| Background:Interstitial fibrosis is a hallmark of pathological cardiac remodeling,which leads to heart failure progression following myocardial infarction(MI).However,the underlying mechanisms are not fully understood and the therapies are still limited.Volume sensitive outward rectification chloride channel(VSOR Cl~-channel),also known as volume regulated anion channel(VRAC),is an important regulator of cell volume,proliferation,differentiation,and apoptosis.It is well-known that VSOR Cl~-channels are associated with multiple cardiovascular diseases,such as myocardial ischemia/reperfusion injury,cardiac arrhythmia,and cardiac hypertrophy.SWELL1,an enssential composition protein of VSOR Cl~-channel,has been recognized to regulate multiple intracellular signaling pathway as a signaling transduction molecule.It has been reported that SWELL1 participates in the regulation of the B cell development,neurotransmitter releasing,and insulin resistance.So far,it is totally unknown whether SWELL1 regulates post-MI cardiac fibrosis.Methods:(1)Adult male C57BL6J mice were subjected to Sham or MI operation.The cardiac expression levels of SWELL1 protein and mRNA in the border zone and in the remote zone were determined by Western blot and RT-PCR at different time-points after MI.Cardiac fibroblasts were isolated from Sham and MI operated mice at day 28 following the operation.The expression levels of SWELL1 in cardiac fibroblasts were determined by Western blot and RT-PCR.(2)Adenovirus carrying SWELL1 siRNA(Ad-SWELL1 siRNA)or scramble RNA(Ad-Scramble)was delivered into cardiac tissues via intra-myocardial injection.At day 7after the injection,mice were subjected to Sham or MI operation.During 28 days post-MI,the survival conditions were monitored.At day 28 post-MI,cardiac function was evaluated by echocardiography.Cardiac fibrosis was evaluated by masson trichrome staining and sirus red staining.The expression levels of?-SMA,a specific marker protein of activated myofibroblast,were determined by both Western blot and immunofluorescence staining.Cardiac collagen I,collagen III,Fibronectin,and connective tissue growth factor(CTGF)mRNA levels were determined by RT-PCR.(3)Primary cardiac fibroblasts were isolated and cultured in vitro.Ad-Scramble or Ad-SWELL1 siRNA was separately transfected into cardiac fibroblasts.The expression levels of?-SMA were determined by Western blot and immunofluorescence staining.The proliferation of cardiac fibroblasts was evaluated by CCK-8 assay.The expression levels of collagen I,collagen III,Fibronectin,and CTGF were determined by RT-PCR.The pro-fibrotic capabilities of cardiac fibroblasts were determined by collagen contraction assay.(4)Primary cardiac fibroblasts were isolated and cultured in vitro.Lentivirus overexpressing SWELL1(LV-SWELL1)or control lentivirus(LV-Control)was separately transfected into cardiac fibroblasts.The expression levels of?-SMA were determined by Western blot and immunofluorescence staining.The proliferation of cardiac fibroblasts was evaluated by CCK-8 assay.The expression levels of collagen I,collagen III,Fibronectin,and CTGF were determined by RT-PCR.The pro-fibrotic capabilities of cardiac fibroblasts were determined by collagen contraction assay.(5)Ad-Scramble and Ad-SWELL1 siRNA were transfected into cardiac fibroblasts.The total RNA was isolated and was analyzed by high throughput RNA-sequencing(RNA-Seq).Next,differentially expressed genes between two groups were functionally categorized by GO and KEGG assay.(6)The JAK2 specific inhibitor WP-1066 or the STAT3 specific inhibitor Stattic was administrated into LV-Control or LV-SWELL1 transfected cardiac fibroblasts.The protein levels of?-SMA were determined by Western blot and immunofluorescence staining.The proliferation of cardiac fibroblasts was evaluated by CCK-8 assay.The expression levels of collagen I,collagen III,Fibronectin and CTGF were determined by RT-PCR.The pro-fibrotic capabilities of cardiac fibroblasts were determined by collagen contraction assay.Results:(1)Compared with Sham group,the protein and mRNA expression levels of SWELL1 in the border zone increased at day 7 and day 14 post-MI(all P<0.05).The protein and mRNA expression levels of SWELL1 in the remote zone increased at day 14and day 28 after MI(all P<0.05).Compared with sham-operated group,the protein and mRNA levels of SWELL1 in cardiac fibroblasts isolated from MI-operated mice were upregulated(all P<0.05).