The Role Of JAK2/STAT3 Signaling Pathway And Autophagy In Daunorubicin-induced Cardiac Myocyte Hypertrophy

Background Myocardial hypertrophy is an important stage in the development of many cardiac diseases.It is characterized by functional deterioration from compensation to decompensation and structural derangement from reversibility to irreversibility.If not effectively intervened in the early stage of the disease process it will lead to irreversible damages including myocyte death and myocardial fibrosis,resulting eventually in cardiac dysfunction,congestive heart failure,and even sudden cardiac death.An important approach to preventing and treating such heart failure is to relieve or reverse the myocardial hypertrophy,a crucial step in reducing the occurrence of hypertrophy-related cardiovascular diseases and mortality.Unfortunately,the molecular mechanisms and related signal transduction pathways of myocardial hypertrophy remain incompletely understood.Investigation of the mechanism of cardiac hypertrophy is therefore an important topic yielding both theoretical value and clinical significance in the field of cardiovascular medicine.Daunorubicin(DNR),one of the most commonly used anthracycline class chemotherapeutic agents,has a strong antitumor effect against a variety of both hematopoeitic and solid malignancies.Many studies have shown that DNR can cause a dose-dependent cardiac toxicity.Since DNR-induced injury to the heart may be blunted by a compensatory hypertrophy in the early stage of drug application,serious irreversible damages ensue only with increasing dose accumulation in vivo,leading eventually to advanced heart failure and death.Indeed,the major limitation of DNR in clinical application is the well-established,significant cardiotoxicity.Preventing myocardial cell hypertrophy may therefore be a critical step in reducing DNR-induced cardiac complications.Currently,studies on the DNR-induced cardiac hypertrophy remain scarce.The Janus kinase2/signal transducer and activator of tran-scriptions-3(JAK2/STAT3)signaling,an important non-receptor tyrosine kinase activated cell signaling pathway,has gained significant attention in recent years.Mounting studies have established that JAK2/STAT3 signaling participates extensively in the pathogenesis of certain cardiovascular diseases.Activation of STAT3 can lead to the myocardial hypertrophy but the specific mechanism remains unclear.Can DNR induce myocardial hypertrophy by activation of the JAK2 / STAT3 signaling pathway? This question is yet to be answered.Numerous studies have found that myocardial hypertrophy is primarily due to increased cardiomyocyte volume.The pathological remodeling involves not only the increase of cellular proteins,but also activation of the autophagy and proteasome degradation system.Autophagy refers to the intracellular process of transporting degenerated,damaged or aging organelles and proteins to the lysosome for enzymatic digestion.Under normal circumstances,autophagy can help maintaining cell metabolism and survival.Sustained low levels of autophagy is a protective mechanism against cardiomyocyte stress.However,excessive autophagy activity can induce normal cell damage,causing non-apoptotic cell death.While autophagy is known to be closely involved in the pathogenesis of cardiovascular diseases,its specific role and molecular mechanism are not clear.Whether autophagy promotes or inhibits the occurrence and development of cardiovascular diseases remains yet to be addressed.This question constitutes another important focus for ongoing and future investigation.Based on the above research background,we hypothesized that in the early stage DNR-induced cardiac toxicity was mediated by activation of the JAK2 / STAT3 signaling pathway and inhibition of the cellular autophagy activity,both can lead to cardiomyocyte hypertrophy.Information from our study provides new perspectives and theoretical basis for the targeted clinical interventions aimed at reducing the anthracycline cardiotoxicity,further improving the efficacy of prevention and treatment of a deadly side effect from a commonly used,most effective cancer therapy agent.Objective By observing the DNR effect on in vitro cultured rat H9C2 cells,investigate whether the DNR induced cardiomyocyte hypertrophy and through what way cause cardiomyocyte hypertrophy.Therefore provide new theoretical basis for safe and effective clinical application of DNR to prevent and reduce the heart damage.Methods 1.H9C2 rat myocardial cells were cultured in vitro.Experiments were divided into 6 groups.(1)Control group;(2)DNR group;(3)RAPA + DNR group;(4)RAPA group;(5)AG490 + DNR group;(6) AG490 group.2.Using Image-Proplus Image software to analyze the DNR-induced morphologic changes including cell surface areas in cultured H9C2 myocardial cells.3.To analyze the effect of DNR on total protein synthesis in individual H9C2 cells.This was calculated by dividing the protein quantification with the number of cells counted.4.Using Western blot analysis to quantify DNR-induced changes in individual protein profiles known to be involved in the hypertrophy signaling and autophagy process.These proteins include c-Myc,?-SMA,LC3-II,Atg7,JAK2,STAT3,p-JAK2 and p-STAT3.Results 1.Myocyte Morphology Changes: DNR-treated H9C2 cells demonstrated a significant increase in cell surface area comparing with the control group(p< 0.01).RAPA and AG490 treated H9C2 cells showed a significant decrease in cell surface area when comparing with the DNR group(p < 0.01).2.Changes in Cellular Protein Synthesis: Comparing with the control group,DNR induced a significant increase in total protein synthesis in individual H9C2 cells(p <0.01).Cells treated with either RAPA and AG490 showed a significant decrease in the amount of total proteins(p <0.01).3.Changes in Individual Proteins in Signal Transduction and Autophagy Pathway: Western blot analysis demonstrated that compared with the control group,DNR induced a significant increase in c-Myc,?-SMA,P-JAK2 and P-STAT3 protein expressions,and decrease in LC3-II and Atg7 protein expressions(p <0.05 or p <0.01).No significant changes in total JAK2 and STAT3 were detected(p > 0.05).Comparing with the DNR group,H9C2 cells treated with AG490 +DNR or AG490 alone both demonstrated a significant decrease in c-Myc,?-SMA,P-JAK2 and P-STAT3 protein expressions that were previously increased by DNR(p <0.05 or p <0.01).The total JAK2 and STAT3 had no statistically significant changes(p> 0.05).H9C2 cells treated with either RAPA +DNR or RAPA alone both had significant decrease in c-Myc and ?-SMA protein expressions that were previously increased by DNR(p < 0.01).RAPA +DNR or RAPA treatment also significantly increased LC3-II and Atg7 protein expressions that were previously decreased by DNR(p <0.05 or p <0.01).Conclusions DNR can induce cardiomyocyte hypertrophy which is likely mediated by activating JAK2/STAT3 signaling pathway and decreasing autophagy activity..