Loss of mitochondrial pyruvate transport initiates cardiac glycogen accumulation and heart failure

Abstract

Heart failure involves metabolic alterations including increased glycolysis despite unchanged or decreased glucose oxidation. The mitochondrial pyruvate carrier (MPC) regulates pyruvate entry into the mitochondrial matrix, and cardiac deletion of the MPC in mice causes heart failure. How MPC deletion results in heart failure is unknown. We performed targeted metabolomics and isotope tracing in wildtype (fl/fl) and cardiac-specific Mpc2-/- (CS-Mpc2-/-) hearts after in vivo injection of U-¹³C-glucose. Failing CS-Mpc2-/- hearts contained normal levels of ATP and phosphocreatine, yet these hearts displayed increased enrichment from U-¹³C-glucose and increased glycolytic metabolite pool sizes. ¹³C enrichment and pool size was also increased for the glycogen intermediate UDP-glucose, as well as increased enrichment of the glycogen pool. Glycogen levels were increased ~6-fold in the failing CS-Mpc2-/- hearts, and glycogen granules were easily detected by electron microscopy. In young, non-failing CS-Mpc2-/- hearts, increased glycolytic ¹³C enrichment occurred, but glycogen levels remained low and unchanged compared to fl/fl hearts. Inhibiting glycogen synthase with MZ-101 reduced cardiac glycogen levels and improved heart failure. Feeding a ketogenic diet to CS-Mpc2-/- mice reversed the heart failure and normalized the cardiac glycogen and glycolytic metabolite accumulation. Cardiac glycogen levels were also elevated in mice infused with angiotensin-II, and both the cardiac hypertrophy and glycogen levels were improved by ketogenic diet. Thus, loss of MPC in the heart causes glycogen accumulation and heart failure, while inhibition of glycogen synthesis or a ketogenic diet can reverse both the glycogen accumulation and heart failure.

Keywords: heart failure; ketogenic diet; mitochondria; pyruvate.

Fig. 1:. Heart Failure in CS-Mpc2−/− mice is not due to energetic stress.

a Heart weight/tibia length (HW/TL) in CS-Mpc2−/− and fl/fl littermates, n=6. b Representative gross images of excised fl/fl and CS-Mpc2−/− hearts. Scale bar = 5 mm. c Heatmap of differentially expressed genes identified by RNA sequencing of hearts from fl/fl and CS-Mpc2−/− littermates, n=3. Most prominently dysregulated KEGG pathways upregulated and downregulated are listed. d-n mass spectrometry normalized peak area pool sizes of adenosine triphosphate (ATP) (d), adenosine diphosphate (ADP) (e), adenosine monophosphate (AMP) (f), phosphocreatine (g), creatine (h), phosphocreatine/creatine (PCr/Cr) ratio (i), PCr/ATP ratio (j) oxidized nicotinamide adenine dinucleotide (NAD⁺) (k), reduced nicotinamide adenine dinucleotide (NADH) (l), oxidized nicotinamide adenine dinucleotide phosphate (NADP⁺) (m), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) (n) in hearts of fl/fl and CS-Mpc2−/− littermates that had been injected i.p. with U-¹³C-glucose, n=11–12. Data are presented as mean±SD. Data were evaluated by unpaired, two-tailed Student’s t-test with Welch correction.

Fig. 2:. Failing CS-Mpc2−/− hearts display increased glycolysis and accumulate glycogen.

a Schematic of U-¹³C-glucose labeling of glycolytic metabolites and glycogen, created with BioRender. b-i ¹³C % enrichment of glucose (b), glucose-6-phosphate (G6P) (c), fructose bisphosphate (FBP) (d), dihydroxyacetone phosphate (DHAP) (e), phosphoenolpyruvate (PEP) (f), pyruvate (g), lactate (h), and alanine (i) in hearts from fl/fl and CS-Mpc2−/− mice after injection with U-¹³C-glucose, n=11–12. j Cardiac glycogen levels measured biochemically in fl/fl and CS-Mpc2−/− littermates, n=16. k Representative transmission electron microscopy images at 5,000X magnification of the cardiac papillary muscle of fl/fl and CS-Mpc2−/−, white arrowhead denotes glycogen granules. Scale bars = 1 μm. l-m ¹³C % enrichment (l) and pool size (m) of uridine diphosphate (UDP)-glucose, n=11–12. n-o ¹³C % enrichment of the glucose within glycogen for fully labeled M6 (n), and fully unlabeled M0 (o), n=11–12. Data are presented as mean±SD. Data were evaluated by unpaired, two-tailed Student’s t-test with Welch correction. See also Supplementary Figs. 1–4.

Fig. 3:. Glucose uptake is increased, and glycogen synthesis should be inhibited in CS-Mpc2−/− hearts.

a Glucose uptake assessed by 2-deoxyglucose in the present or absence of insulin in cardiac muscle fibers from fl/fl and CS-Mpc2−/− littermates, n=9–12. b Western blot images of phosphorylated (serine-473) and total protein kinase B (AKT) and the ratio of quantified densitometry for pAKT/AKT from hearts of fl/fl and CS-Mpc2−/− mice injected with either saline vehicle or 5mU/g insulin. c-g Western blot images of phosphorylated glycogen synthase (pGS) serine-640, total glycogen synthase (GS), muscle glycogen phosphorylase (PYGM), glycogenin-1, mitochondrial pyruvate carrier (MPC) 1, MPC2, and -tubulin (c), and the normalized densitometry quantification for MPC1/tubulin (d), MPC2/tubulin (e), pGS/total GS (f), and glycogenin-1/tubulin (g) in hearts of fl/fl and CS-Mpc2−/− littermates, n=4. Data are presented as mean±SD. Data in a were evaluated by two-way analysis of variance (ANOVA) with Tukey post-hoc multiple-comparisons test. Data in b were evaluated by unpaired, two-tailed Student’s t-test with Welch correction due to only n=2 in saline groups. Data in d-g were evaluated by unpaired, two-tailed Student’s t-test with Welch correction.

