Modulation Of MicroRNA-9 On Cardiac Remodeling Following Myocardial Infarction In Mice

Background Myocardial infarction(MI)remains the most common cause of cardiac morbi dity and mortality in the world.Following MI,insufficient oxygen/blood supply,as well as oxidative stress accumulation,leads to irreparable loss of cardiomyocytes,and this is further exacerbated by toxic substances released from dead cells.In addition,these immediate damages also trigger left ventricular(LV)remodeling,which eventually results in functional decomposition and hea rt failure.Thus,maintaining sufficient cardiomyocytes and favorable infarct microenvironment is essential for preservation of heart structure and function.Follistatin-like 1(Fstl1)protects against cardiomyocyte injury over a broad spectrum of cardiac conditions including myocardial infarction.It is worthy of note that although cardiac Fstl1 is obviously elevated in post-MI microenvironment,its cardioprotective role is still restricted to a limited extent considering the frequency and severity of adverse cardiac remodeling following MI.We therefore propose that intrinsic Fstl1 suppressor may exist in the ischemic myocardium and its silence may further assist post-MI recovery.Micro RNAs(miRNAs)are a class of ubiquitously expressed,non-coding RNAs with a length of 17-24 nucleotides;they func tion as gene silencer by inhibiti ng their translation and/or by promoti ng degradation of thei r m RNA.We therefore hypothesize that intrinsic Fstl1-suppressing miRNAs may exist in the heart and their neutralization may further alter cardiac remodeling process and benefit recovery from MI.Methods and Results Here,we predict the miRNA targeting Fstl1 through three main online software(Target Scan,micro RNA and miRDB).The results have showed that miR-9 is predicted as one of the potential miRNAs that target Fstl1.The expression of miR-9 is decreased in MI tissue and reversely correlated with Fstl1 expression.Luciferase activity assay further confirmed Fstl1 as a putative target of miR-9.In addition,forced expression of miR-9 in H9c2 cells is concurrent with the diminished expression of Fstl1 and vice versa.This indicates that miR-9 negatively regulates Fstl1.We establish hypoxia cell model in order to further analyze the mechanism of miR-9 in hypoxic cells.Examine the cell apoptosis rate,release of lactate dehydrogenase(LDH),accumulation level of reactive oxygen species(ROS)and the producti on of ATP by DAPI staining,LDH release assay kit,DCF fluorescence staining and luciferase after transfecting miR-9 mimics/ inhibitor into the hypoxia cell model.Transfection of miR-9 mimics in hypoxic H9c2 cells promotes cardiac cell death,LDH release,and ROS accumulation.This suggests that miR-9 exacerbates hypoxic-induced cell damage.The transfection of miR-9 inhibitor resulted in the opposite effect,which supported the view from the other side.Establish the myocardial infarction model by ligaturing the left anterior descending on C57BL/6 mice.Importantly,in vivo silencing of miR-9 by a specific antagomir in a murine acute MI model effectively preserves post-MI heart function,attenuates fibrosis and macrophage infiltration.Further studies demonstrated that antagomir treatment stabilizes Fstl1 expression as well as blocks cardiac cell death and ROS generation.Finally,we transfect miR-9 inhibitor + si Fstl1/si NC to hypoxia H9c2 cells in order to verify whether miR-9 regulates hypoxic injury through Fstl1.It was suggested that decreasing the expression of miR-9 and Fstl1 in cells simultaneously could reverse the inhibition of hypoxic injury of cells by decreasing the expression of miR-9 in cells partially.miR-9 inhibitor protects against hypoxic challenge through targeting Fstl1.Conclusion In summa ry,these findings identified miR-9 as a mediator of hypoxic injury in cardiomyoblasts and miR-9 suppression prevents cardiac remodeling after acute MI,providi ng a potential strategy for early treatment against myocardial infarction.