FOXO1-mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) decreases glucose oxidation and impairs right ventricular function in pulmonary hypertension: therapeutic benefits of dichloroacetate

Abstract

Pyruvate dehydrogenase kinase (PDK) is activated in right ventricular hypertrophy (RVH), causing an increase in glycolysis relative to glucose oxidation that impairs right ventricular function. The stimulus for PDK upregulation, its isoform specificity, and the long-term effects of PDK inhibition are unknown. We hypothesize that FOXO1-mediated PDK4 upregulation causes bioenergetic impairment and RV dysfunction, which can be reversed by dichloroacetate. Adult male Fawn-Hooded rats (FHR) with pulmonary arterial hypertension (PAH) and right ventricular hypertrophy (RVH; age 6-12 months) were compared to age-matched controls. Glucose oxidation (GO) and fatty acid oxidation (FAO) were measured at baseline and after acute dichloroacetate (1 mM × 40 min) in isolated working hearts and in freshly dispersed RV myocytes. The effects of chronic dichloroacetate (0.75 g/L drinking water for 6 months) on cardiac output (CO) and exercise capacity were measured in vivo. Expression of PDK4 and its regulatory transcription factor, FOXO1, were also measured in FHR and RV specimens from PAH patients (n = 10). Microarray analysis of 168 genes related to glucose or FA metabolism showed >4-fold upregulation of PDK4, aldolase B, and acyl-coenzyme A oxidase. FOXO1 was increased in FHR RV, whereas HIF-1 α was unaltered. PDK4 expression was increased, and the inactivated form of FOXO1 decreased in human PAH RV (P < 0.01). Pyruvate dehydrogenase (PDH) inhibition in RVH increased proton production and reduced GO's contribution to the tricarboxylic acid (TCA) cycle. Acutely, dichloroacetate reduced RV proton production and increased GO's contribution (relative to FAO) to the TCA cycle and ATP production in FHR (P < 0.01). Chronically dichloroacetate decreased PDK4 and FOXO1, thereby activating PDH and increasing GO in FHR. These metabolic changes increased CO (84 ± 14 vs. 69 ± 14 ml/min, P < 0.05) and treadmill-walking distance (239 ± 20 vs. 171 ± 22 m, P < 0.05). Chronic dichloroacetate inhibits FOXO1-induced PDK4 upregulation and restores GO, leading to improved bioenergetics and RV function in RVH.

Fig 1. Acute dichloroacetate improves RV metabolism in FHR ex vivo

In an isolated LV working heart preparation: A. GO is reduced in FHR versus CTR and infusion of dichloroacetate (DCA) (1mM for 40 minutes) increases GO in both CTR (age-matched Sprague-Dawley rats) and FHR. B. Glycolysis is increased in FHR and dichloroacetate tends to reduce this. C. FAO is reduced in FHR hearts. Dichloroacetate further decreases FAO in FHR and CTR. D. Proton production from uncoupled glucose metabolism, which is increased in FHR, is decreased by dichloroacetate. E&F. Total TCA cycle acetyl CoA production and ATP production are reduced in FHR hearts, and are increased by dichloroacetate. G. Dichloroacetate increases the TCA cycle acetyl CoA produced by GO in both CTR and FHR. H. ATP production from GO, which is reduced in FHR, is increased by dichloroacetate.

Fig 2. Chronic dichloroacetate treatment reverses RVH in FHR in vivo

A&B. Histology and the statistical bar graph show that the RV myocytes hypertrophy in FHR is reduced by chronic dichloroacetate treatment (6 months of dichloroacetate 0.75g/l of drinking water). C. The reduced PAAT corrected for HR in FHR is increased by dichloroacetate treatment, reflecting a decrease in pulmonary hypertension. D–F. TAPSE, CO and stroke volume (SV), which are reduced in FHR, are increased by dichloroacetate. G. RVSP tends to increase in FHR and is unchanged by dichloroacetate. H. The reduced cardiac output measured by cardiac catheterization in FHR is increased in FHR+DCA. I. The reduced treadmill waking distance seen in FHR versus CTR is increased by DCA treatment.

Fig 3. Metabolic dysfunction in isolated RV myocytes in FHR

A. Images of freshly isolated RV cardiomyocytes from CTR, FHR and FHR+DCA rats. B&C. GO and FAO is reduced in RV myocytes in FHR. D. Glycolysis is increased in FHR and dichloroacetate tends to reduce glycolysis in RV myocytes. E. GO/glycolysis is reduced in FHR and dichloroacetate tends to increase this ratio in isolated RV myocytes.

Fig 4. Expression profiling of genes related to glucose metabolism highlights the dysregulation of PDK4 in FHR

A. Volcano plot from rat glucose metabolism PCR Array shows PDK4 mRNA is increased in FHR RV. B&C. Chronic dichloroacetate treatment normalizes PDK4 mRNA in FHR. D. The table shows genes that significantly changed (4-fold) in FHR.

Fig 5. Chronic dichloroacetate treatment does not change fatty acid metabolism gene expression profile

Volcano plot for rat fatty acid metabolism shows: A. Increased acyl-coenzyme A oxidase 2 mRNA in FHR RV. B. Dichloroacetate did not change expression of fatty acid metabolism-related genes in FHR. D. The genes that were significantly changed (2-fold) in FHR are listed.

Fig 6. Pyruvate dehydrogenase activity is increased by chronic dichloroacetate treatment in FHR RV

In rats treated with dichloroacetate or control water for 6 months: A. Immunostaining shows that the increased PDK4 protein expression (red) is reduced by dichloroacetate. Dystrophin (green) is used to mark the myocytes membrane and nuclei are stained with DAPI (blue). B&D. Representative immunoblot and mean data normalized to actin expression shows that both PDK2 and PDK4 are increased in the FHR RV. Dichloroacetate decreases PDK4 expression and tends to decrease PDK2 expression. E&F. Representative images and mean data showing reduced PDH activity (band density) in FHR RV. Dichloroacetate treatment increases PDH activity in the RV in FHR.

Fig 7. Forkhead box protein O1 (FOXO1) is increased in FHR RV

A&B. Representative Immunoblot and mean data normalized to actin shows that FOXO1 protein is increased in FHR RV. Dichloroacetate normalizes FOXO1. C&D. Immunostaining of Phospho-FOXO1 Thr24 (an inactivated form of FOXO1) shows that cytosolic expression is reduced in FHR and is restored by chronic dichloroacetate.

Fig 8

PDK4 protein is increased and inactive FOXO1 is decreased in RV tissue in Human PAH. Representative images (each panel is from 1 patient) and mean values from human RV microarray showing the upregulation of PDK4 (A&B) and downregulation of inactive FOXO1 (P-FOXO1 Thr24) (C&D) in human PAH RVs vs. age- and gender-match Control.