| Many of the cellular mechanisms responsible for cardiac cell death during ischemia and subsequent reperfusion have been elucidated, while others remain largely unknown. The sphingolipid-signaling pathway is one such mechanism by which cell death during ischemia/reperfusion (IR) may play a role. Exposure of cells to certain stressful stimuli, such as TNF-α and IR, causes activation of neutral sphingomyelinase (nSMase). Downstream effectors of sphingolipid-signaling pathway have been shown to be involved in apoptosis in the cardiomyocyte. This dissertation concentrated on the location, presence and role of sphingolipid-signaling proteins like factor associated with neutral sphingomyelinase activation (FAN) and nSMase in HR induced cell death in the cultured cardiomyocyte. The novel protein, rat FAN, was discovered and along with nSMase1 was shown to be expressed in the cardiomyocyte. Furthermore, FAN and nSMase1 were found to have substantial co-localization using immunofluorescence and confocal microscopy. Importantly, TUNEL analysis determined that FAN and nSMase1 play a critical role in the signaling pathway leading to cell death in the hypoxia/reoxygenated cardiomyocyte. The role of nSMase was additionally demonstrated through the use of an nSMase1 inhibitor, which decreased HR induced caspase-3 activity. Although nSMase2 was discovered in the rat heart, it was not found to play a role in HR injury. These results suggest a model for HR mediated cell death by which FAN activates nSMase1 leading to ceramide production and subsequent cell death.; Two novel biochemical findings are also presented. Both nSMase1 and nSMase2 were discovered to be Triton insoluble integral membrane proteins. Additionally, these enzymes were kinetically characterized and shown to behave as allosteric enzymes, with EC50s for SM of 18.17 μM and 21.39 μM, respectively.; Finally, neither HR induced cell-death, nor the proteins, FAN and nSMase1, was discovered to be present in caveolae, while EDG1 and SCaMPER were revealed to co-localize with cav-3, suggesting that caveolae in cardiomyocytes may organize only certain sphingolipid-signaling components. |
