TNF-α-mediated caspase-8 activation induces ROS production and TRPM2 activation in adult ventricular myocytes

TRPM2 is a Ca2+-permeable cationic channel of the transient receptor potential (TRP) superfamily that is linked to apoptotic signalling. Its involvement in cardiac pathophysiology is unknown. The aim of this study was to determine whether the pro-apoptotic cytokine tumour necrosis factor-alpha (TNF-alpha) induces a TRPM2-like current in murine ventricular cardiomyocytes. Adult isolated cardiomyocytes from C57BL/6 mice were exposed to TNF-alpha (10 ng/mL). Western blotting showed TRPM2 expression, which was not changed after TNF-alpha incubation. Using patch clamp in whole-cell configuration, a non-specific cation current was recorded after exposure to TNF-alpha (I-TNF), which reached maximal steady-state amplitude after 3 h incubation. I-TNF was inhibited by the caspase-8 inhibitor z-IETD-fmk, the antioxidant N-acetylcysteine, and the TRPM2 inhibitors clotrimazole, N-(P-amylcinnamoyl) anthranilic acid and flufenamic acid (FFA). TRPM2 has previously been shown to be activated by ADP-ribose, which is produced by poly(ADP-ribose) polymerase 1 (PARP-1). TNF-alpha exposure resulted in increased poly-ADP-ribosylation of proteins and the PARP-1 inhibitor 3-aminobenzamide inhibited I-TNF. TNF-alpha exposure increased the mitochondrial production of reactive oxygen species (ROS; measured with the fluorescent indicator MitoSOX Red), and this increase was blocked by the caspase-8 inhibitor z-IETD-fmk. Clotrimazole and TRPM2 inhibitory antibody decreased TNF-alpha-induced cardiomyocyte death. These results demonstrate that TNF-alpha induces a TRPM2 current in adult ventricular cardiomyocytes. TNF-alpha induces caspase-8 activation leading to ROS production, PARP-1 activation, and ADP-ribose production. TNF-induced TRPM2 activation may contribute to cardiomyocyte cell death.