OSM Mitigates Post-infarction Cardiac Remodeling And Dysfunction By Up-regulating Autophagy Through Mst1 Suppression

Ischemic heart disease takes responsibility of the most death worldwide and has increased frequency.It causes almost 1.8 million deaths annually,or 20% of all deaths in Europe.Cardiac dysfunction is one of the common and well-known complications for acute myocardial infarction(AMI).Advances in reperfusion therapy,such as the rapid adoption of percutaneous coronary intervention(PCI)and intensive pharmacotherapy,may lead an overall trend for a reduction in mortality after AMI over the past three decades,thus lowering the subsequent risk of developing post-infarction cardiac remodeling and dysfunction.Myocardial infarction is one of the most common causes leading to severe chronic heart failure with adverse remodeling of the left ventricle which is characterized by cavity dilatation and cardiac dysfunction.The 1-year mortality among the hospitalized patients with heart failure(HF)following AMI remains substantial at 45.5% in 2010 with a number having been rising since 2007.In China,according the data in 2013,the prevalence of chronic heart failure reaches up to 0.9%,but the therapeutic strategies are still challenging.The process of heart failure after myocardial infarction is rather complicated.Studies reported that cardiomyocyte necrosis,excessive cardiomyocyte apoptosis,secretion of various cytokines,interstitial fibrosis,and non-myocyte dynamics within infarct tissues are all playing important roles in the development of cardiac dysfunction after MI.Autophagy is a physiological process which can degrade and recycle damaged protein and organelles maintaining organelle function and protein quality.Autophagy is proofed to be protective in many myocardial disorders.Studies suggested that insufficient autophagy may cause functional myocardial impairment and lead to cardiac dysfunction.Mst1(mammalian Ste20-like kinase 1),a serine-threonine kinase,is a critical component of the Hippo signaling pathway which has multiple functions including regulating autophagy,apoptosis and organ size.Persistent activation of Mst1 in the heart triggers dilated cardiomyopathy(DCM),whereas suppression of endogenous Mst1 protects cardiac function from pathological stresses.Moreover,Mst1 causes cardiac dysfunction in mice after myocardial infarction by suppressing autophagy.Oncostatin M(OSM),a well-known inflammatory cytokine,belongs to the interleukin-6(IL-6)family.Our previous study demonstrated that in diabetic condition OSM exerts protective roles against cardiac ischemia/reperfusion(I/R)injury through regulating apoptosis,insulin sensitivity and mitochondrial biogenesis by binding the O? receptor.Further study also shown OSM regulating autophagy protects cardiac function after myocardial infarction.However,the detailed mechanism underlying OSM-mediated beneficial effects against MI is still unclear.This study aimed to uncover the potential mechanism through which OSM rescues cardiac dysfunction after MI by regulating autophagy.?Objective?: 1.To investigate the effects of OSM on the cardiac function after MI.2.In vitro,neonatal cardiomyocytes were isolated and cultured followed by OSM and hypoxia treatment.Autophagic tools,such as GFP-LC3 adenovirus and bafilomycin A1,were used to detect the level of autophagy after those treatments to evaluate the effects of OSM on autophagic flux.3.To confirm that OSM exerts protective effects in an O?-dependent manner.O? receptor was knocked out in mice and mice were subjected to OSM treatment and MI surgery.Cardiac remodeling and cardiac function were measured.4.To investigate the role of OSM/O?/Mst1 pathway in OSM protecting cardiac function after MI,Mst1 was knocked out and overexpressed in mice.?Methods? 