title: TNF-α-mediated caspase-8 activation induces ROS production and TRPM2 activation in adult ventricular myocytes.
abstract: OBJECTIVE
  TRPM2 is a Ca(2+)-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-α (TNF-α) induces a TRPM2-like current in murine ventricular cardiomyocytes.
  
  
  RESULTS
  Adult isolated cardiomyocytes from C57BL/6 mice were exposed to TNF-α (10 ng/mL). Western blotting showed TRPM2 expression, which was not changed after TNF-α incubation. Using patch clamp in whole-cell configuration, a non-specific cation current was recorded after exposure to TNF-α (ITNF), which reached maximal steady-state amplitude after 3 h incubation. ITNF 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-α exposure resulted in increased poly-ADP-ribosylation of proteins and the PARP-1 inhibitor 3-aminobenzamide inhibited ITNF. TNF-α 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-α-induced cardiomyocyte death.
  
  
  CONCLUSIONS
  These results demonstrate that TNF-α induces a TRPM2 current in adult ventricular cardiomyocytes. TNF-α 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.
authors.affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Roberge
authors.affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Roussel
authors.affiliation: Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden Department of Medicine, Karolinska Institutet, Stockholm, Sweden Department of Cardiology, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Andersson
authors.affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
authors.forename: Albano C
authors.initials: AC
authors.lastname: Meli
authors.affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
authors.forename: Bastien
authors.initials: B
authors.lastname: Vidal
authors.affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Blandel
authors.affiliation: Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Johanna T
authors.initials: JT
authors.lastname: Lanner
authors.affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Le Guennec
authors.affiliation: Department of Physical Therapy, School of Health Sciences, Ariel University, Ariel, Israel.
authors.forename: Abram
authors.initials: A
authors.lastname: Katz
authors.affiliation: Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Håkan
authors.initials: H
authors.lastname: Westerblad
authors.affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Lacampagne
authors.affiliation: INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France jeremy.fauconnier@inserm.fr.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Fauconnier
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053181:Caspase 8 / Q000378:metabolism*; D003022:Clotrimazole / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D050052:TRPC Cation Channels / Q000378:metabolism; D050053:TRPM Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D017382:Reactive Oxygen Species; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D050053:TRPM Cation Channels; C544443:TRPM2 protein, mouse; D014409:Tumor Necrosis Factor-alpha; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505526:Casp8 protein, mouse; D053181:Caspase 8; D003022:Clotrimazole
keywords: Cardiac; Cell death; Ischaemia–reperfusion; TNF-α; TRPM2