Abstract 18619: Scaffold Protein CARD11 Modulates Receptor Mediated Apoptosis in Cardiomyocytes Following Myocardial Infarction
Background: The CARD scaffold family members, named for containing the common caspase-recruitment domain, are important modulators of inflammatory and apoptotic signalling. CARD11, a membrane bound scaffold, is essential for the production of cytokines in the immune cellular response but is present in heart and lung tissue, where its effects are unknown, and described to associate with apoptotic players such as BCL10 and caspase-8. Due to the complex nature of cardiac response to myocardial infarction (MI), we hypothesize that CARD11 can act as a modulator and integrator of receptor mediated apoptotic pathways following cardiac tissue injury.
Methods & Results: CARD11 expression was increased in neonatal cardiomyocytes by general stress induction by H2O2 (25μM) and death receptor activation by Fas agonist Jo2 (1μg/ml) and TNF-alpha (50ng/ml) in isolated neonatal cardiomyocytes. Annexin V (AV) and propidium iodide (PI) double staining demonstrated decreased levels of apoptotic cells (AV+/PI+) following H2O2, Jo2 and TNF-alpha treatments in CARD11-/- cardiomyocytes to that of placebo levels compared to CARD11+/+ cells, as analyzed by flow cytometry. Pretreatment with caspase inhibitor zVAD-FMK (25μM) blocked this increase in apoptosis. Replacement of CARD11 through plasmid transfection restored the apoptotic phenotype in CARD11-/- cells and it was susceptible to zVAD-FMK inhibition. Experimental MI was created in CARD11-deficient mice (CARD11-/-) and wild-type littermate controls (CARD11+/+) by left coronary artery ligation. Survival over 28 days was significantly improved in CARD11-/- mice which exhibited lower rates of rupture and reduced infarct size post MI. TUNEL staining identified a lower percentage of apoptotic cells in the border zone of CARD11-/- hearts post-MI. Western blot analysis displayed decreased levels of cleaved caspase-3, -8 and -9, as well as pro-apoptotic mitochondrial proteins Apaf1, cytochrome-C and Bid.
Conclusion: CARD11 up-regulation provides a scaffold for the formation of death receptor signaling through caspase-8 to promote cell death via apoptosis and its removal to increased cardiomyocyte survival in the MI environment.
- © 2013 by American Heart Association, Inc.