Mechanism of De-energized Mitochondria Membrane Potential Formation in Permeabilized Cardiac Myocytes

Abstract

The biophysical characteristics in de-energized mitochondria are not known very well. In this study, we carried out the investigation into the membrane potential formation at de-energized states of mitochondria. NADH, FAD and ΔΨm were monitored simultaneously using permeabilized cardiac myocytes of the rat. We showed de-energized mitochondria in absence of mitochondrial substrates still have a membrane potential and it was about 56 ± 5 mV. To pursue the mechanism of that, firstly we tried the possibility of the involvement of mito-KATP channel. We tested effects of Pi /ATP, KATP channel blockers/opener, and K+ replacement with meglumine on the de-energized mitochondria. The addition of Pi and ATP could dramatically hyperpolarized about 62 ± 7.7 mV. However, all agents blocking KATP channel did not show any significant effects. Interestingly, Diazoxide (DZX), KATP channel opener, and Oligomycin A (OligoA), F1-F0-ATPase inhibitor, itself could depolarize the Ψm in basal condition about 11 ± 1 mV and 46.5 ± 6 mV respectively, and the addition of Pi could reverse the effect. The ATP induced the hyperpolarization was occurred with DZX but was fully inhibited by OligoA. K+ replacement with meglumine slowed down the speed of ATP induced hyperpolarization and made it transient. However, the change NMDG to K+ could reverse and showed the full effect of ATP on Ψm. From these results, KATP channel and reverse mode of F1-F0-ATPase by the residual ATP hydrolysis might contribute the formation of the resting Ψm in de-energized mitochondria even though the mechanism of formation of the resting Ψm is still not clear. The K+ environment seemed to be important for the activity of F1-F0-ATPase.

Key Words: Ψm ,mito-KATP channel, ATP, Diazoxide, Oligomycin A