Abstract 1624: Neural Cell Adhesion Molecule Is Upregulated By Metabolic Stress And Enhances The Cardiomyocyte Survival Rate Through Neurite-cardiomyocyte Communication
Screening for cell-surface proteins under metabolic stress may lead to a better understanding of the cell-to-cell interaction involved in the pathophysiology of cardiac diseases. We performed a signal sequence trap in combination with a functional cloning method, using H9C2 rat cardiac myoblasts. We used the human CD2 cell-surface antigen fused in-frame to a neomycin-resistant gene as a reporter in the retroviral gene trap method. Since the signal sequence of CD2 cDNA terminates in a splice acceptor site, the cell-surface expression of the CD2-neo fusion protein would rely on the acquisition of a signal sequence from an endogenous gene. We screened for cell-surface proteins whose expression was enhanced by oligomycin, a metabolic stress agent, by flow-cytometric analysis using FITC-conjugated anti-CD2 antibody. One of the genes identified was neural cell adhesion molecule (NCAM, CD56), a major regulator of neurite outgrowth in the nervous system. Flow cytometry analysis revealed that oligomycin treatment also significantly upregulated cell-surface expression of NCAM in primary rat cardiac myocytes that had been treated with oligomycin. An immunohistochemical analysis in a mouse MI model revealed that NCAM was strongly expressed in residual cardiac myocytes and surrounding sprouting neuron fibers in the MI region, whereas it was detectable only at the intercalated disc in a non-infarcted area or in sham-operation mouse. To investigate the role of NCAM in the interaction between neurons and myocytes, GFP-positive PC12 rat pheochro-mocytoma cells were cultured on cardiomyocytes and their neurite length was evaluated. PC12 cultured on NCAM-overexpressing and control myocytes extended significantly longer neurites than those cultured on NCAM-knocked down myocytes (14.4±1.33 vs. 8.08±0.60 vs. 3.86±0.47μm, P<0.01), suggesting that NCAM expressed in cardiac myocytes enhanced neurite outgrowth from PC12. Moreover, the survival rate of cardiomyocytes cultured with PC12 cells was significantly reduced when NCAM was knocked down (Troponin T level in the culture medium; 9.07±0.291 vs. 42.4±16.54 ng/ml) . Thus, the upregulation of NCAM in stressed myocytes may be involved in neuron-cardiac myocyte interaction and enhances cardiomyocyte survival.