title: Mitochondria as targets for nitric oxide-induced protection during simulated ischemia and reoxygenation in isolated neonatal cardiomyocytes.
abstract: BACKGROUND
  As shown previously, exposure to NO donors initiates protective mechanisms in cardiomyocytes that persist after removal of the donor, a form of pharmacological preconditioning. Because NO also affects mitochondrial respiration, we studied the effect of NO on mitochondrial Ca(2+) uptake.
  
  
  RESULTS
  Neonatal rat ventricular myocytes in primary culture were exposed to 1 hour of simulated ischemia and 1 hour of reoxygenation (sI/R). Pretreatment with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (1 mmol/L for 90 minutes), followed by washing and incubation for 10 to 30 minutes, reduced sI/R-induced cell death to 25.4% compared with control (propidium iodide exclusion assay, P<0.001). Short (10-second) exposures to SNAP reversibly suppressed mitochondrial respiration without a detectable change in mitochondrial potential. In contrast, treatment with SNAP for 90 minutes caused a modest but sustained mitochondrial depolarization, as judged by JC-1 fluorescence. SNAP pretreatment limited cellular Ca(2+) overload during ischemia (fura-2 ratio rose to 226+/-40% versus 516+/-170% of baseline, n=5, P<0.05) and prevented loss of cell membrane integrity during reoxygenation. SNAP pretreatment also significantly reduced the ability of mitochondria to accumulate Ca(2+) in the face of a similar cytosolic Ca(2+) load (peak rhod-2 fluorescence 133+/-4% versus 166+/-7% of baseline at similar fluo-3 levels, P=0.0004, n=52 and 25, respectively).
  
  
  CONCLUSIONS
  Pretreatment with an NO donor induces a modest, sustained mitochondrial depolarization and protects cardiomyocytes from sI/R injury. The demonstrated reduction in mitochondrial Ca(2+) uptake possibly reduces cytosolic Ca(2+) overload, providing a likely mechanism for NO-induced protection.
authors.affiliation: Departments of Cardiology, Kings College London, St Thomas' Hospital , University College London, UK.
authors.forename: R D
authors.initials: RD
authors.lastname: Rakhit
authors.forename: M H
authors.initials: MH
authors.lastname: Mojet
authors.forename: M S
authors.initials: MS
authors.lastname: Marber
authors.forename: M R
authors.initials: MR
authors.lastname: Duchen
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019194:Ischemic Preconditioning; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D017202:Myocardial Ischemia / Q000150:complications / Q000503:physiopathology*; D015428:Myocardial Reperfusion Injury / Q000209:etiology / Q000517:prevention & control; D009569:Nitric Oxide / Q000502:physiology; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D010100:Oxygen / Q000494:pharmacology*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; D009569:Nitric Oxide; D010396:Penicillamine; D010100:Oxygen; D002118:Calcium