Effect of KATP channel activators and inhibitors on skeletal muscle mitochondrial function
Authors: SANCHEZ‐DUARTE ELIZABETH , TRUJILLO XOCHITL , HUERTA MIGUEL , ORTIZ‐AVILA OMAR , CORTES‐ROJO CHRISTIAN , SAAVEDRA‐MOLINA ALFREDO , RODRIGUEZ‐OROZCO ALAIN , MONTOYA‐PEREZ ROCIO 
Published: November 2023

During muscle contraction, myofibrils consume ATP, and during prolonged activity oxygen demands increases. Mitochondrion plays an important role as the main source of energy production in the muscle cell. Mitochondrial ATP‐sensitive potassium channels (mitoKATP) has been proposed by its participation in several signaling routes that promotes the production of ATP, and modifying the energy flow through the electron transport system, affecting the transfer of energy between mitochondria and cellular ATPases. It has been reported that the action of inhibitors and activators of mitoKATP channels besides acting on the channels, could also have effects on different structures at mitochondrial level. Therefore, the aim of this work was to explore the effects of KATP channel activators and inhibitors on oxygen consumption and on the activity of the electron transport chain (I–IV) in isolated mitochondria derived from chicken skeletal muscle.

Methods

Mitochondria from pectoralis muscle of 2‐week‐old Arbor Acres chickens were isolated by differential centrifugation. The rate of oxygen consumption was determined at room temperature using a Clark‐type oxygen electrode coupled to an oxygen monitor YSI 5300 and a computer for data acquisition. The determinations started after adding 10 mM glutamate/malate as respiratory substrate for complex I (state 4) and 1 mM ADP was added to determine oxygen consumption in the phosphorylating state (state 3). On the other hand, the measurement of the enzymatic activities of respiratory chain complexes (I–IV) were assayed spectrophotometrically in a Perkin Elmer Lambda 18UV/vis spectrophotometer using 0.5 mg/mL mitochondria resuspended in 50 mM KH2PO4 buffer. NADH‐oxidoreductase (complex I) activity was evaluated by measuring the oxidation of NADH at an absorbance of 340 nm; Succinate‐DCIP oxidoreductase (complex II) activity was measured at 600 nm by following the reduction of 2,6‐dichlorophenolindophenol (DCIP). Antimycin A‐sensitive succinate‐cytochrome oxidoreductase (complex III) activity was followed by measuring at 550 nm the reduction of cytochrome. Cytochrome oxidase (complex IV) activity was evaluated by measuring the oxidation of reduced cytochrome at 550 nm. To explore the effects of the experimental drugs (KATP channel inhibitors and activators) on mitochondrial function, was employed: glibenclamide (20 μM), 5‐hidroxidecanoate (500 μM), diazoxide (30 μM), nicorandil (10 μM), cromakalim and levcromakalim (10 μM).

Both KATP channel inhibitors and activators produced changes in oxygen consumption, being the higher inhibition (60 ± 4.7%) of oxygen consumption in presence of the KATP channel blocker glibenclamide (20 μM); their effects on mitochondrial function affects the enzymatic activity of complexes II, III and IV. This suggests that the effect of agonists and antagonists of mitoKATP channel could be on the electron transport chain and not exclusively on the channel.

Support or Funding Information

RMP‐CIC2015