1.Mice in C57BL/6 background were randomly divided into the following groups: a)Sham group(Sham);b)Myocardial infarction group(MI);c)Myocardial infarction+OSM group(MI+OSM).2.MI model was conducted by ligating the LAD of the coronary artery.Before the surgery,OSM were injected intraperitoneally twice a day for 14 days.Cardiac function was measured by using echocardiogram.3.At 4 weeks after MI,Masson Trichrome staining was used to detect the cardiac remodeling.4.Heart tissues were harvested after 4 weeks of MI.Ultrastructural morphology of cardiomyocytes was detected by transmission electron microscopy.5.Neonatal cardiomyocytes were isolated and cultured in vitro.Cells were divided into the following groups: a)Control group(CON);b)Hypoxia group(H);c)Hypoxia+OSM group(H+OSM).Simulated ischemia was performed in vitro by using hypoxia incubator.6.In order to check the level of autophagy after hypoxia,cardiomyocytes were transduced with GFP-LC3 adenovirus(Ad-GFP-LC3)(MOI: 80:1)before OSM and hypoxia treatment.Images were observed under microscope.7.In physical condition,autophagy can degrade the damaged protein and organelles.We used the p62 and aggresomes staining to detect the level of autophagy indirectly.After 48 h of OSM treatment and 8h of hypoxia,p62 was stained by using primary and secondary antibodies and aggresomes were detected by using a specific kit.Images were observed under confocal microscope.8.O?-/-and O?+/+mice were randomly allocated into the following groups: a)O?-/-+Sham group(O?-/-Sham);b)O?-/+Myocardial infarction group(O?-/-+MI);c)O?-/-+Myocardial infarction+OSM group(O?-/-+MI+OSM);d)O?+/++Sham group(O?+/+ Sham);e)O?+/++Myocardial infarction group(O?+/++MI);f)O?+/++Myocardial infarction+OSM group(O?+/++MI+OSM).9.Neonatal cardiomyocytes were transduced with adenovirus harboring short hairpin RNA targeting O? and divided into following groups: a)O? knockdown group(Ad-sh-O?);b)O? knockdown+Hypoixa group(Ad-sh-O?+H);c)O? knockdown +Hypoxia+OSM group(Ad-sh-O?+H+OSM);d)Control group(Ad-Lac Z);e)Hypoxia group(Ad-Lac Z+H);f)Hypoxia+OSM group(Ad-Lac Z+H+OSM).10.Mst1 Tg,Mst1-/-and Mst1Tg: O?-/-mice were randomly allocated into the following groups: a)Mst1-/-+Sham group(Mst1-/-Sham);b)Mst1-/-+Myocardial infarction group(Mst1-/-+MI);c)Mst1-/-+Myocardial infarction+OSM group(Mst1-/-+MI+OSM);d)Mst1Tg+Sham group(Mst1Tg Sham);e)Mst1Tg+Myocardial infarction group(Mst1Tg+MI);f)Mst1Tg+Myocardial infarction+OSM group(Mst1Tg+MI+OSM);g)Mst1Tg:O?-/-+Myocardial infarction+OSM group(Mst1Tg:O?-/-+MI+OSM).11.Mst1 was knocked down and overexpressed in neonatal cardiomyocytes by using adenovirus which express a short hairpin(sh)RNA targeting Mst1 and harbor Mst1 c DNA(Ad-sh-Mst1,Ad-Mst1).Cells were divided into following groups: a)Mst1 knockdown group(Ad-sh-Mst1);b)Mst1 knockdown + Hypoxia group(Ad-sh-Mst1+H);c)Mst1 knockdown+Hypoxia+OSM group(Ad-sh-Mst1+H+OSM);d)Mst1 overexpression group(Ad-Mst1);e)Mst1 overexpression+Hypoxia group(Ad-Mst1+H);f)Mst1 overexpression+Hypoxia+OSM group(Ad-Mst1+H+OSM);g)Mst1 overexpression:O? knockdown+Hypoxia+OSM group(Ad-Mst1+Ad–sh –O?+H+OSM).12.After 4 weeks of MI surgery,heart tissues were harvested and lysed by using RIPA buffer.Neonatal cardiomyocytes were lysed after OSM treatment and hypoxia.The expression of protein was detected by Western blotting.13.After OSM treatment and hypoxia,the rate of apoptosis of cardiomyocytes was detected by using TUNEL kit.TUNEL-positive cells were counted under microscope.14.Mitochondrial membrane potential in cardiomyocytes was detected by JC-1 staining.?Results? 1.OSM improves cardiac function and alleviates left ventricle remodeling in mice after MI surgery.2.OSM increases autophagy level and reduces the accumulation of p62/aggresomes in cardiomyocytes after hypoxia.Besides,OSM inhibited the phosphorylation of Mst1.3.O? receptor knockout abolishes the protective effects of OSM on improving cardiac performance after MI.4.OSM increases autophagy flux in cardiomyocytes after hypoxia through O? receptor.5.OSM alleviates cardiac dysfunction and cardiac remodeling after MI by inhibiting Mst1.6.OSM increases autophagy flux in cardiomyocytes after hypoxia through Mst1 inhibition.7.Inhibition of autophagy by using 3-MA abrogates the protective effects of OSM after MI,indicating OSM exerts cardiac-protective effects through autophagy after MI.?Conclusion? In this study,our data show that OSM protected cardiac function and alleviated postinfarction cardiac remodeling after MI by enhancing cardiomyocyte autophagy through OSM/O?/Mst1 pathway.OSM holds promise as a therapeutic target in treating HF after MI through O? receptor by inhibiting Mst1 phosphorylation.