Fig. 4:. Non-failing CS-Mpc2−/− hearts from young mice display increased glycolysis, but no glycogen accumulation.

a-k ¹³C % enrichment of glucose (a), gluocse-6-phosphate (G6P) (b), fructose bisphosphate (FBP) (c), dihydroxyacetone phosphate (DHAP) (d), 2/3-phosphoglycerate (PG) (e), phosphoenolpyruvate (PEP) (f), pyruvate (g), lactate (h), alanine (i), and uridine diphosphate (UDP)-glucose (j), and the UDP-glucose normalized pool size (k) in hearts of young non-failing CS-Mpc2−/− and fl/fl littermates injected i.p. with U-¹³C-glucose, n=8–10. l Heart glycogen content measured biochemically in young non-failing CS-Mpc2−/− and fl/fl littermates, n=6–7. m Representative transmission electron microscopy images at 15,000X magnification of the cardiac papillary muscle in young fl/fl and CS-Mpc2−/− littermates. Scale bars = 1 μm. Data are presented as mean±SD, and were analyzed by unpaired, two-tailed Student’s t-test with Welch correction. See also Supplementary Figs. 5–6.

Fig. 5:. Inhibition of glycogen synthase with MZ-101 reduces glycogen and improves heart failure in CS-Mpc2−/− mice.

a-b Blood glucose (a) and blood lactate (b) measured after a 2 hour fast from fl/fl and CS-Mpc2−/− mice treated with either vehicle or 100 mg/kg MZ-101 twice daily for 2 weeks, n=7–12. c-e Heart (c), soleus (d), and liver (e) glycogen content measured biochemically in fl/fl and CS-Mpc2−/− mice treated with either Veh or MZ-101, n=7–12. f Heart weight to tibia length ratio (HW/TL) of fl/fl and CS-Mpc2−/− mice treated with either Veh or MZ-101, n=7–12. g Representative H&E stained images of mid-ventricular short axis sections of fl/fl and CS-MPC2−/− mice treated with either Veh or MZ-101. Scale bars = 1 mm. h-i Cardiac gene expression for Nppa and Nppb markers of heart failure from fl/fl and CS-Mpc2−/− mice treated with either vehicle or 100 mg/kg MZ-101 twice daily for 2 weeks, n=7–12. j Representative western blot images for phosphorylated and total glycogen synthase 1 and α-Tubulin, and normalized quantified densitometry for phosphorylated/total glycogen synthase, n=4 per group. Data are presented as mean±SD, and were analyzed by unpaired, two-tailed Student’s t-test with Welch correction.

Fig. 6:. Ketogenic diet normalizes the CS-Mpc2−/− heart failure and glycogen accumulation.

a Heart weight to tibia length ratio (HW/TL) of fl/fl and CS-Mpc2−/− mice fed either low fat (LF) or ketogenic diet (KD), n=4–7. b Heart glycogen content measured biochemically in fl/fl and CS-Mpc2−/− mice fed either a LF diet or KD, n=4–7. c Representative transmission electron microscopy images at 5,000X magnification of the cardiac papillary muscle of fl/fl and CS-Mpc2−/− mice fed LF or KD. Arrowheads denote glycogen granules. Scale bars = 1 μm. d Western blot images of phosphorylated glycogen synthase (serine 640) and total glycogen synthase from hearts of fl/fl or CS-Mpc2−/− mice fed either LF or KD, n=2. e-j ¹³C % enrichment of glucose (e) and the glucose within glycogen (f), and the normalized pool size of glucose-6-phosphate (G6P) (g), pyruvate (h), uridine diphosphate (UDP)-glucose (i), and glycogen (j) in fl/fl and CS-Mpc2−/− mice that were fed LF or KD and injected i.p. with U-¹³C-glucose, n=6–7. Data are presented as mean±SD, and were analyzed by two-way analysis of variance (ANOVA) with Tukey post-hoc multiple-comparisons test. See also Supplementary Fig. 7.

Fig. 7:. AngII-induced cardiac hypertrophy increases glycogen and is improved by ketogenic diet.

a and b Blood glucose (a) and plasma β-hydroxybutyrate (β-HB) concentrations (b) of saline vehicle or AngII-infused mice fed low fat (LF) diet or ketogenic diet (KD), n=7–10. c Heart weight to tibia length ratio (HW/TL) of saline vehicle and AngII infused mice fed LF or KD, n=7–10. d Cardiac glycogen measured biochemically from mice infused with saline or AngII fed either LF or KD, n=7–10. e and f Normalized gene expression for Mpc1 (e) and Mpc2 (f) in hearts from mice infused with saline or AngII fed LF or KD, n=7–10. g and h representative western blot images of phosphorylated glycogen synthase (pGS1), total glycogen synthase (GS1), glycogenin-1, and -tubulin (g), and the normalized densitometry quantified for pGS/GS (h) from hearts of mice infused with saline or AngII fed LF or KD, n=4. Data are presented as mean±SD, and were analyzed by two-way analysis of variance (ANOVA) with Tukey post-hoc multiple-comparisons test.