The Role Of Voltage-dependent Anion Channel 1 In Cardiac Fibrosis Induced By Pressure Overload

Background:Cardiac fibrosis is a common pathological process,happened in acute or chronic stimuli such as myocardial infarction,hypertension,and neurohumoral factor activation.The main manifestations of fibrotic hearts are cardiomyocyte hypertrophy and cardiac fibroblast phenotypic switching.Excessive proliferation and activation of cardiac fibroblasts lead to a deposition of extracellular matrix,increasing the stiffness of cardiac tissue matrix,destroying the structure of myocardium,and dysregulation the contraction of myocardium.Eventually the deposited ECM damages the systolic and diastolic function of heart.The activation of cardiac fibroblasts not only reflects the situation of myocardial cell injury,but also is directly involved in the pathogenesis of myocardial dysfunction.Therefore,a deep understanding of the pathophysiological mechanism of cardiac fibroblast activation from a new perspective will facilitate the exploration of effective therapeutic strategies for myocardial fibroblasts activation and cardiac fibrosis,and is also potentially important for clinical practice.In recent years,metabolic remodeling has received much attention from researchers as an emerging important pathogenic process of fibrosis in a variety of organs.Dysregulation of fatty acid metabolism leads to accumulation of lipids,and lipotoxicity,which can also cause impaired denaturation of ECM.Imbalances in fatty acid oxidation causes cell apoptosis,dedifferentiation,and fibrosis.Mitochondria-bound endoplasmic reticulum membrane（MAM）is a tightly bound domain between the endoplasmic reticulum（ER）and mitochondria that plays an important role in maintaining lipid homeostasis.As one of the most abundant proteins on MAM,VDAC1 plays a critical role in mediating Ca²⁺signaling,reactive oxygen species（ROS）generation,and ER stress.Studies have shown that in liver fibrosis,intervention of the expression of VDAC1 affects the levels of enzymes related to fatty acid synthesis and metabolism,thereby significantly alleviating liver fibrosis,suggesting that VDAC1 may have a regulatory role in fatty acid oxidation during fibrosis.However,the role of VDAC1 in cardiac fibrosis as well as cardiac fibroblast activation,has not yet been reported.Therefore,this study was designed to explore the expression of VDAC1 in cardiac fibrosis and activation of cardiac fibroblasts,as well as the levels of key enzymes in fatty acid oxidation related pathways during this process,and further explore how VDAC1 regulates cardiac fibroblasts activation,as well as its roles in altering fatty acid oxidation related pathways.Objective:1.To observe the expression of VDAC1 in cardiac fibrosis in vivo and cardiac fibroblasts activation in vitro.2.To clarify the role of VDAC1 in the activation of cardiac fibroblasts and the effect of exogenous supplementation of VDAC1 on cardiac fibrosis.3.To explore the effect of intervention of VDAC1 on fatty acid metabolism and its related enzymes,and whether VDAC1 is involved in the process of regulating cardiac fibrosis and cardiac fibroblast activation by regulating fatty acid metabolism.4.To assess the clinical translational potential of VDAC1 as a therapeutic target for cardiac fibrosis.Materials and Methods:1.Animal experiments:A mouse model of pressure overload-induced fibrosis was established,and the expression of VDAC1 and the levels of fatty acid metabolism-related proteins were detected.（1）Cardiac fibrosis model of mice was established by transthoracic aortic constriction（TAC）.Echocardiography was performed at 4 weeks after operation to assess the changes of cardiac structure and cardiac function.HE,Masson and Sirius red staining were used to observe myocardial hypertrophy and collagen deposition.RT-q PCR and Western-blot were used to detect the expression of fibrotic markers and myocardial hypertrophy makers in the heart tissue of mice.（2）In cardiac fibrosis mice model,the level of VDAC1 in fibrosis heart tissues was detected by RT-q PCR,Western-blot and immunohistochemical staining.Meanwhile,the levels of proteins related to fatty acid metabolism in fibrosis heart tissues of mice were detected.（3）To construct an adeno-associated virus（AAV）specifically overexpressing VDAC1 in activated cardiac fibroblasts,and the TAC mice were administrated with this AAV via tail vein.Echocardiography was performed at 4 weeks after operation to assess the changes of cardiac structure and cardiac function in mice.Myocardial hypertrophy and collagen deposition were observed by HE,Masson and Sirius red staining.The therapeutic effect of specific overexpression of VDAC1 in cardiac fibroblasts on cardiac fibrosis was evaluated by detecting the expression of fibrotic markers in heart tissue by RT-q PCR and Western-blot.