Abstract 632: Caspase-12 Cleavage and Cardiomyocyte Apoptosis Following Activation Of Ca++/Calmodulin Kinase IIδ(CaMKIIδ)-p38 MAPK By Anti-β1-adrenergic Receptor (AR) Antibody
Background We have recently shown that autoimmune cardiomyopathy induced by β1- adrenergic receptor (AR) peptide immunization in rabbits is associated with enhanced phosphorylation of CaMKIIδ and p-38 MAPK, activation of endoplasmic reticulum (ER) stress and caspase-12 cleavage, and cardiomyocyte apoptosis. These changes are thought to be induced by anti-β1AR antibody on the post-receptor CaMKIIδand MAPK system, but a cause-and-effect relationship has not been established.
Methods To study the direct effect of anti-β1AR antibody on the CaMKIIδ-p-38 MAPK-ER stress and caspase-12 pathway, we carried out experiments in cultured neonatal rat cardiomyocytes using purified IgG obtained from rabbits immunized with the β1AR peptide. The myocytes were treated with the β1AR IgG for up to 48 h, and studied for myocyte apoptosis (TUNEL), cleavage of caspase-12 and caspase-3, GRP78, CHOP, and phosphorylation of CaMKIIδ and p38 MAPK (Western blot and histoimunoflurescence). We also studied the functional roles of CaMKIIδ, p38 MAPK and caspase-12 using selective inhibibors of CaMKIIδ(KN93), p38-MAPK (SB203580), and caspase-12 (siRNA).
Results Table⇓ below shows that β1AR IgG activated CaMKIIδ and p-38 MAPK in myocytes at 6 h, and that this was associated with increased cleavage of both caspase-3 and caspase-12, upregulated GRP78 and CHOP, and cell apoptosis at 48 h. CaMKIIδ inhibitor abolished both ER stress response and cell apoptosis produced by β1-AR IgG. Similar effects were produced by the p38 inhibitor, but it had no effect on CaMKIIδ phosphorylation. Caspase-12 siRNA attenuated the cleavage of caspase-12 and apoptosis produced by β1AR IgG, but with no effects on either CaMKIIδ or p38 MAPK activation.
Conclusion The results indicate that the β1AR antibody exerts an apoptotic effect on cardiomyocytes through activation of CaMKII-p38 MAPK-ER stress pathway. ER-resided caspase-12 activation plays a pivotal role in the downstream pro-apoptotic pathway.