Published online before print August 11, 2005, doi: 10.1161/01.RES.0000181171.65293.65
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Submitted on May 31, 2005
Revised on July 29, 2005
Accepted on August 1, 2005
Impairment of Diazoxide-Induced Formation of Reactive Oxygen Species and Loss of Cardioprotection in Connexin 43 Deficient Mice
Frank R. Heinzel ;
From the Institut für Pathophysiologie (F.R.H., Y.L., X.L., K.B., A.B., R.S., G.H.), Universitätsklinikum Essen, Germany; Servicio de Cardiologia (D.G.-D.), Hospital Vall d’Hebron, Barcelona, Spain; and the Dipartimento di Chimica Biologica (F.D.L.), Universita di Padova, Italy.
* To whom correspondence should be addressed. E-mail: gerd.heusch{at}uni-essen.de.
Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous connexin 43 deficient (Cx43+/-) mice. Because connexin 43 (Cx43) is expressed in cardiomyocyte mitochondria and mitochondrial Cx43 content is increased with ischemic preconditioning, we now tried to identify a functional defect at the level of the mitochondria in Cx43+/- mice by use of diazoxide. Diazoxide stimulates the mitochondrial formation of reactive oxygen species (ROS) and menadione which generates superoxide at multiple intracellular sites; both substances elicit cardioprotection through increased ROS formation. ROS formation in response to the potassium ionophore valinomycin was also measured for comparison. Menadione (2 µmol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild-type (WT) and Cx43+/- cardiomyocytes. In contrast, diazoxide (200 µmol/L) increased ROS formation by 43±10% versus vehicle in WT, but only by 18±4% in Cx43+/- cardiomyoctes (P<0.05). Two hour-simulated ischemia and oxygenated, hypo-osmolar reperfusion reduced viability as compared with normoxia (WT: 7±1% versus 39±2%, Cx43+/-: 8±1% versus 40±3%, P<0.01). Although menadione protected WT and Cx43+/- cardiomyocytes, diazoxide increased viability (17±2%, P<0.01) in WT, but not in Cx43+/- (9±1%). Menadione (37 µg/kg i.v.) before 30 minutes coronary occlusion and 2 hour reperfusion reduced infarct size in WT and Cx43+/- mice (24±4% versus 24±5%). In contrast, diazoxide (5 mg/kg i.v.) reduced infarct size in WT (35±4% versus 55±3% of area at risk, P<0.01), but not in Cx43+/- mice (56±2% versus 54±3%). Cardiomyocytes of Cx43+/- mice have a specific functional deficit in ROS formation in response to diazoxide and accordingly less protection.
Key words: ischemia • signal transduction
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