(2)Intra-myocardial injection of Ad-SWELL1 siRNA significantly reduced SWELL1 protein and mRNA expression in the heart(all P<0.05).Ad-SWELL1 siRNA injection increased post-MI survival rate(76.6%vs 66.6%).SWELL1 knockdown ameliorated cardiac dysfunction after MI as indicated by higher left ventricular ejection fractions(LVEF)and lower left ventricular end-diastolic diameters(LVEDD)and left ventricular end-systolic diameters(LVESD)(all P<0.05)?Masson trichrome staining and sirus red staining showed that SWELL1 knockdown reduced interstitial fibrosis and collagen deposition following MI(P<0.05).Western blot and immunofluorescence staining showed that SWELL1 knockdown inhibited the transactivation of cardiac fibroblasts into myofibroblasts as indicated by reduced?-SMA protein expression(all P<0.05).(3)Ad-SWELL1 siRNA significantly reduced SWELL1 expression in primary cardiac fibroblasts(P<0.05).Western blot and immunofluorescence staining showed that Ad-SWELL1 siRNA inhibited the protein expression of?-SMA in TGF-?1stimulated cardiac fibroblasts(all P<0.05).SWELL1 knockdown also inhibited TGF-?1induced cardiac fibroblast proliferation as determined by CCK-8 assay.Ad-SWELL1siRNA decreased the mRNA expression levels of collagen I,collagen III,fibronectin,and CTGF(all P<0.05).Collagen contraction assay showed that Ad-SWELL1 siRNA obviously inhibited the pro-fibrotic capabilities of primary cardiac fibroblasts(P<0.05).(4)LV-SWELL1 significantly increased SWELL1 protein expression in cardiac fibroblasts(P<0.05).Western blot and immunofluorescence staining showed that SWELL1 overexpression enhanced the expression of?-SMA in TGF-?1 stimulated cardiac fibroblasts(all P<0.05).SWELL1 overexpression promoted TGF-?1 stimulated cardiac fibroblast proliferation as determined by CCK-8 assay.LV-SWELL1 also upregulated the mRNA expression of collagen I,collagen III,fibronectin,and CTGF(all P<0.05).Collagen contraction assay showed that LV-SWELL1 obviously promoted the pro-fibrotic capabilities of cardiac fibroblasts(all P<0.05).(5)RNA-Seq showed that there were 2087 differentially expressed genes between Ad-Scramble and Ad-SWELL1 siRNA group.1025 genes were upregulated and 1062genes were downregulated.The GO and KEGG assay showed that the most different signaling pathways between these two groups were TNF signaling pathway,PI3K-Akt signaling pathway,JAK-STAT signaling pathway,IL-17 signaling pathway and MAPK signaling pathway.There were 37 differentially expressed genes in JAK-STAT signaling pathway.7 genes were upregulated and 30 genes were downregulated in Ad-SWELL1siRNA group.The expression levels of Jak2,Socs3,Il6 and Pim1,the important downstream targets of JAK2-STAT3 signaling pathway,were significantly downregulated(all P<0.05).RT-PCR also identified that the mRNA levels of Jak2,Socs3,Il6 and Pim1 were decreased in Ad-SWELL1 siRNA group(all P<0.05).(6)SWELL1 knockdown inhibited JAK2-STAT3 signaling pathway in the post-MI cardiac tissues,as indicated by reduced protein expression levels of JAK2 and p-STAT3(Tyr705)(all P<0.05).In contrast,SWELL1 overexpression upregulated JAK2 and p-STAT3(Tyr705)protein levels in cultured cardiac fibroblasts(all P<0.05).The JAK2specific inhibitor WP-1066 and the STAT3 specific inhibitor Stattic significantly blocked transactivation,proliferation,and collagen and pro-fibrotic gene expression in SWELL1overexpressed cardiac fibroblasts(all P<0.05).Conclusion:The present study has demonstrated that:(1)Cardiac SWELL1 is upregulated in the post-MI cardiac tissues and in cardiac fibroblasts;(2)SWELL1 plays a crucial role in the regulation of cardiac fibrosis following MI and cardiac-specific SWELL1 knockdown ameliorates post-MI cardiac fibrosis and ventricular dysfunction;and(3)SWELL1regulates cardiac fibroblast transactivation,proliferation,and collagen and pro-fibrotic gene expression via JAK2-STAT3 signaling pathway.Taken together,our data suggest that targeting SWELL1 may be a promising and effective therapeutic strategy for cardiac fibrosis and heart failure. |
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