Skeletal Muscle Pyruvate Dehydrogenase Activity Follows the Cellular Energetic Status during Contraction
Authors: Kasper Jonathan D , Lewis Matthew T , Gudziak Gregory L , Meyer Ronald A , Wiseman Robert W 
Published: November 2023

Pyruvate dehydrogenase (PDH) is a mitochondrial matrix enzyme that regulates glucose oxidation in skeletal muscle. The activation status of PDH is regulated via reversible phosphorylation catalyzed by a set of pyruvate dehydrogenase phosphatases (PDP) and kinases (PDK) each with their own allosteric regulators. The PDKs are sensitive to energetically important substrates (ATP:ADP, NADH:NAD+, Acetyl‐CoA:CoA) whereas PDP activity is determined primarily through calcium all of which change dramatically during exercise. PDH dephosphorylation and the subsequent increase in activity is brought about during contraction by both a reduction in the cellular energetic status and increased calcium resulting in coordinated inhibition of PDK and stimulation of PDP activity respectively. However because many of these modulators co‐vary during contraction, the relative roles of muscle energetic status or calcium cycling on PDH activity are difficult to discern. We hypothesized that by reducing mitochondrial density that the sustainable energetic load and calcium cycling could be temporally separated. To deconvolve the relative control strength of energetics versus calcium cycling in activating PDH, adult male Wistar rats were treated with methimazole (MMI), a thyroid hormone synthesis inhibitor, for four weeks in their drinking water (0.025% w/v) to reduce skeletal muscle mitochondrial density by 50%. Phosphorus nuclear magnetic resonance spectroscopy (PMRS) was used to determine the muscle energetic status (Pi, PCr, ATP) in vivo using a custom built probe and force transducer. Parallel benchtop experiments were performed to obtain muscle samples for determination of PDH activity at each frequency using a radioisotopic tracer assay. In each experiment the twitch contractile intensity of the gastrocnemius muscle was controlled in vivo through surgically implanted electrodes adjacent to the sciatic nerve. Control rats were stimulated at 0.5Hz and 1.0Hz intensities corresponding to 50 and 100% of the aerobic threshold while MMI‐treated rats were stimulated at 0.25Hz and 0.5Hz intensities which were halved based on the measured drop in citrate synthase activity (50%). The resting PCr content in both control and MMI‐treated animals were identical. Further at 0.5 Hz 30% of PCr was hydrolyzed irrespective of treatment group. There were no discernable differences in intracellular pH. At 100% of the aerobic threshold 55% of PCr was hydrolyzed irrespective of treatment group. The time constant of PCr recovery was significantly longer in the MMI‐treatment group consistent with the decreased mitochondrial density. Interestingly, when PDH activity was plotted against the steady state energetics (ΔPCr % initial), there was no significant difference in slope between the control and MMI groups. The MMI group was stimulated at half the contractile intensity (and by extension a significant reduction in calcium cycling), yet the relationship between PDH activity and steady state energetics in the MMI group is no different than controls. Therefore these data suggest that PDH activity is primarily regulated through changes in the cellular energetic status during increased contractile activity and not through changes in cytosolic calcium cycling.Support or Funding InformationSupported by NIH DK 095210