Calcium Activation of Oxidative Phosphorylation in Skeletal Muscle Mitochondria

Abstract

Skeletal muscle work rate can rise ∼100-fold above rest. Thus, the ability of myocytes to maintain homeostasis is severely challenged during exercise. This challenge may be partially met through calcium activation of both energy supply and demand. This study focused on energy supply by examining calcium activation of oxidative phosphorylation in skeletal muscle mitochondria. Mitochondrial membrane potential (ΔΨ), NAD/NADH ratio, and cytosolic phosphorylation potential (ΔGATP) were measured at anoxia, resting (State 4), intermediate, and maximal (State 3) respiration, in isolated porcine skeletal muscle mitochondria. Intermediate respiration was elicited with a creatine kinase energetic clamp to set the PCr/Cr ratio, and thus, ATP/ADP ratio. All measurements were made with glutamate+malate in calcium-depleted mitochondria. State 3 and 4 NADH increased linearly with varying calcium (17 - 31% and 50 - 67% reduced for State 3 and 4, respectively), as did State 3 respiration (204 - 420 nmol O2/min/nmol cytochrome a,a3). State 4 respiration increased slightly (31 - 45 nmol O2/min/nmol cytochrome a,a3), and there was no correlation with State 3 or 4 ΔΨ and calcium. For PCr/Cr ratios of 0.5 - 2.0, respiration ranged from 1/3 - 2/3 of State 3, changing linearly with ΔΨ, NAD/NADH, ΔGATP, NAD/NADH-ΔΨ, NAD/NADH-ΔGATP, and ΔΨ-ΔGATP. The slopes of these relationships (conductances) increased 1.7 - 2.8-fold with calcium, and NADH (32 - 45% reduced vs. 60 - 73% reduced) and ΔΨ (−189 to −193 mV vs. −197 to −203 mV) also increased. Finally, calcium added at anoxia resulted in a ∼35% increase in NADH reduction level. These data suggest that calcium increases NADH production and conductances of the cytochrome chain and/or ATP production/transport. This balanced activation of oxidative phosphorylation supports a six-fold increase in flux (State 4 to PCr/Cr=0.5) while maintaining NAD/NADH and ΔΨ at near resting values.