Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice

Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous connexin 43 deficient ( Cx43 (+/-) ) mice. Because connexin 43 ( Cx43) is localized 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 and menadione. Diazoxide stimulates the mitochondrial formation of reactive oxygen species ( ROS) and menadione 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 mu mol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild- type ( WT) and Cx43 (+/-) cardiomyocytes. In contrast, diazoxide ( 200 mu mol/ 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 similar to +/- 1%). Menadione ( 37 mu g/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 similar to +/- 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.