The K_ATP channel opener diazoxide protects cardiac myocytes during metabolic inhibition without causing mitochondrial depolarization or flavoprotein oxidation

Abstract

1 The K ATP channel opener diazoxide has been proposed to protect cardiac muscle against ischaemia by opening mitochondrial K ATP channels to depolarize the mitochondrial membrane potential, DC m . We have used the¯uorescent dye TMRE to measure DC m in adult rat freshly isolated cardiac myocytes exposed to diazoxide and metabolic inhibition. 2 Diazoxide, at concentrations that are highly cardioprotective (100 or 200 mM), caused no detectable increase in TMRE¯uorescence (n=27 cells). However, subsequent application of the protonophore FCCP, which should collapse DC m , led to large increases in TMRE¯uorescence (4300%). 3 Metabolic inhibition (MI: 2 mM NaCN+1 mM iodoacetic acid (IAA) led to an immediate partial depolarization of DC m , followed after a few minutes delay by complete depolarization which was correlated with rigor contracture. Removal of metabolic inhibition led to abrupt mitochondrial repolarization followed in many cells by hypercontracture, indicated by cell rounding and loss of striated appearance. 4 Prior application of diazoxide (100 mM) reduced the number of cells that hypercontracted after metabolic inhibition from 63.7+4.7% to 24.2+1.8% (P50.0001). 5-hydroxydeanoate (100 mM) reduced the protection of diazoxide (46.8+2.7% cells hypercontracted, P50.0001 vs diazoxide alone). 5 Diazoxide caused no detectable change in¯avoprotein auto¯uorescence (n=26 cells). 6 Our results suggest that mitochondrial depolarization and¯avoprotein oxidation are not inevitable consequences of diazoxide application in intact cardiac myocytes, and that they are also not essential components of the mechanism by which it causes protection.

Mitochondrial K channels in cell survival and death

“…However, Lawrence et al [66] recently reported that no change in ΔΨ m or flavoprotein oxidation was observed when diazoxide was added under normoxic conditions. However, they observed protection by diazoxide against chemical cell death produced by iodoacetate and cyanide.…”

Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes

“…2,3 These observations favor an involvement of the mitoK ATP channel in the preconditioning response, although an independent effect of K ATP channel modulators on mitochondrial metabolism cannot be ruled out. [12][13][14] To understand the different roles of sarcK ATP and mitoK ATP channels in cardiac myocytes, the molecular composition of each needs to be resolved. In this study a panel of isoform specific, site-directed antibodies against K ATP channel subunit peptides was produced to investigate the cellular localization of K ATP channel subunit isoforms in isolated ventricular myocytes by (i) confocal microscopy and (ii) subcellular fractionation and Western blotting.…”

“…This condition was associated with defects in vascular sarcolemmal K ATP channels, rather than their cardiac counterparts. 30 In view of these results and reservations concerning the use of flavoprotein fluorescence to monitor mitoK ATP channel activity [12][13][14] , the confirmation of the presence and potential rôle of a cardiac mitoK ATP channel remains elusive.…”

“…6 Moreover, the insensitivity of Kir6.1/SUR2A channels to either diazoxide or 5HD argues against this combination in mitoK ATP channel structure. 6 In view of some reservations over the validity of the association of potassium channel modulator-induced changes in flavoprotein fluorescence with mitoK ATP channel activity, [12][13][14] care is required in attributing a pharmacological profile to the mitoK ATP channel and, therefore, all three of the K ATP channel subunits identified in mitochondria herein may contribute to its structure.…”

Mitochondrial depolarization following hydrogen sulfide exposure in erythrocytes from a sulfide-tolerant marine invertebrate

“…Although it might be assumed that inhibition of oxidative phosphorylation would directly decrease ⌬⌿m, this is not necessarily true. For example, azide and cyanide in vitro decrease ⌬⌿m in some mammalian cells (Feeney et al, 2003;Jensen et al, 2002;Prabhakaran et al, 2002), but other mammalian cells show only a small reduction in ⌬⌿m during cyanide exposure unless inhibitors of glycolysis are added simultaneously (Lawrence et al, 2001). With regard to sulfide exposure, one study has reported that isolated rat hepatocytes exposed to 0.5·mmol·l -1 sulfide in vitro lost over 50% of ⌬⌿m within 1·h, and that this effect was reduced when the cells were supplemented with glycolytic substrate (Eghbal et al, 2004).…”