（4）The VDAC1-based peptide R-Tf-D-LP4 was synthesized,and administrated in TAC model of mice by tail vein every three days（10 mg/kg）.Echocardiography was performed at 4 weeks after operation to assess the changes of cardiac structure and cardiac function in mice.Myocardial cell hypertrophy and collagen deposition were observed by WGA,Masson and Sirius red staining.The therapeutic effect of peptides on cardiac fibrosis was evaluated by detecting the changes of myocardial hypertrophy and fibrotic markers in myocardial tissue by RT-q PCR and Western-blot.The expression of VDAC1 in heart tissue were observed by immunohistochemical staining.The levels of proteins related to fatty acid metabolism in cardiac tissue of mice with fibrosis were detected by RT-q PCR assay.2.In vitro experiments:induction of myocardial fibroblast activation,and detection of expression of VDAC1 expression and level of the fatty acid metabolism-related proteins.（1）Cardiac fibroblast activation was induced:Rat neonatal cardiac fibroblasts were isolated and cultured in vitro and treated with transforming growth factor-β1（TGF-β1,10 ng/m L）for 24 h to induce activation.The expression of fibrotic markers were detected by RT-q PCR and Western-blot.The fluorescence intensity ofα-SMA were observed by confocal laser scanning microscopy（Confocal）.The cell proliferation rate was measured by Ed U incorporation assay,the cell migration rate was measured by scratch assay,and the capacity contraction was measured by collagen gel contraction assay.These detection aims to identify the activation phenotype of cardiac fibroblasts.（2）To determine the role of VDAC1 in the activation of cardiac fibroblasts,the expression of endogenous VDAC1 in cardiac fibroblasts was silenced with specific siRNA（100 n M）,or VDAC1 was overexpressed with adenoviral vector（MOI=100）,followed by TGF-β1 stimulation of cardiac fibroblasts for 24 h.The effect of inhibiting or overexpressing VDAC1 on the activation of cardiac fibroblasts was observed.（3）To explore the mechanistic pathway by which VDAC1 regulates the activation process of cardiac fibroblasts:Quantitative mass spectrometry（LC-MS/MS）was used to detect the changes of various intracellular fatty acids during myocardial fibroblast activation,and electron microscopy was used to observe the changes in the number of lysosomes around the mitochondria of the cells.Flow cytometry and RT-q PCR were used to detect the levels of fatty acid metabolism-related proteins during cardiac fibroblast activation.Confocal was used to observe the level of fatty acidβ-oxidation（FAOBlue fluorescence intensity）during cardiac fibroblast activation,and the co-location of VDAC1 and CPT1a.（4）To determine the role of fatty acid oxidation during cardiac fibroblasts activation,after pretreatment of myocardial fibroblasts with inhibitors of CPT1a（Etomoxir,4M）and AMPK agonists（AICAR,0.25 m M）,TGF-β1 was given for stimulation for 24 h.The effect of inhibiting or activating the activity of CPT1a on myocardial fibroblast activation was observed.（5）To determine whether VDAC1 regulates cardiac fibroblast activation by affecting fatty acid oxidation-related pathways,flow cytometry and fluorescence microscopy were used to observe the expression of CD36,CPT1a,intracellular reactive oxygen species（ROS）content and fatty acidβ-oxidation levels during cardiac fibroblast activation after intervention with VDAC1.RT-q PCR was used to detect the effect of activating or inhibiting the activity of CPT1a on cardiac fibroblast activation while intervening with VDAC1.Results:1.VDAC1 expression was decreased in the heart tissue of mice in the TAC group,as well as in TGF-β1-induced activated cardiac fibroblasts.（1）Mice in the TAC group developed cardiac hypertrophy and fibrosis,and the m RNA and protein levels of VDAC1 in the heart tissue were significantly reduced compared with those in the sham group（Sham）.（2）TGF-β1 induced a significant decrease in the m RNA and protein levels of VDAC1 in activated cardiac fibroblasts compared to the control.2.Specific silencing of VDAC1 expression promotes the activation of cardiac fibroblasts,and overexpression of VDAC1 can alleviates the activation of cardiac fibroblasts.Overexpression of VDAC1 by targetting cardiac fibroblasts alleviated cardiac function impairment and cardiac fibrosis induced by pressure overload.