Abstract P053: Impaired Autophagosome Removal in Cardiomyocytes Triggers Programmed Necrosis in Mouse Hearts
Programmed cell death includes apoptosis and programmed necrosis. The latter is also known as necroptosis. A well-recognized feature of necrosis is the loss of membrane integrity of the dying cell, resulting in inflammatory responses at the surrounding tissue. Cells undergoing apoptosis, however, maintain their membrane integrity very well and hence do not trigger inflammation. These features are effectively captivated to detect necrotic cells. By assessing Evans Blue Dye (EBD) uptake and CD45+ leukocytes, we previously detected massive cardiomyocyte (CM) necrosis, before increased apoptosis was evident, in mouse hearts deficient of COP9 signalosome (CSN) subunit 8 (Csn8) (Circ Res 2011; 108:40–50). We show here that the detection of necrosis is confirmed by immuno-detection of endogenous IgG inside the CM. We found that virtually all EBD positive cells were also mouse IgG positive whereas some of the IgG positive CMs, as expected, did not show increased EBD uptake, suggesting that probing intracellular IgG offers a simpler and even more sensitive method for detecting necrotic CMs in vivo. Notably, we were able to demonstrate that both the EBD uptake assay and the immuno-detection of intracellular IgG clearly have a resolution at the single cell level. We then explored the mechanism by which Csn8/CSN deficiency causes necrosis. Autophagosomes are accumulated due to a defect in their fusion with lysosomes in mouse hearts with perinatal CM-restricted Csn8 knockout (CR-Csn8KO) but the accumulation is heterogeneous among CMs likely due to a temporal heterogeneity of Csn8 gene ablation and/or a spatial difference in the microenvironment and functionality of different CMs. A remarkably higher percentage of CMs with greater autophagosome accumulation were undergoing necrosis than those with less or without autophagosome accumulation. Importantly, chronic lysosomal inhibition caused CM necrosis in wild type mice. Moreover, the protein levels of RIP1, RIP3 kinases and the RIP1-bound RIP3 were all significantly increased in the CR-Csn8KO heart. Taken together, these novel findings strongly suggest that impaired autophagosome removal causes CM necrosis in Csn8/CSN deficient hearts through likely activating the RIP1-RIP3 mediated necroptotic pathway.
- © 2011 by American Heart Association, Inc.