title: Inhibition of phosphodiesterases leads to prevention of the mitochondrial permeability transition pore opening and reperfusion injury in cardiac H9c2 cells.
abstract: OBJECTIVE
  We tested if inhibition of phosphodiesterases (PDEs) with IBMX (1-methyl-3-isobutylxanthine) can modulate the mitochondrial permeability transition pore (mPTP) opening by inactivating glycogen synthase kinase 3β (GSK-3β).
  
  
  METHODS
  H9c2 cells were exposed to 600 μM H(2)O(2) for 20 min to cause the mPTP opening. Mitochondrial membrane potential (ΔΨm) was assessed by imaging cells loaded with tetramethylrhodamine ethyl ester (TMRE). Cell viability was measured with propidium iodide (PI) fluorometry using a fluorescence reader. Ischemia/reperfusion injury was induced by exposing cells to ischemic solution for 90 min followed by 30 min of reperfusion.
  
  
  RESULTS
  IBMX reduced loss of ΔΨm caused by H(2)O(2), indicating that inhibition of PDEs can prevent the mPTP opening. However, IBMX could not inhibit the pore opening in cells transfected with the constitutively active GSK-3β (GSK-3β-S9A) mutant, suggesting a critical role of GSK-3β in the action of IBMX. IBMX also reduced reperfusion injury in a GSK-3β dependent manner. In support, IBMX increased GSK-3β phosphorylation at Ser(9), an effect that was reversed by both the PKA inhibitor H89 and the PKG inhibitor KT5823. In support, IBMX activated both PKA and PKG. IBMX failed to prevent the loss of ΔΨm in the presence of H89 or PKA siRNA. Similarly, both KT5823 and PKG siRNA reversed the protective effect of IBMX.
  
  
  CONCLUSIONS
  Inhibition of PDEs prevents the mPTP opening by inactivating GSK-3β through PKA and PKG. GSK-3β is a common downstream target of PKA and PKG. Inhibition of PDEs may be a useful approach to prevent reperfusion injury.
authors.affiliation: Department of Anesthesiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Chanoit
authors.forename: Juan
authors.initials: J
authors.lastname: Zhou
authors.forename: SungRyul
authors.initials: S
authors.lastname: Lee
authors.forename: Rachel
authors.initials: R
authors.lastname: McIntosh
authors.forename: Xiangjun
authors.initials: X
authors.lastname: Shen
authors.forename: David A
authors.initials: DA
authors.lastname: Zvara
authors.forename: Zhelong
authors.initials: Z
authors.lastname: Xu
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology; D000071679:Glycogen Synthase Kinase 3 beta; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects*; D000083162:Mitochondrial Permeability Transition Pore; D015428:Myocardial Reperfusion Injury / Q000517:prevention & control*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D051381:Rats
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D010726:Phosphodiesterase Inhibitors; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D015056:1-Methyl-3-isobutylxanthine