Cardiac fibrosis is a common pathway to heart injury and failure,where continued activation of cardiac fibroblasts(CFs)during myocardium damage causes excessive deposition of the extracellular matrix and thus increase...Cardiac fibrosis is a common pathway to heart injury and failure,where continued activation of cardiac fibroblasts(CFs)during myocardium damage causes excessive deposition of the extracellular matrix and thus increases matrix stiffness.Increasing evidence has shown that stiff matrix plays an important positive role in promoting CF differentiation and cardiac fibrosis,with several signaling factors medicating CF mechanotransduction already identified.However,key moleculesthat perceive matrix stiffness to regulate CF differentiation remain to be fully defined.Recently,Hippo pathway transcriptional coactivators,i.e.,Yes-associated protein(YAP)and transcriptional coactivator with PDZ-binding motif(TAZ),have been found to work as mechanical signal transductors.Importantly,it has shown that YAP plays important roles in various types of fibrosis.Despite these findings,the role of YAP in CF mechanotransduction and cardiac fibrosis still remains elusive.Moreover,several several types of GPCRs have also been found to enable cells to sense mechanical cues,however,the relationship between these GPCRs and YAP in cell mechanotransduction is still not clear.Our recent work demonstrated that blocking of angiotensin II type 1 receptor(AT1R,the first GPCRs found to be mechanosensors)with losartan significantly inhibited the differentiation of CFs to myofibroblasts induced by stiff substrate.Taken these findings into account,we speculate that YAP may work as an important downstream signaling molecule of AT1R in mediating matrix stiffness-induced CF differentiation.In this work,we first characterized the expression of YAP in normal control(NC)and myocardial infarct(Ml)tissues of rats by using immunohistochemistry,immunofluorescence and Western blot analysis.We then investigated the role of YAP in matrix stiffness-induced CF differentiation in vitro by culturing CFs on mechanically tunable gelatin hydrogels.Finally,we explored the relationship between YAP and AT1R in CF mechanotransduction by selective transfection and inhibition experiments.The expression of YAP andα-SMA in cultured CFs were evaluated with immunofluorescence staining,Western blot and real-time quantitative PCR analysis.Immunohistochemical analysis revealed that both YAP andα-SMA significantly increased in Ml tissue compared with NC tissue.The expression and nuclear localization of YAP increased in CFs cultured on stiff matrix.YAP-deficient CFs cultured on soft and stiff matrix both showed decreased expression ofα-SMA.Meanwhile,YAP-overexpressing CFs cultured on soft and stiff matrix both showed increased expression ofα-SMA.Blocking of AT1R decreased the expression levels ofα-SMA and YAP and thus affected the responses of CFs to matrix stiffness.To sum up,our results identified an important role of YAP in mediating matrix stiffness-induced CF differentiation and also established the YAP pathway as an important signaling branch downstream of AT1R in CF mechanotransduction.This study may help to better understand the mechanism of fibrotic mechanotransduction and inspire the development of new approaches for treating cardiac fibrosis.展开更多
基金financially supported by the National Natural Science Foundation of China ( 11872298, 11602191,11532009)the China Postdoctoral Science Foundation ( 2018M631141)+1 种基金the Natural Science Basic Research Plan in Shaanxi Province of China ( 2017JM1026)the Shaanxi Postdoctoral Science Foundation,and the Fundamental Research Funds for the Central Universities ( Z201811336)
文摘Cardiac fibrosis is a common pathway to heart injury and failure,where continued activation of cardiac fibroblasts(CFs)during myocardium damage causes excessive deposition of the extracellular matrix and thus increases matrix stiffness.Increasing evidence has shown that stiff matrix plays an important positive role in promoting CF differentiation and cardiac fibrosis,with several signaling factors medicating CF mechanotransduction already identified.However,key moleculesthat perceive matrix stiffness to regulate CF differentiation remain to be fully defined.Recently,Hippo pathway transcriptional coactivators,i.e.,Yes-associated protein(YAP)and transcriptional coactivator with PDZ-binding motif(TAZ),have been found to work as mechanical signal transductors.Importantly,it has shown that YAP plays important roles in various types of fibrosis.Despite these findings,the role of YAP in CF mechanotransduction and cardiac fibrosis still remains elusive.Moreover,several several types of GPCRs have also been found to enable cells to sense mechanical cues,however,the relationship between these GPCRs and YAP in cell mechanotransduction is still not clear.Our recent work demonstrated that blocking of angiotensin II type 1 receptor(AT1R,the first GPCRs found to be mechanosensors)with losartan significantly inhibited the differentiation of CFs to myofibroblasts induced by stiff substrate.Taken these findings into account,we speculate that YAP may work as an important downstream signaling molecule of AT1R in mediating matrix stiffness-induced CF differentiation.In this work,we first characterized the expression of YAP in normal control(NC)and myocardial infarct(Ml)tissues of rats by using immunohistochemistry,immunofluorescence and Western blot analysis.We then investigated the role of YAP in matrix stiffness-induced CF differentiation in vitro by culturing CFs on mechanically tunable gelatin hydrogels.Finally,we explored the relationship between YAP and AT1R in CF mechanotransduction by selective transfection and inhibition experiments.The expression of YAP andα-SMA in cultured CFs were evaluated with immunofluorescence staining,Western blot and real-time quantitative PCR analysis.Immunohistochemical analysis revealed that both YAP andα-SMA significantly increased in Ml tissue compared with NC tissue.The expression and nuclear localization of YAP increased in CFs cultured on stiff matrix.YAP-deficient CFs cultured on soft and stiff matrix both showed decreased expression ofα-SMA.Meanwhile,YAP-overexpressing CFs cultured on soft and stiff matrix both showed increased expression ofα-SMA.Blocking of AT1R decreased the expression levels ofα-SMA and YAP and thus affected the responses of CFs to matrix stiffness.To sum up,our results identified an important role of YAP in mediating matrix stiffness-induced CF differentiation and also established the YAP pathway as an important signaling branch downstream of AT1R in CF mechanotransduction.This study may help to better understand the mechanism of fibrotic mechanotransduction and inspire the development of new approaches for treating cardiac fibrosis.
