Early preneoplastic cells (sup+) exhibit increased susceptibility to apoptosis, which is lost in late stage preneoplastic cells (sup-). Sup+ cells, which undergo apoptosis when cultured in low serum, show little or no DNA binding activity by NF-kB either in 10% or 0.2% serum. In contrast sup- cells, which are resistant to apoptosis in low serum, show a sustained constitutive activation of NF-kB. The constitutive activation of NF-kB observed in sup- cells is not due to loss of IkBa. We considered that the activation of NF-kB in sup- cells might be secondary to an increase in cytosolic Ca2+, since sup- cells have a cytosolic Ca2+ level that is double that in sup+ cells. In support of a role for Ca2+, lowering cytosolic Ca2+ in sup- cells by addition of the cell permeable Ca2+ chelator BAPTA-AM reduced cytosolic Ca2+ by ~ 31% relative to untreated sup- cells, concomitant with a 65% reduction in NF-kB DNA binding activity and a reduction in IkB kinase activity. In sup- cells in low serum, BAPTA-AM induced a lowering of cytosolic Ca2+, NF-kB binding activity, and IkB kinase (IKK) activity, and also resulted in a significant (~50%) increase in caspase-3 activity. Furthermore, in sup+ cells, raising extracellular Ca2+ resulted in activation of IkB kinase and enhanced NF-kB DNA binding activity. Using proteasome and calpain inhibitors, we determined that the basal activity of NF-kB in sup- cells is largely proteasome independent, but sensitive to calpain inhibitors. Taken together these data suggest that the elevated Ca2+ in sup- cells causes a modest activation of IKK which likely contributes to the enhanced basal activation of NF-kB in sup- cells; however the predominant effect of Ca2+ appears to be mediated by Ca2+ enhanced degradation by calpain. We also investigated the role of apoptotic signaling pathways in the pathogenesis of human heart failure. Human left ventricular myocardium was obtained from explanted idiopathic cardiomyopathic hearts from patients with end-stage heart failure undergoing heart transplantation. Non-failing left ventricular myocardium was obtained from donor hearts that could not be used for transplantation. Failing myocardium showed caspase 3 activation consistent with a role for apoptosis in heart failure. This activation of apoptosis is not due to a decrease in bcl-2 protein levels, as failing hearts showed increased levels of bcl-2 in western blots. Consistent with a role for apoptosis in heart failure, in failing hearts we observed a significant decrease in phosphorylation of BAD at serine 112. There was no difference in total BAD protein levels between failing and non-failing hearts. Phosphorylation of BAD inhibits its binding to, and inactivation of, the anti-apoptotic bcl-2; thus a decrease in phospho-BAD would be pro-apoptotic. Protein phosphatase 1 (PP1), 2A (PP2A), and 2B (PP2B) activity was assessed by measuring the release of 33P from labeled substrate. Failing hearts exhibited a significant increase in PP2A and PP2B phosphatase activity, phosphatases that dephosphorylate BAD, providing a mechanism for the decrease in BAD phosphorylation. Taken together, these data are consistent with the hypothesis that phosphatase activity is increased in failing hearts leading to decreased phosphorylation of BAD and activation of apoptosis. We used DNA microarray profiling to investigate changes in expression of genes involved in apoptosis that occur in human idiopathic dilated cardiomyopathic hearts that had progressed to heart failure. We observed altered gene expression consistent with a pro-apoptotic shift in the TNF-a signaling pathway. Specifically we found decreased expression of the TNF-a and NF-kB induced anti-apoptotic genes such as GADD45-beta, Flice inhibitory protein (FLIP) and TNF induced protein 3 (A20). We also found decreased expression of several mitochondrial heat shock proteins. This study identifies several pathways that are altered in human heart failure and des new targets for therapy. In another project, we tested whether cardiac specific overexpression of the anti-apoptotic Bcl-2 overexpression modifies metabolism including ischemic acidosis during ischemia/reperfusion. We studied isolated perfused hearts from mice overexpressing human Bcl-2 (BCL, n=6) and from their wild-type littermates (WT, n=8), subjected to 24 minutes of global ischemia at 37 degrees followed by 2 hours of reperfusion. High-energy phosphates and intracellular pH were measured by P-31 NMR spectroscopy, while contractile function was measured by a balloon in the left ventricle connected to a pressure-transducer. At baseline there were no significant differences in intracellular pH (BCL: 7.27+/-0.01 vs. WT: 7.28+/-0.04) or contractile function (BCL: 114.0+/-6.4 cmH2O vs. WT: 114.6+/-2.8), including +/-dP/dt between WT and BCL hearts (p0.05). During ischemia, the fall in pH was significantly reduced in BCL (6.49+/-0.06) compared with WT hearts (6.26+/-0.07, p<0.05). Creatine phosphate (PCr) and ATP both declined during ischemia, and at the end of ischemia there were no significant differences between the groups. At 2 hours of reperfusion, percent recovery of left ventricular developed pressure (78.9+/-7.8%) and +/-dP/dt (+3.7+/-0.4 and -2.4+/-0.3 cmH2O/ms) were significantly improved in BCL hearts compared with WT hearts (38.0+/-3.1%; +/-dP/dt=+2.1+/-0.3 and -1.2+/-0.2 cmH2O/ms; p<0.05). Infarct size measured with TTC staining at 2 hours of reperfusion was also significantly reduced in BCL (18+/-3%) compared with WT hearts (38+/-5%, p<0.05). Postischemic recovery of PCr was significantly better in BCL (85+/-5% of pre-ischemic level) than in WT hearts (60+/-4%, p<0.05). Since it has been suggested that mitochondrial F0F1-ATPase H+ pump is required for apoptotic cell death pathway, we investigated whether inhibition of this pump with oligomycin would affect intracellular pH during ischemia. Addition of oligomycin prevented the fall in pH during ischemia, suggesting that BCL may inhibit the F0F1-ATPase to achieve reduced acidification during ischemia. In conclusion, Bcl-2 overexpression reduced cytosolic acidification, which may be partly mediated by mitochondrial F0F1-ATPase activity, and led to improved recovery of function and PCr and smaller infarcts after reperfusion.
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