Tyrosine kinases inhibitors block Fas-mediated deleterious effects in normoxic and hypoxic ventricular myocytes

Experimental evidences suggest an important role for Fas receptor activation in a wide range of myocardial pathologies, which are associated with a variety of arrhythmias and mechanical dysfunction. Our recent studies have shown that Fas-mediated arrhythmias and mechanical disturbances of ventricular myocytes can be accounted for by activation of the phospholipase C-1,4,5-inositol triphosphate-intracellular calcium release (PLC-1,4,5-IP ₃-[Ca²⁺]_i) cascade, which is responsible for attenuating the transient outward current (I_to) and augmenting the L-type Ca²⁺ current (I_Ca,L). We have also shown that whereas ventricular myocytes are resistant to Fas-mediated apoptosis, hypoxia predisposes myocytes to apoptosis induced by Fas activation by shifting the balance between pro-apoptotic and anti-apoptotic proteins towards the former. Since protein tyrosine phosphorylation has been shown to be involved in Fas signaling, we have hypothesized that inhibiting tyrosine kinases will attenuate Fas-mediated effects in ventricular myocytes in normoxic and hypoxic conditions. Therefore, we tested the effect of the tyrosine kinases inhibitors, genistein (50 μmol/l) and herbimycin A (50 μg/ml), on the abovementioned Fas-mediated effects in cultured neonatal rat ventricular myocytes (NRVM) and in freshly isolated adult murine ventricular myocytes. Fas receptor was activated by incubating NRVM with recombinant Fas ligand (rFasL, 50 ng/ml) and enhancing antibody (1 μg/ml), or by incubation of murine ventricular myocytes with the Fas-activating antibody Jo2 (10 μg/ml). The major findings were that genistein prevented Fas-mediated increase in 1,4,5-IP₃ production in NRVM (quantified by ion-exchange chromatography): 216 ± 41 counts per minute (cpm) in control, 605 ± 184 cpm in FasL-treated cardiomyocytes and 137 ± 51 cpm in rFasL + genistein-treated cultures. Accordingly, genistein or herbimycin A abolished the diastolic [Ca ²⁺]_i-rise (as measured by fura-2 fluorescence) and arrhythmogenic activity in both rat and murine ventricular myocytes, and the Fas-mediated changes in I_to and I_Ca,L in murine ventricular myocytes. Importantly, genistein attenuated Fas-mediated apoptosis in hypoxic (22 h in 1% O₂) NRVM; the apoptotic ratios as measured by DAPI fluorescence assay were: 4.6 ± 1.0% in control, 12.5 ± 3.0% in rFasL and 7.3 ± 1.6% in rFasL + genistein-treated NRVM. This prevention of apoptosis by tyrosine kinases blockade was accompanied by inhibition of hypoxia-induced increased Fas expression and decreased expression of the anti-apoptotic protein xIAP. In conclusion, our findings indicate that tyrosine phosphorylation is involved in Fas signaling in ventricular myocytes, and can, therefore, serve as a novel potential target for attenuating Fas-mediated dysfunction in normoxic and hypoxic myocardium.