The Role And Mechanism Of Retinol-binding Protein 4 In Pathological Cardiac Hypertrophy In Mouse Cardiomyocytes

Background:With the aging of our population,the prevalence of cardiovascular diseases is increasing rapidly.Heart failure has been becoming the leading cause of mortality and morbidity for cardiovascular diseases.Insulin resistance plays a major role in pathological cardiac hypertrophy,which is a fundamental determinant of heart failure.Heart failure in turn promotes insulin resistance and increases the risk for diabetes.The vicious cycle determines significant mortality in patients with heart failure.Retinol-binding protein 4(RBP4)is an adipokine that contributes to insulin resistance.Emerging evidences have also linked elevated RBP4 to cardiovascular diseases;however,the exact role of RBP4 in the pathological cardiac hypertrophy remains unclear and needs to be determined.Objective:In the present study,we aimed to investigate the effect and underlying mechanisms of RBP4 in pathological cardiac hypertrophy,as well as the role of RBP4 in the vicious cycle of insulin resistance and pathological cardiac hypertrophy.Methods and Results:Pathological cardiac hypertrophy was induced by transverse aortic constriction(TAC).Serum and adipose RBP4 levels were detected by enzyme linked immunosorbent assay(ELISA)and quantitative real-time PCR(qRT-PCR),respectively.Our data showed that serum and white adipose tissue(WAT)RBP4 levels were increased in mice with pathological cardac hypertrophy,and were positively correlated with the extent of cardiac hypertrophy.Angiotensin-?(Ang-?)levels were also elevated in TAC model mice,and were positively correlated with serum RBP4 levels.The causative role of Ang-? in increasing RBP4 levels was further investigated in vivo and in vitro.In mice treated with Ang-II infusion using osmotic pumps,serum and WAT RBP4 levels were also increased.qRT-PCR and western blot showed that incubating primary white adipocytes with Ang-II stimulated RBP4 mRNA and protein expression,and the effects were abolished by Losartan,a specific Ang-II receptor antagonist.To determine whether RBP4 may have direct effects on cardiomyocytes to induce pathological hypertrophy,primary cardiomyocytes were treated with different doses of recombinant RBP4(25,50,100?g/ml).Three methods including cell surface measurements by immnofluorescence,protein to DNA ratio and hypertrophic gene markers were used to assess cardiomyocyte hypertrophy.The results showed that RBP4 stimulation dose-dependently increased cell size,protein/DNA ratio,and hypertrophic gene markers including atrial natriuretic peptide(Anp),brain natriuretic peptide(Bnp),myosin,heavy chain 7(Myh7)in cardiomyocytes.Mechanistically,qRT-PCR and ELISA showed that RBP4 stumulation dose-dependently induced the mRNA expression and scerection of pro-inflammatory cytokines,including tumor necrosis factor-a(TNF-?),interleukin-6(IL-6),monocyte chemotactic protein 1(MCP-1),and IL-1?,as well as the expression of genes involved in oxidative stress,including NADPH oxidase 2(Nox2),Nox4),nuclear factor E2-like 2(Nrf2),NADPH dehydrogenase,quinone 1(Nqol),and heme oxygenase 1(Hol).Consistently,intracellular reactive oxygen species(ROS)production was elevated in RBP4-treated cardiomyocytes by using DCFH-DA as a fluorescent probe.The elevated levels of both ROS gene markers and production were attenuated by antioxidant N-acetylcysteine(NAC).Moreover,NAC pretreatment mitigated RBP4-induced cytokine expression and hypertrophic responses in cardiomyocytes.Further experiments showed that RBP4 treatment increased the expression of Toll-like receptor 4(Tlr4)and myeloid differentiation primary response gene 88(Myd88)in cardiomyocytes.TLR4 inhibitor TAK242 abolished RBP4-induced expression of pro-inflammatory cytokine Tnf-a,11-6,Mcp-1,and Il-1? as well as hypertrophic markers Anp,Bnp and Myh7.In cardiomyocytes isolated from Tlr4 and Myd88 knockout mice.RBP4-stimulated cytokine expression and secretion,as well as ROS production were significantly attenuated.Importantly,the increased cell size,protein/DNA ratio and hypertrophic markers induced by RBP4 were mitigated in cardiomyocytes isolated from Tlr4 and Myd88 knockout mice compared with those from wild type mice.To further investigate the role of RBP4 in insulin resistance during pathological cardiac hypertrophy,blood glucose and insulin levels were measured.Serum RBP4 levels were positively correlated with HOMA-IR(homeostatic model of insulin resistance),an index of insulin resistance.2-NBDG based analysis of glucose uptake showed that RBP4 stimulation significantly impaired insulin-stimulated glucose uptake in the cardiomyocytes isolated from wild type mice,but not in the cardiomyocytes from Tlr4 knockout mice.Consistently,RBP4 decreased the mRNA and protein levels of GLUT4 in cardiomyocytes.Remarkably,Tlr4 knockdown normalized the GLUT4 expression in RBP4 treated cardiomyocytes.Conclusion:Our data showed that RBP4 is an adipokine that contributes to both insulin resistance and pathological cardiac hypertrophy.The effects are mediated by inducing cardiac inflammation through activating TLR4/MyD88 pathway.Therefore,RBP4 may be a central component in the vicious cycle of insulin resistance and heart failure.Lowering RBP4 might break the vicious cycle and benefit both insulin sensitivity and heart function.