Role Of SOCS1/3 Signaling Pathway And Autophagy In Doxorubicin-induced Hypertrophy Of H₉C₂ Cardiomyocytes

BackgroundIt is widely expected that patients with various malignancies would require ever increasing treatment options including chemotherapy,targeted immunotherapy,and radiation therapy.Chemotherapy certainly remains to be a common treatment of many malignancies.Currently,anthracycline antibiotics are among the commonly used antitumor drugs.Doxorubicin?Dox?is a prototypical anthracycline which has been the focus of study in this class of antitumor agents ever since its clinical application.Dox has been used to treat a variety types of the most common malignancies including breast cancer,lung cancer,cancers from the gastroenterology,urology,hematology,as well as the musculoskeletal systems,with well-established positive outcome and clinical prospective.However,Dox can accumulate easily in the liver and heart causing organ toxicity.This is a major limitation to its clinical application due to the development of irreversible functional and structural organ damage.The chronic or long term Dox-induced cardiotoxicity mainly manifests as congestive heart failure.Early detection,prevention,and treatment of Dox cardiotoxicity will likely become the critical step in its future clinical application.One of the early manifestations of heart failure is cardiomyocyte hypertrophy at the cellular level.In fact,a number of studies have shown that JNK/Smad3 signaling pathway plays an important role in cardiac hypertrophy and heart failure.Discovered in 1962 cell autophagy occurs during both physiological and pathological processes.The specific role of autophagy in the development of cardiomyocyte hypertrophy is incompletely understood,and the subcellular mechanisms also remain to be fully elucidated.Given the above research background we hypothesized that Dox may induce cardiomyocyte hypertrophy in the early stage of its cardiotoxicity;the cellular and molecular mechanisms likely involve activation of JNK/Smad3signaling pathway and modulation of autophagy activity.Information derived from this study will facilitate early detection and effective treatment of anthracycline cardiotoxicity,therefore broaden its future clinical application.Objectives1.To investigate whether doxorubicin can induce cardiomyocyte hypertrophy2.To define the role of JNK/Smad3 signaling and autophagy in doxorubicin induced cardiotoxicity.MethodsCultured rat H₉C₂ cardiomyocytes were used in our study.Experiments were divided into 5 different sections.Experiments in section one were designed to test if Dox could induce cardiomyocyte hypertrophy.Studies were performed in two groups:Control and Dox-treatment.The cell surface areas were calculated by ImageJ.Changes in single cell protein content were calculated by measured total protein concentration divided by total cell count.Western blot was used to detect the expressions of cardiomyocyte hypertrophy marker proteins C-Myc andá-SMA.Expression of myocardium hypertrophy marker protein ANP was analyzed by cell immunohistochemistry.Experiments in section two were designed to test if myocyte autophagy could be affected by Dox,with data from 2 groups being included:the control and the Dox treatment.The expression of autophagy protein LC3 and Atg7 in cardiomyocytes were detected and analyzed by Western Blot.Experiments in section three were conducted to test if the JNK/Smad3 signaling pathway could be involved in the Dox-induced hypertrophy,also with a 2-group design:Control and Dox treatment.Expression changes in hypertrophic signaling proteins including P-JNK?T-JNK?P-Smad3?T-Smad3 were detected and analyzed using Western Blot.Experiments in section four were designed to further test the role and signal transduction of autophagy in Dox-induced cardiomyocyte hypertrophy by obtaining and comparing data from 4subgroups:Control,Dox treatment,Dox+RAPA?an enhancer of autophagy?,and RAPA alone.Cell surface area,total cell protein,cardiomyocyte hypertrophy marker protein C-Myc andá-SMA,and the expression of ANP in cardiac hypertrophy were measured,calculated,detected,and analyzed as described in Section one.The expressions of autophagy protein LC3 and Atg7 in myocardium were detected by Western Blot as described in Section two.Experiments in section five were designed to further test the involvement of hypertrophic signal transduction pathway in Dox-induced cardiotoxicity.Data from 4 groups were collected and compared:Control,Dox treatment,Dox+SP?an inhibitor of JNK/Smad3 signaling?,and SP alone.Cell surface area,total cell protein,cardiomyocyte hypertrophy marker protein C-Myc andá-SMA,the expressions of P-JNK,T-JNK,P-Smad3 and T-Smad3,the expression changes of JNK/Smad3 were measured,calculated,detected,and analyzed using imaging,protein quantification,Western blot,and immunohistochemistry techniques as described above.Results1.Dox treatment caused significant cardiomyocyte hypertrophy:Comparing with the control group,the cell surface area of Dox treatment group increased significantly?mean+/-SD;p<0.05?.The total cell protein?mean+/-SD,p<0.05?and the expression of C-Myc,á-SMA and ANP also increased significantly?mean+/-SD,p<0.05?.2.Dox significantly inhibited cardiomyocyte autophagy activity:Comparing with the control group,the expressions of autophagy protein LC3 and Atg7 in the Dox group decreased significantly?mean+/-SD,p<0.05?.3.Dox induced a significantly increase in the phosphorylation of JNK/Smad3 signaling pathway in the cultured cardiomyocytes:Comparing with the control group,the expression of P-JNK and P-Smad3increased significantly?mean+/-SD,p<0.05?although the T-JNK and T-Smad3 showed no significant changes?mean+/-SD,p>0.05?.4.Dox induced decrease in autophagy activity could be reversed by an autophagy enhancer RAPA:Comparing with the Dox treatment group,Dox+RAPA treatment showed a significant decrease in cell surface area?mean+/-SD,p<0.05?.The total amount of single cell proteins?mean+/-SD,p<0.05?and the expressions of the hypertrophic marker proteins C-Myc,á-SMA and ANP also decreased significantly?mean+/-SD,p<0.05?.These experiments strongly suggest that Dox induced inhibition in autophagy contributes to the myocyte hypertrophy,and this process could be reversed by an autophagy enhancer.5.Dox induced cardiomyocyte hypertrophy involves activation of the JNK/Smad3 signal pathway.Comparing with the Dox group,the Dox+SP?the JNK/Smad3 signal pathway inhibitor?treatment still increased the expressions of P-JNK and P-Smad3?mean+/-SD,P<0.05?while the T-JNK and T-Smad3showed no significant change?mean+/-SD,p>0.05?.Dox+SP group also had decreased cell surface area?mean+/-SD,p<0.05?,the total cell protein decreased?mean+/-SD,p<0.05?and the expressions of the hypertrophy marker proteins C-Myc?mean+/-SD,p<0.05?,á-SMA?mean+/-SD,p<0.05?,and ANP?mean+/-SD,p<0.05?.Conclusion1.Dox can induce cardiomyocyte hypertrophy,which could represent the manifestation of early drug toxicity;2.The cellular and molecular mechanisms likely involve activation of the pro-hypertrophic JNK/Smad3signaling pathway and inhibition of the myocyte autophagy.