Abstract 4020: Antibodies Against KChiP2 Induce Necrosis in Isolated Cardiomyocytes: Role of Uncoupling Protein 2
Background: Disturbances of humoral immunity have been described in patients with dilated cardiomyopathy (DCM). Antibodies against Kv channel-interacting proteins (KChIPs) may be associated with heart failure. Uncoupling protein 2 (UCP2) is a proton carrier that uncouples ATP synthesis and is located at the inner membrane of mitochondria. It has been shown that UCP2 modulates cell viability of adult rat cardiomyocytes.
Methods: Isolated rat cardiomyocytes were treated with antibodies against the C-terminal part of KChIP2 (80 pmol/ml). Caspase-3 and -9 activities (after 6 hours) and mitochondrial membrane potential ΔΨm (after 24 hours) were determined by flow cytometry. After 24 hours, necrotic and apoptotic cells were distinguished by staining with Hoechst 33258 and propidium iodide. mRNA and proteins were isolated by standard procedures and quantified by reverse transcription PCR and Western blotting, respectively, relative to beta-actin.
Results: Treatment for 24 hours did not induce changes in mitochondrial membrane potential, ΔΨm. Caspase-3 and -9 activities were not altered as well. The anti-KChIP2-treated cell population consisted of 75±3% necrotic, 2±1% apoptotic, and 13±2% viable cells. In contrast, cells treated with experimental buffer were viable to 86±1%. UCP-2 protein expression was increased by treatment with antibodies against KChIP2 (145±19%, n=5, p<0.05) compared to control cells (100%). In contrast, UCP-2 expression was down-regulated at the mRNA level (60±12%, n=5, p<0.01) in antibody-treated cells.
Conclusions: Antibodies against KChIP2 enhanced cell death rate of isolated rat cardiomyocytes probably due to necrosis which was accompanied by up-regulation of UCP2 protein expression. This may contribute to the pathogenesis and progression of heart failure.