Next we aimed to investigate if
Next, we aimed to investigate if the cardiac apoptosis induced by CO-HFD, LA, T1DM or hyperglycemia involves up-regulation and activation of Fas/FasL-mediated cell death and if such effects are mediated by ROS and/or Ca+2/calcineurin/NFAT signaling axis. Similar to previous reports, , , , , , , , , we have found a significant increase in levels of both Fas and FasL (both soluble and membranous) as well as their downstream target, caspase-8, in the LVs of diabetic rats as well cardiomyocytes cultured in HG-media for 24 h. Kalivendi et al. (2005) have shown that the increase in intracellular ROS and Ca+2 levels are potent stimulators of NFAT nuclear accumulation and transcriptional activity in the cardiac 3065 via direct activation of calcineurin. Supporting to these findings, LVs of T1DM-induced rats, as well as LV cardiomyocytes cultured in HG media, showed a significant increase in the cardiac or cardiomyocytes levels ROS, intracellular Ca2+, and calcineurin activity and have higher nuclear levels of NFAT4. These data confirm our initial hypothesis and confirm that activation of this apoptotic axis is activated during a later stage of T1DM or after exposure to hyperglycemia, in vitro. To confirm our results, we further pre-incubated the cardiomyocytes with NFAT or calcineurin inhibitors (NFAT-I and FK506, respectively) before being re-cultured in GH. Indeed, with both inhibitors, levels of FasL, caspase-8, and caspase-3 were significantly decreased up to their normal levels observed in LG media. In addition, cell survival was significantly increased up to 85% with both treatments, thus confirming that hyperglycemia-induced Fas/FasL-mediated apoptosis is dominant over that of the intrinsic origin. This could be correct given that higher mRNA and proteins levels of Fas and FasL in the hearts, as well as the high soluble Fas (sFAs) antigen, were reported in animal and patients with HF , , . On the other hand and associated with no alteration in intracellular Ca2+ levels and calcineurin activity, we have found stable levels of mRNA and protein levels of FasL as well as the nuclear/cytoplasmic ratio of NFAT-4 in the LVs of CO-HFD-fed rats or in cultured cardiomyocytes treated with LA and grown in LG media. These findings dissipate the involvement of Ca+2/NFAT/FasL from the apoptotic role of LA on cardiomyocytes. However, we have found a significant increase in mRNA and protein levels of Fas under both conditions. Interestingly, LA potentiated cell death induced by rFasL and rendered the cardiomyocytes were more sensitive to rFAsL-induced cell death, as shown by the highest increase in caspase-8 and caspase-3 as well as the highest ratio of cell death, as compared to both individual treatments. This is basically can be explained by the higher Fas expression in LA-treated cell. To examine the mechanism by which CO-HFD or LA increases mRNA, we aimed to investigate if ROS, the only observed variable affected by CO-HFD or LA treatments, is responsible for this effect. As expected, pre-incubating the cells with NAC significantly abolished the stimulatory effect of LA on Fas, caspase-8 and caspase-3 and enhanced cell survival to almost normal levels, thus confirming that ROS is a major inducer of Fas in CO-HFD-treated rats or LA-treated cells. Supporting our findings, other studies have shown that hypoxia and doxorubicin, individually up-regulates Fas mRNA in neonatal and isolated cardiomyocytes by a mechanism that involves ROS , . Soluble FasL can be released from the cell surface by metalloproteinase-mediated cleavage of membrane-bound FasL . Currently, soluble Fas and FasL (sFas/sFasL) are believed to be biochemical surrogates and independent predictors of cardiovascular events in the population . However, their precise effects on the heart of animals or humans are still unclear. In hypertensive pateints and in patient with acute coronary disease (ACD), sFas and sFasL level were significantly increased and were correlated with carotid intima-media thickness and severity of the disease , . In ACD as well as in T2DM patients, studies have suggested that sFasL does not efficiently induce apoptosis but rather it blocks mFasL-mediated apoptosis and induce the proinflammatory and chemotactic activity of immune cells - , . Also, sFas has been shown to protect against atherosclerotic complications as it is able to inhibit the apoptosis of many cells, including leukocytes and vascular smooth muscle cells . Although this study have examined the cardiac expression of Fas and FasL, further investigation should include the effect of hyperglycemia and CO-HFD on circulatory levels of soluble Fas and FasL and to examine their cardiac effects. In fact, the roles of sFas and FasL in the diabetic hearts remain elusive and conflicting results exist. Indeed, sFasL were significantly increased ,  or decreased  in the serum of T2DM patients. Unfortunately, we didn't measure levels of sFas and sFasL in this study and correlate their effects, which should be considered in future work under similar experimental conditions.