Myofibroblast-specific Deficiency Of LSD1 Attenuates Myocardial Injury Induced By Doxorubicin And The Underlying Mechanisms

Background and Objective:Doxorubicin is an anthracycline anticancer drug,its clinical application is compromised by cardiotoxicity.The cardiotoxic mechanisms of doxorubicin mainly include oxidative stress,release of inflammatory mediators,apoptosis and fibrosis,but the exact mechanisms have not been fully elucidated.Doxorubicin caused cardiomyocytes apoptosis and necrosis,damaged cardiomyocytes release a lot of fibrogenic active factors and induced fibroblast activation to secrete a large number of extracellular matrix proteins,then the fibrotic scar tissue gradually replaced apoptotic cardiomyocytes,and eventually progressed to dilated cardiomyopathy.More and more evidence showed that epigenetic especially histone modification is closely related to the process of myocardial fibrosis.Histone lysine specific demethylase 1（LSD1）can specifically remove the mono-and demethylation of H3K4 and H3K9.Studies suggested that LSD1 plays an important regulatory role in cardiac development,at present the relationship between doxorubicin-induced myocardial injury and LSD1 has not been reported.This study used activated fibroblasts specific LSD1 knockout mice to explore the role of doxorubicin-induced myocardial injury and its mechanism.Methods and Results:1.Myofibroblast-specific deficiency of LSD1 attenuates myocardial injury induced by doxorubicin in mouse and the underlying mechanismsLSD1^fl/fl mice were hybridized with myofibroblast specific cre mice containing the RFP reporter gene(Postn^MCMR26^RFP).The genomic DNA was extracted and Postn^MCMR26^RFP/LSD1^fl/fl genotypes were screened by PCR.The basline Cardiac functions measured by echocardiography of 8-week-old Postn^MCMR26^RFP mice and Postn^MCMR26^RFPLSD1^fl/fl mice were same.The mice were divided into Postn^MCMR26^RFP（Control）group and Postn^MCMR26^RFPLSD1^fl/fl（KO）group,The myocardial injury model was established by intraperitoneal injection of doxorubicin（DOX,3mg/kg/w,for 6 weeks,the cumulative dose was 18 mg/kg）,while the Postn^MCMR26^RFP（Control）group was given the corresponding volume of saline.All groups were fed with tamoxifen food from two days before first administration to the end of the experiment（total of 8 weeks）.6 weeks after the first DOX administration,the expression of LSD1 was significantly reduced in the myofibroblasts isolated from the KO DOX group compared with the Control DOX group.The results showed that myofibroblast specific LSD1 knockout was successfully achieved in the hearts of Postn^MCMR26^RFPLSD1^fl/fl mice.24 hours after administration in the 6th week,the blood samples was collected to separate serum and serum myocardial injury markers LDH,CK-MB,MDA activity and antioxidant SOD activity were detected.The results showed that the activity of serum myocardial injury markers in the Control DOX group were significantly increased,and the content of antioxidant was significantly decreased.Compared with the Control DOX group,the abnormal changes of myocardial injury markers and antioxidant in the KO DOX group were significantly alleviated.After 8 weeks,the ejection fraction（EF%）and fractional shortening（FS%）were measured by echocardiography.The body weight（BW）and the ratio of heart weight to tibia length（HW/TL）were measured.The results showed that compared with the Control DOX group,the decrease of cardiac function and the decrease of HW/TL and BW induced by DOX were significantly attenuated in KO DOX group.TUNEL staining was used to detect the apoptosis of cardiomyocytes.HE staining and Masson staining were used to evaluate the pathological changes and collagen deposition.Vimentin andα-SMA immunofluorescence were used to detect the activation of cardial fibroblasts.The results showed that compared with the Control Saline group,the Control DOX group had significant cardiomyocytes apoptosis,obvious vacuolation of cytoplasm,disordered arrangement of myocardial fiber tissue,significantly increased fibrosis areas,and significantly increased fluorescence intensity of Vimentin andα-SMA.DOX induced myocardial injury in KO DOX group were significantly attenuated compared with the Control DOX group,indicating that myofibroblast-specific deficiency of LSD1 significantly reduced cardiomyocytes apoptosis,cardiac fibroblasts activation and fibrosis induced by DOX.Furthermore,after 8 weeks,we detected the proteins expression related to cardiac fibrosis and TGF-β/MAPK pathway in mice.The results showed that compared with the Control DOX group,the increase of CollagenⅠ,CollagenⅢ,α-SMA expression and the increase of TGF-β1,p-JNK,p-ERK and p-p38 expression induced by DOX were significantly alleviated in KO DOX group,indicating that myofibroblast-specific deficiency of LSD1 alleviated DOX-induced myocardial injury and fibrosis,and its mechanism may be related to the inhibition of TGF-β/MAPK pathway.2.Effect of LSD1 knockdown on myocardial fibroblast activation induced by DOX treated cardiomyocytes supernatant and its mechanismThe neonatal rats cardiomyocytes（NRCMs）were treated with DOX.It was found that the expression of CollagenⅠ,CollagenⅢandα-SMA proteins in the neonatal rats cardiac fibroblasts（NRCFs）were significantly up-regulated,suggesting the activation of cardiac fibroblasts.This may be related to the increase of TGF-β1 secreted by DOX treated cardiomyocytes.At the same time,the expression of LSD1 was up-regulated in the activated cardiac fibroblasts,suggesting that LSD1 was involved in the activation of cardiac fibroblasts induced by the supernatant of DOX treated cardiomyocytes.LSD1 was knocked down in cardiac fibroblasts transfected with siRNA,then cultured fibroblasts with the supernatant of DOX treated cardiomyocytes.The results showed that the increased expression of CollagenⅠ,CollagenⅢ,α-SMA and TGF-β/MAPK pathway proteins TGF-β1,p-JNK,p-ERK and p-p38 were significantly alleviated.It is suggested that LSD1 knockdown in cardiac fibroblasts alleviated the activation of fibroblasts induced by DOX caused cardiomyocyte injury.and its mechanism may be related to the inhibition of TGF-β/MAPK pathway.Conclusion:1.This study firstly suggested that myofibroblast-specific deficiency of LSD1attenuates myocardial injury induced by doxorubicin.2.In vitro,LSD1 knockdown alleviated the activation of cardiac fibroblasts caused by DOX induced cardiomyocyte injury.3.The protective mechanism of myofibroblast-specific deficiency of LSD1 on DOX induced myocardial injury may be related to the inhibition of the TGF-β/MAPK signaling pathway.