Lysine acetylation regulates mitochondrial pyruvate carrier activity and cardiac pyruvate oxidation

Abstract

Background/Objective: The normal heart can acutely switch fuel sources based on delivery. While this is beneficial in physiological scenarios, this flexibility is often lost in cardiac pathologies. During fasting, blood glucose levels drop and increased free fatty acid delivery causes the heart to shut off glucose/pyruvate oxidation and increase fat oxidation. This balance, known as the Randle cycle, has mainly been attributed to regulation of pyruvate dehydrogenase (PDH) activity via PDH phosphorylation. Hypothesis: Decreased pyruvate transport into the mitochondria via posttranslational modifications of the mitochondrial pyruvate carrier (MPC) plays a role in regulating cardiac pyruvate oxidation. Methods: Wildtype mice were allowed to consume normal chow ad libitum or were fasted for 24 hours. After euthanasia, plasma and hearts were collected and left-ventricular muscle fibers were saponin permeabilized and mitochondrial respiration measured in an Oroboros Oxygraph O2k. Cardiac protein lysates were immunoprecipitated with anti-acetyl-lysine beads, and western blotted using standard procedures. A bioluminescent resonance transfer (BRET) assay to detect conformational changes caused by pyruvate/inhibitor binding the MPC was used, and lysine mutant MPC2 constructs were generated by site-directed mutagenesis. Lastly, MPC2-/- H9C2 cardiomyocytes were generated by CRISPR, and respiration measured by Seahorse bioanalyzer in these cells after transfection with WT or lysine mutant MPC2 constructs. Results: 24h fasting reduced blood glucose and insulin concentrations, and increased circulating free fatty acids. Cardiac mitochondrial oxidation of pyruvate/malate was decreased, while oxidation of fatty acids (palmitoyl-CoA+carnitine) was increased in fasted hearts. Western blotting of cardiac lysates showed the expected increase in phosphorylation of PDH-E1α in fasted hearts, known to decrease PDH activity. Immunoprecipitating cardiac lysates with anti-acetyl-lysine beads and blotting for MPC2 suggested increased acetylation of MPC2 in fasted hearts. Using a model structure of the MPC, we have recently proposed and validated lysine 49 (K49) of MPC2 as a critical pyruvate binding site within the MPC. To assess the possible importance of K49 acetylation we mutated K49 to glutamine to mimic acetylation and observed that this K49Q-MPC2 mutant was no longer able to bind pyruvate or competitive inhibitors in a MPC BRET assay. We next created MPC2 knockout H9C2 cardiomyocytes by CRISPR-Cas9 and observed complete loss of MPC2 and MPC1 expression in these cells which decreased pyruvate respiration compared to wildtype H9C2s. Overexpression of wildtype MPC1 and MPC2 constructs increased pyruvate respiration in these MPC-/- cells, while overexpression of the MPC2-K49Q mutant was unable to improve pyruvate respiration. Conclusion: Fasting results in decreased cardiac oxidation of pyruvate in part by acetylation of the MPC and decreased mitochondrial pyruvate transport. National Institutes of Health and SLU institutional funding. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.