Title Globular adiponectin , acting via AdipoR 1 / APPL 1 , protects H 9 c 2 cells from hypoxia / reoxygenation-induced apoptosis

Cardiomyocyte apoptosis is an important remodeling event contributing to heart failure and adiponectin may mediate cardioprotective effects at least in part via attenuating apoptosis. Here we used hypoxia-reoxygenation (H/R) induced apoptosis in H9c2 cells to examine the effect of adiponectin and cellular mechanisms of action. We first used TUNEL labeling in combination with laser scanning cytometry to demonstrate that adiponectin prevented H/R-induced DNA fragmentation. The anti-apoptotic effect of adiponectin was also verified via attenuation of H/R-induced phosphatidylserine exposure using annexin V binding. H/R-induced apoptosis via the mitochondrial-mediated intrinsic pathway of apoptosis as assessed by cytochrome c release into cytosol and caspase-3 activation, both of which were attenuated by adiponectin. Mechanistically, we demonstrated that adiponectin enhanced anti-oxidative potential in these cells which led to attenuation of the increase in intracellular reactive oxygen species (ROS) caused by H/R. To further address the mechanism of adiponctins antiapoptotic effects we used siRNA to efficiently knockdown adiponectin receptor (AdipoR1) expression and found that this attenuated the protective effects of adiponectin on ROS production and caspase 3 activity. Knockdown of APPL1, an important intracellular binding partner for AdipoR, also significantly reduced the ability of adiponectin to prevent H/Rinduced ROS generation and caspase 3 activity. In summary, H/R-induced ROS generation and activation of the intrinsic apoptotic pathway was prevented by adiponectin via AdipoR1/APPL1 signaling and increased anti-oxidant potential. Citation: Park M, Youn B, Zheng X-l, Wu D, Xu A, et al. (2011) Globular Adiponectin, Acting via AdipoR1/APPL1, Protects H9c2 Cells from Hypoxia/ReoxygenationInduced Apoptosis. PLoS ONE 6(4): e19143. doi:10.1371/journal.pone.0019143 Editor: Rakesh K. Srivastava, The University of Kansas Medical Center, United States of America Received December 2, 2010; Accepted March 20, 2011; Published April 28, 2011 Copyright: 2011 Park et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Heart and Stroke Foundation of Canada via a Grant-in-Aid to GS. GS and DW also acknowledge support from The Canadian Institutes of Health Research (CIHR) China Collaborative grant, MP holds a Natural Sciences and Engineering Research Council of Canada (NSERC) postgraduate studentship award. Work by XLZ was partially supported by Canadian Institutes of Health Research (RT734076) and XLZ is the recipient of Senior Scholar Award of Alberta Heritage Foundation for Medical Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gsweeney@yorku.ca