Tnfα Alters Mitochondrial Function And Ca2+ Homeostasis In Ventricular Cardiomyocytes: A Key Role For Caspase-8 Activation

Tumor necrosis factor α (TNFα), a pro-inflammatory cytokine, is associated with major cardiomyopathy. In the heart, TNFα binding to the TNF receptor 1 (TNFR1) has been implicated in TNFα mediating negative inotropic effects as well as apoptosis. TNFα-TNFR1 activates caspase-8 which leads to caspase-3 activation either directly or following mitochondrial disruption. Here we investigated whether caspase-8-induced mitochondrial dysfunction could lead to TNFα-induced alterations of Ca2+ homeostasis. All experiments were performed on freshly isolated rat ventricular cardiomyocytes using multi-photons or confocal microscope. One hour of TNFα application (10 ng/ml) activates caspase-8 as well as caspase-3 measured with carboxyfluroscein-derived specific probes. TNFα depolarized mitochondrial membrane potential (measured with TMRM), and increased mitochondrial superoxyde production (measured with MitoSox). In the mean time, mitochondrial Ca2+ decreased, preceding an elevation in resting cytosolic Ca2+ fluorescence (Rhod-2 and Fluo-4 measurements respectively) and an increase in spontaneous ryanodine receptors activities (sparks frequency). Alternatively, on field stimulated cells (0.5 Hz), TNFα decreased Ca2+ transients' amplitude and SR load. TNFα-mediated alteration in SR Ca2+ function was normalized by antioxidant (NAC; 20 mM). In addition, a broad-spectrum caspase inhibitor (Q-VD-oph; 10 μM) or specific caspase-8 inhibitors (TRP801 and z-IETD-fmk; 10 μM), blocked TNFα effects both on mitochondria and Ca2+ handling. On an ischemia-reperfusion model, intra-peritoneal injection of TRP801, 15 min minutes prior reperfusion, prevented long term morpho-functional remodeling. In conclusion, caspase-8 activity appears to mediate TNFα-induced mitochondrial dysfunction which in turn alters global Ca2+ handling independently of caspase-3 activation. Caspase-8 inhibition presents a potential therapeutic target.