（1）Specific silencing of the endogenous level of VDAC1 with siRNA aggravates the activation degree of cardiac fibroblasts and enhances their proliferation,migration and contractility on the basis of TGF-β1 stimulation.（2）Specific overexpression of VDAC1 with adenoviral vectors reduces the activation of cardiac fibroblasts and attenuates their proliferation,migration and contractility on the basis of TGF-β1 stimulation.（3）In vivo,overexpression of VDAC1 with adeno-associated virus administered with a specific promoter of activated cardiac fibroblasts was able to improve cardiac fibrosis and the impaired cardiac function induced by pressure overload in mice.Compared with the TAC group,the cardiac function of mice in AAV group was significantly improved,the LVIDd and LVPWd were reduced,and the IVSd was increased after specific overexpression of myocardial fibroblasts VDAC1.Masson staining showed reduced collagen fiber deposition.3.VDAC1 regulates the process of cardiac fibroblast activation through CPT1a-mediated fatty acid metabolism.（1）During the activation of cardiac fibroblasts,a variety of medium and long chain fatty acids accumulate in the cytoplasm,and the expression of fatty acid transport,synthesis and metabolism-related enzymes is dysregulated.A.The content of a variety of higher fatty acids was increased in TGF-β1-induced activated cardiac fibroblasts compared with untreated controls,and pathway enrichment analysis showed that the cardiac fibroblast activation process involved the intracellular transport of fatty acids.B.In TGF-β1-induced activated cardiac fibroblasts,the number of lysosomes around mitochondria was reduced（observed by transmission electron microscopy）,the fluorescence intensity of FAOBlue was decreased,the expression of enzymes related to fatty acid transport and metabolic pathways（CD36,CPT1a,PGC1,PPAR-,PPAR-）was decreased,and the expression of enzymes related to fatty acid synthesis pathways（ACC1,ACC2,FASN）was increased.（2）Specific overexpression of VDAC1 in cardiac fibroblasts can up-regulate the expression of CD36 and CPT1a,which are key fatty acid transport enzymes,increase the level of fatty acid oxidation,and reduce intracytoplasmic ROS accumulation;while silencing the expression of VDAC1 can down-regulate the expression of CD36,inhibit fatty acid oxidation,and lead to intracytoplasmic ROS accumulation.（3）Inhibition of CPT1a with the specific inhibitor Etomoxir（4M）promoted the activation of cardiac fibroblasts and increased their capacity of proliferation,migration and contraction on the basis of TGF-β1 stimulation.Activation of CPT1a with AICAR（0.25 m M）can reduce the degree of activation of cardiac fibroblasts and attenuate their capacity of proliferation,migration and contraction on the basis of TGF-β1 stimulation.（4）On the basis of overexpression of VDAC1,and then inhibition of the activity of CPT1a diminished the protective effect on myocardial fibroblast activation;while on the basis of silencing VDAC1 expression,and then inhibition of the activity of CPT1a further aggravated myocardial fibroblast activation.4.In animal therapeutic experiments,supplementation of mice with TAC surgery with the exogenous VDAC1-derived peptide R-Tf-D-LP4 ameliorated impaired cardiac function and cardiac fibrosis induced by pressure overload.（1）Compared with TAC group,the diastolic diameter（LVIDd）and posterior wall thickness（LVPWd）of interventricular septum thickness（IVSd）were reduced,the score（EF）was increased,and the cardiac function injury was reduced in mice treated with peptide.（2）The degree of cardiac fibrosis was improved in mice given peptide therapy compared with the TAC group:A.Masson staining showed a decrease in myocardial collagen fiber deposition,and HE staining showed a decrease in the hypertrophic area of cardiomyocytes.B.The m RNA and protein expression of myocardial tissue fibrosis markers（CTGF,POSTN and COL-1）were decreased and the m RNA expression of CPT1a,PGC1 and PPAR-αwere increased in heart tissue.C.The results of HE staining of mouse liver,kidney,and spleen did not show significant damage.Conclusion:VDAC1 plays a protective role in pressure overload-induced cardiac fibrosis and TGF-β1-induced fibroblasts activation,and its protective role may be exerted by affecting the pathways related to CPT1a-mediated fatty acid metabolism.Exogenous supplementation of VDAC1-based peptide R-Tf-D-LP4 can improve the damage of heart function and cardiac fibrosis in mice caused by pressure overload.VDAC1 might be a potential treatment target of cardiac fibrosis.