The Effect And Mechanism Of STEAP3 On Cardiac Hypertrophy

According to the World Health Organization,approximately 18 million people worldwide die of cardiovascular disease each year,and this number is expected to climb to 23 million by 2030.Cardiovascular disease has become a major public health problem endangering human health.At present,with the improvement of social living standards and the development of aging,there are as many as 290 million patients with cardiovascular disease in our country.This common chronic non-communicable disease has become a major enemy of the health of middle-aged and even young people.Cardiac hypertrophy is an independent risk factor for cardiovascular disease,and its pathological processes mainly include myocardial hypertrophy,apoptosis and fibrosis,which are activated and inhibited by complex signal pathways.At present,although ?-blockers,angiotensin-converting enzyme inhibitors and other drugs that improve pathological cardiac hypertrophy and ventricular remodeling have been widely used,heart failure the incidence of the end point of cardiac hypertrophy is still high.Therefore,intensive study on the molecular mechanism and development of cardiac hypertrophy could provide new therapeutic targets for clinically antagonizing pathological cardiac remodeling and heart failure.Six-transmembrane epithelial antigen of prostate 3(STEAP3),as a metal reductase,plays an important role in maintaining iron homeostasis in human and STEAP3 is involved in regulating important biological events such as cell proliferation,apoptosis,inflammation,and fibrosis.The signaling pathway and iron homeostasis regulated by STEAP3 in cells are closely related to the mechanism of cardiac hypertrophy.However,there is no report on the role and mechanism of STEAP3 in cardiac hypertrophy.Therefore,this study explores the effects of STEAP3 on cardiac hypertrophy from the perspective of animals,cells,and transcriptomics.And we also studied the molecular mechanism of STEAP3 on cardiac hypertrophy systematically through transcriptomics analysis,signal pathway analysis,and protein interaction screening.(1)Transverse aortic constriction(TAC)surgery was used to construct a mouse pathological cardiac hypertrophy model,and phenylephrine(PE)was used to stimulate the neonatal rat cardiomyocytes(NRCM)to establish an in vitro myocardial hypertrophy model.The result shows that STEAP3 expression was down-regulated in TAC-induced mice and PE-stimulated NRCM compared with the sham control group.The results suggest that STEAP3 may be involved in the development of pathological cardiac hypertrophy.(2)We cultured NRCM in vitro and infected them with recombinant adenovirus Adsh STEAP3.The significant reduction of STEAP3 expression in NRCM indicates successful transfection of adenovirus.Then PE stimulation was used to construct a model of cardiomyocyte hypertrophy.Immunofluorescence staining and cardiomyocytes surface area statistics showed that compared with the control group,STEAP3 knockdown significantly increased cardiomyocytes,and the mRNA levels of cardiac hypertrophy markers ANP and Myh7 were significantly different.The results suggest that STEAP3 can directly inhibit cardiac hypertrophy through regulating cardiomyocytes induced by PE.(3)We established STEAP3 knockout mice(STEAP3-KO)through TAC surgery,a model of cardiac hypertrophy induced by pressure overload.Through cardiac function analysis and histomorphology analysis,we found that compared with WT-TAC mice,STEAP3-KO-TAC mice showed significant cardiac dilatation,decreased myocardial contractility,worsened cardiac function,and increased cardiac fibrosis.While compared with mice in the WT-Sham group,the cardiac function and tissue morphology of the STEAP3-KO-Sham group did not change significantly,suggesting that STEAP3 knockout did not cause pathological changes of the heart,but aggravates the degree of cardiac hypertrophy and myocardial fibrosis during pressure overload-induced cardiac hypertrophy.