after ISO treatment.2)ISO-stimulated expression of YAP,p-YAP,BIM and Gal-3 were abolished in the dn Mst1-TG hearts.3)By using bioinformatics methods,we identified a total of 8,619 differentially expressed genes in Mst1-TG heart with 4,080 genes up-regulated and 4,539 genes down-regulated.Of the 666 YAP target genes expressed in this experiment according to publicly available Ch IP-seq data,262 were up-regulated and 179 were down-regulated.Gene set enrichment analysis shown upregulation of YAP target genes.4)YAP knockdown(si RNA transfection)significantly increase the expression of BIM and Gal-3 in H9c2 cells.5)The expression of three YAP target genes(CTGF,Ankrd1 and Birc5)was consistently upregulated in the hearts of ISO-treated mice or Mst1-TG mice.Upregulated expression of these YAP target genes,except Birc5 in the Mst1-TG background,was significantly blunted by Gal-3 gene deletion.4.ISO induces activation of Hippo pathway and expression of Gal-3 and BIM through c AMP/PKA signalling 1)PKA inhibitors H89 or PKI 14-22 abolished ISO-induced YAP phosphorylation and expression of both BIM and Gal-3 in H9c2 cells.2)Treatment of H9c2 cells with the adenylate cyclase activator Forskolin significantly increased Mst1 expression,YAP phosphorylation and expression of BIM and Gal-3.Conclusions ?-AR stimulation results in c AMP/PKA-dependent upregulation of Mst1 and hyper-phosphorylation of YAP,indicating activation of the Hippo signalling pathway.Cardiac ?-AR stimulation leads to upregulation of BIM and Gal-3 that is dependent on Mst1-activity.This study indicates the ?-AR/Hippo signalling pathway mediated upregulated expression of BIM and Gal-3 in the heart.Our results imply that targeting ?-AR/Hippo/BIM-Gal-3 signalling pathway could form a new therapeutic approach to the treatment of cardiac injury and heart failure.