The K-ATP channel opener diazoxide protects cardiac myocytes during metabolic inhibition without causing mitochondrial depolarization or flavoproteinoxidation

1 The K-ATP channel opener diazoxide has been proposed to protect cardiac m uscle against ischaemia by opening mitochondrial KATP channels to depolariz e the mitochondrial membrane potential, Delta Psi (m). We have used the flu orescent dye TMRE to measure Delta Psi (m) in adult rat freshly isolated ca rdiac myocytes exposed to diazoxide and metabolic inhibition. 2 Diazoxide, at concentrations that are highly cardioprotective (100 or 200 muM), caused no detectable increase in TMRE fluorescence (n=27 cells). How ever, subsequent application of the protonophore FCCP, which should collaps e Delta Psi (m) led to large increases in TMRE fluorescence (> 300%), 3 Metabolic inhibition (MI: 2 mm NaCN+ 1 mm iodoacetic acid IAA) led to an immediate partial depolarization of Delta Psi (m) followed after a few minu tes delay by complete depolarization which was correlated with rigor contra cture. Removal of metabolic inhibition led to abrupt mitochondrial repolari zation followed in many cells by hypercontracture, indicated by cell roundi ng and loss of striated appearance. 4 Prior application of diazoxide (100 muM) reduced the number of cells that hypercontracted after metabolic inhibition from 63.7 +/-4.7% to 24.2 +/-1. 8% (P <0.0001). 5-hydroxydeanoate (100 muM) reduced the protection of diazo xide (46.8 +/-2.7% cells hypercontracted, P <0.0001 vs diazoxide alone). 5 Diazoxide caused no detectable change in flavoprotein autofluorescence (n =26 cells). 6 Our results suggest that mitochondrial depolarization and fla voprotein oxidation are not inevitable consequences of diazoxide applicatio n in intact cardiac myocytes, and that they are also not essential componen ts of the mechanism by which it causes protection.