(4)Cardiac hypertrophy was induced in STEAP3-KO mice by TAC surgery.Transcriptomics analysis of their cardiac tissues showed that 394 genes were up-regulated and 363 genes were down-regulated in mouse hearts from STEAP3-KO group compared with the wild-type(WT)group.Hierarchical cluster analysis and q PCR verification found that STEAP3 knockout caused significant transcriptional reprogramming.After STEAP3 knockout,cardiac hypertrophy and cardiac fibrosis-related genes were significantly up-regulated.Cardiac hypertrophy markers ANP,BNP,Myh7 and the mRNA levels of fibrosis markers including Col1a1,Col3a1,and Ctgf were significantly up-regulated.Furthermore,the expression of protein synthesis-related genes in heart increased after STEAP3 knockout.These findings suggested that STEAP3 gene knockout promotes the expression of cardiac hypertrophy and fibrosis-related genes and protein synthesis.(5)In order to clarify the downstream signaling pathways of STEAP3 regulating pathological cardiac hypertrophy,we further analyzed the enrichment of KEGG signaling pathways in cardiac transcriptome differential genes after TAC surgery in STEAP3-KO group and WT control group.In the STEAP3-KO group,the cardiac hypertrophy-related pathways such as the MAPK signaling pathway and PI3K-Akt signaling pathway were significantly changed.Western blot results showed that compared with the WT and STEAP3-KO Sham groups,the levels of phosphorylation of key kinases MEK1/2,ERK1/2,JNK1/2,and p38 in MAPK signaling pathway were significantly increased in the WT and STEAP3-KO group mice after TAC 4 weeks,suggesting that MAPK signaling pathway was activated in pathological cardiac hypertrophy.After TAC surgery,the absence of STEAP3 significantly increased the phosphorylation activation of MEK1/2 and ERK1/2without affecting the phosphorylation activation of JNK1/2 and p38.To further discuss the regulatory effect of STEAP3 on the MAPK signaling pathway in cardiomyocytes,we interfered with STEAP3 knockdown in NRCM by the adenovirus Adsh STEAP3.Then PE was used to stimulate cardiomyocytes to establish an in vitro model of cardiomyocyte hypertrophy.The results show that STEAP3 deletion increased the activation of MEK1/2and ERK1/2 after PE induction which consistent with the result in vivo.The above results suggest that STEAP3 can regulate the occurrence of cardiac hypertrophy by inhibiting the MEK1/2-ERK1/2 signaling of the MAPK signaling pathway in cardiomyocytes.(6)The STEAP3 interaction protein complex was enriched by immunoprecipitation and detected by mass spectrometry.It was found that Rac1 is a potential target of STEAP3.We have been verified by extracellular GST-pull down experiments and intracellular Co-IP experiments.Laser scanning confocal microscopy showed that STEAP3 and Rac1 were mainly co-localized in the cytoplasm.These observations indicate that STEAP3 can directly interact with Rac1 in the cytoplasm.Co-IP experiments also suggest that most regions of Rac1 including the C-terminal region of Rac1(Rac1?162-192aa)and the insert region of Rac1(Rac1?124-135aa)interacted with STEAP3.(7)Inhibition of the activation of Rac1 by adding Rac1 inhibitor NGC23766 to the STEAP3 knockdown NRCM blocked PE-triggered activation of MEK1/2 and ERK1/2 but also reversed the knockdown of STEAP3 on MEK1/2-ERK1/2 activation.Furthermore,the constitutively active mutant Rac1(G12V)significantly activated MEK1/2 and ERK1/2 and greatly reduced the inhibitory effect of overexpression STEAP3 on MEK1/2-ERK1/2activation in NRCM.Immunofluorescence showed that the protective effect of STEAP3 on cardiac hypertrophy was significantly inhibited by Rac1(G12V)overexpression,Rac1(G12V)also significantly increased the mRNA levels of cardiac hypertrophy markers ANP and Myh7,whereas blocking STEAP3 expression inhibited these hypertrophy marker genes.Theses observations demonstrate that the inhibitory effect of STEAP3 on the activation of MEK/ERK signaling pathway mainly depends on Rac1.In summary,these study confirmed that STEAP3 deficiency promoted the development of cardiac hypertrophy through in vivo and in vitro experiments and transcriptomics analysis.Further analysis revealed that STEAP3 regulated cardiac hypertrophy through the MAPK signal pathway.Through protein interaction analysis and molecular biology verification,we found that STEAP3 inhibited cardiac hypertrophy and cardiac hypertrophy by directly regulating Rac1 and inhibiting the MEK1/2-ERK1/2 signaling pathway.This study broadens our understanding of the molecular mechanisms by which STEAP3 affects cardiac hypertrophy,and suggests that targeting STEAP3 may be an effective treatment strategy for pathological cardiac hypertrophy.