IL-10 Attenuates ER-Stress Induced Apoptosis in Doxorubicin Treated Cardiomyocytes.

Despite its antitumor efficacy, use of Doxorubicin (Dox) is limited by its cardiotoxic side effects. In Dox-induced cardiomyopathy (DIC), a causal role of oxidative stress (OS) has gained a lot of traction. Among other subcellular changes, Dox causes vacuolization of the cytoplasm and dilation of endoplasmic reticulum (ER). Dox also causes an excessive production of reactive oxygen species as well as imbalance in redox state both of which can affect ER homeostasis and protein folding. An accumulation of unfolded/misfolded proteins triggers the unfolded protein response (UPR) in the ER, which activates transcriptional and translation pathways involving: a) Protein-kinase like endoplasmic reticulum kinase (PERK); b) activating transcription factor 6α (ATF6α); and c) inositol requiring kinase1α (IRE1α) (Fig.1). Thus leading to the activation of ER chaperons, protein foldases and induce the expression of various genes, including X-box binding protein 1 (XBP1). UPR plays an important role in the attenuation of protein translation, upregulation of chaperones and antioxidant expression, and thus maintains ER homeostasis. However, if UPR fails to restore ER homeostasis, ER stress-initiated apoptotic signaling pathways are activated via CCAAT/enhancer homologous protein (CHOP), caspase-12 activation, phosphorylation of c-JUN NH2-terminal kinase (JNK) and pro-apoptotic gene expression. Understanding the role of these pathways of the ER under Dox treatment will highlight potential targets for new interventions as an adjunct therapy to restore ER functioning. In this study on isolated rat cardiomyocytes, we analyzed ER-stress markers, spliced XBP1 mRNA, and ER-apoptotic proteins. We also assessed protective effects of Interleukin-10 (IL-10) against Dox initiated ER-induced apoptosis and overall ER-stress. Dox significantly decreased the viability of cardiomyocytes and upregulated ER stress markers (GRP78, GRP94, and PDI). Interestingly, the exogenous administration of IL-10, one hour prior to the exposure of cardiomyocytes to Dox for 24hrs, was found to be effective in suppressing the expressions of ER-stress markers as well as ER-related apoptotic proteins and preserved cell viability. Additionally, significant changes in downstream targets of IRE1α, XBP1 in IL-10 treatment were recorded. IL-10 significantly increased the expression of unspliced XBP1 (U), an inhibitor of spliced XBP1(S) particularly in Dox-treated cells. This was followed by a reduction in the UPR mediated cell death in cardiomyocytes confirmed by a decrease in expression of GRP78, GRP94, and spliced XBP1 (S). IRE1 α arm of UPR initiates phosphorylation of JNK, which was reduced by IL-10 in Dox treated cells. Dox also initiated activation of procaspase-12, which is known to activate caspase-3 and apoptosis. IL-10 significantly reduced the activation of procaspase-12 and cell death. These data suggest that IL-10 provides protection against Dox-induced cardiotoxicity by restoring ER homeostasis.