Differential activation of TCA cycle activity and PDH flux by dichloroacetate in skeletal muscle measured by hyperpolarized [2-13C,3-2H3]pyruvate

Despite the central role of skeletal muscle bioenergetics in whole-body metabolic health, assessing mitochondrial oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity in vivo remains a major challenge. While hyperpolarized [1- 13 C]pyruvate has been used to probe pyruvate dehydrogenase (PDH) flux to approximate TCA cycle activity, this approach relies on the unreliable assumption that PDH and TCA cycle fluxes are tightly coupled. Here, we demonstrate that hyperpolarized [2- 13 C,3- 2 H 3 ]pyruvate can track label-incorporation into TCA cycle-derived glutamate in rat skeletal muscle. Following intravenous dichloroacetate administration, we observed a greater increase in hyperpolarized [1- 13 C]acetyl-L-carnitine relative to [5- 13 C]glutamate, suggesting disproportionately increased PDH flux relative to TCA cycle flux. A similar trend was also observed in ex vivo GC-MS analysis of skeletal muscle tissue collected from rats injected with [U- 13 C 3 ]pyruvate. Together, these findings highlight the complex interplay between PDH and TCA cycle fluxes and establish hyperpolarized [2- 13 C,3- 2 H 3 ]pyruvate as a robust agent for assessing mitochondrial metabolism in skeletal muscle.