Effect of mitochondria poisoning by FCCP on Ca2+ signaling in mouse skeletal muscle fibers

Abstract

We have studied the effects of mitochondria poisoning by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) on Ca(2+) signaling in enzymatically dissociated mouse flexor digitorum brevis (FDB) muscle fibers. We used Fura-2AM to measure resting [Ca(2+)](i) and MagFluo-4AM to measure Ca(2+) transients. Exposure to FCCP (2 microM, 2 min) caused a continuous increase in [Ca(2+)](i) at a rate of 0.60 nM/s and a drastic reduction of electrically elicited Ca(2+) transients without much effect on their decay phase. Half of the maximal effect occurred at [Ca(2+)](i) = 220 nM. This effect was partially reversible after long recuperation and was not diminished by Tiron, a reactive oxygen species (ROS) scavenger. FCCP had no effects on fiber excitability as shown by the generation of action potentials. 4CmC, an agonist of ryanodine receptors, induced a massive Ca(2+) release. FCCP diminished the rate but not the amount of Ca(2+) released, indicating that depletion of Ca(2+) stores did not cause the decrease in Ca(2+) transient amplitude. Ca(2+) transient amplitude could also be diminished, but to a lesser degree, by increases in [Ca(2+)](i) induced by repetitive stimulation of fibers treated with ciclopiazonic acid. This suggests an important role for Ca(2+) in the FCCP effect on transient amplitude.

The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers

“…In the companion paper, we have shown that the loss of Ca 2+ from mitochondria determines an increase in the cytosolic Ca 2+ concentration, [Ca 2+ ] i , that rises from less than 100 nM to about 350 nM, measured with Fura-2, not dependent on the presence of external Ca 2+ [8]. Total Fig.…”

“…Recently, we have shown that poisoning the mitochondria with FCCP causes a slow (0.6 nM/s) increase in the basal myoplasmic [Ca 2+ ] i of mouse fast muscle fibers, with a marked effect on the amplitude of Ca 2+ transients [8].…”

“…However, for the case of single twitches or single tetani, successful models for Ca 2+ removal have included SR Ca 2+ ATPase, Parvalbumin and Troponin alone without apparent necessity of mitochondria [4,9,30]. Accordingly, mitochondria poisoning with protonophores, as FCCP, does not appreciably affect Ca 2+ clearance after a tetanus or a single twitch [8,27].…”

Interactions between sarco-endoplasmic reticulum and mitochondria in cardiac and skeletal muscle – pivotal roles in Ca2+ and reactive oxygen species signaling

“…Relevance of SR-mitochondrial Ca 2+ transfer for oxidative metabolism in skeletal muscle When [ATP] was measured using mitochondria-targeted luciferase it was found to be elevated during [Ca 2+ ] m transients that are triggered by membrane depolarization in myotubes, confirming the competence of SR in transferring Ca 2+ to mitochondria to stimulate oxidative metabolism (Jouaville et al, 1999 (Caputo and Bolanos, 2008). Thus, the RyR-mediated mitochondrial transfer of Ca 2+ is relevant for the control of oxidative metabolism and likely allows a local feedback regulation of [Ca 2+ ] c signaling through mitochondria, both in cardiac and skeletal muscle.…”

El acoplamiento excitación-contracción en el músculo esquelético: preguntas por responder a pesar de 50 años de estudio

“…En el caso de las mitocondrias, existen diversas evidencias que señalan su contribución a largo plazo en la homeostasis de Ca 2+ de fibras musculares esqueléticas (63,158,159); no obstante, esto no se acepta universalmente. Por su parte, aunque existen pruebas del funcionamiento del intercambiador Na + -Ca 2+ en el músculo esquelético, su papel en el acoplamiento excitación-contracción ha sido poco estudiado (160)(161)(162).…”