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Mitochondrial pyruvate carriers are required for myocardial stress adaptation

Nature Metabolism volume 2, pages 1248–1264 (2020)Cite this article

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A Publisher Correction to this article was published on 18 November 2020

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Abstract

In addition to fatty acids, glucose and lactate are important myocardial substrates under physiologic and stress conditions. They are metabolized to pyruvate, which enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle metabolism. In the present study, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1−/−) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate and glycogen, and displayed increased protein O-linked N-acetylglucosamine, which was prevented by increasing availability of non-glucose substrates in vivo by a ketogenic diet (KD) or a high-fat diet, which reversed the structural, metabolic and functional remodelling of non-stressed cMPC1−/− hearts. Although concurrent short-term KDs did not rescue cMPC1−/− hearts from rapid decompensation and early mortality after pressure overload, 3 weeks of a KD before transverse aortic constriction was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function.

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Fig. 1: Generation and phenotype of cMPC1−/− mice.
Fig. 2: Substrate metabolism and partitioning in cMPC1−/− hearts.
Fig. 3: Concentrations of metabolic intermediates in perfused cMPC1−/− hearts.
Fig. 4: 13C-labelled enrichment of metabolites in 13C-labelled, substrate-perfused cMPC1−/− hearts.
Fig. 5: KD feeding prevents cardiac dysfunction in cMPC1−/− mice.
Fig. 6: Alternative substrate feeding reverses cardiac dysfunction in cMPC1−/− mice.
Fig. 7: Metabolite profiles in KD hearts.
Fig. 8: Divergent effects of KD feeding on PO-induced cardiac remodelling in cMPC1−/− mice.

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Data availability

All data, apart from the western blots, that support the findings of the present study are available from the corresponding author upon reasonable request. Source data for western blots are provided with this paper.

Change history

  • 18 November 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

This work was supported by: American Heart Association (AHA; grant nos. 16SFRN31810000 to E.D.A. and 15POST22940024 to Y.Z.); Montreal Heart Institute Foundation (CDR); National Institutes of Health (NIH; grant nos. OD019941 to R.M.W., and R01 DK104998 and R00 AR059190 to E.B.T.); T32 (grant no. HL007638 to A.J.R.); American Diabetes Association (grant no. 1-18-PDF-060 (to A.J.R.); and NIH (grant nos. F32 DK101183 to L.R.G., U54DK110858, 1S10OD021505 and 1S10OD018210 to J.E.C., R01HL113057, R01HL132525 and R01HL049244 to E.D.L. and DK091538 to P.A.C.).

Author information

Authors and Affiliations

  1. Fraternal Order of Eagles Diabetes Research Center, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Yuan Zhang, Paul V. Taufalele, Jesse D. Cochran, Jamie Soto, Adam J. Rauckhorst, Lawrence R. Gray, Trevor R. Funari, Rose McGlauflin, Thomas Cassier, Shannon Hitchcock, Kevin Lin, Kevin M. Kato, Jennifer L. Stueve, Lauren Haff, Eric B. Taylor & E. Dale Abel

  2. Division of Endocrinology and Metabolism, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Yuan Zhang & E. Dale Abel

  3. Department of Nutrition, Université de Montréal and Montreal Heart Institute, Montreal, Canada

    Isabelle Robillard-Frayne & Christine Des Rosiers

  4. Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL, USA

    Jonas Maximilian Marx, Patrycja Puchalska, Peter A. Crawford & E. Douglas Lewandowski

  5. Friedrich-Schiller University of Jena, Jena, Germany

    Jonas Maximilian Marx

  6. Mouse Metabolic Phenotyping Core, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Jamie Soto

  7. Metabolomics Core Facility, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Fariba Tayyari, Alvin D. Pewa, Lynn M. Teesch & Eric B. Taylor

  8. Division of Molecular Medicine, Department of Medicine, University of Minnesota, Minneapolis, MN, USA

    Patrycja Puchalska & Peter A. Crawford

  9. Abboud Cardiovascular Research Center, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Kathy Zimmerman, William J. Kutschke, Robert M. Weiss & E. Dale Abel

  10. Department of Biochemistry, School of Medicine, University of Utah, Salt Lake City, UT, USA

    James E. Cox & Jared Rutter

  11. Metabolomics Core Research Facility, School of Medicine, University of Utah, Salt Lake City, UT, USA

    James E. Cox

  12. Howard Hughes Medical Institute, School of Medicine, University of Utah, Salt Lake City, UT, USA

    Jared Rutter

  13. Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Eric B. Taylor

  14. Department of Internal Medicine and Davis Heart and Lung Research Institute, The Ohio State University College of Medicine, Columbus, OH, USA

    E. Douglas Lewandowski

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  1. Yuan Zhang
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Contributions

Y.Z. and E.D.A. designed the research. Y.Z., P.V.T., J.D.C., I.R.-F., J.M.M., J.S., A.D.P., F.T., L.M.T., A.J.R., L.R.G., P.P., T.R.F., R.M., K.Z., W.J.K., T.C., S.H., K.L., K.M.K., J.L.S., L.H., R.M.W. and J.E.C. performed the research. E.B.T. and J.R. provided materials and methodology support. Y.Z., E.D.L., P.A.C., C.D.R. and E.D.A. analysed the data. P.A.C., E.D.L. and C.D.R. contributed to the writing. Y.Z. and E.D.A. wrote the paper.

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Correspondence to E. Dale Abel.

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Extended data

Extended Data Fig. 1 Mitochondrial Characterization of cMPC1-/- hearts.

a, b, MPC protein levels were determined by Western blots in whole heart lysates from 4-week-old cMPC1-/- mice. Images are representative of n = 8 per group. c, Palmitoyl-carnitine driven oxygen consumption by seahorse respirometry in isolated mitochondria (n = 6 both groups). d, Expression of selected electron transport chain (ETC) subunits (from Complex I-V) and VDAC by Western blot in heart lysates from control and cMPC1-/- mice. Images are representative of n = 3 per group. e, mtDNA copy number determined by qPCR analysis and normalized to the nuclear gene RPL13A in 8-week-old cMPC1-/- hearts (Control, 12; cMPC1-/-, 10). f, Representative TEM images of cMPC1-/- hearts from 4 and 8-week-old mice. Images are representative of n = 9 (4-week-old control and cMPC1-/-)/6(8-week-old control)/12(8-week-old cMPC1-/-). g, Quantification of mitochondrial number, volume density and size (4 weeks: Control, 9; cMPC1-/-, 9; 8 weeks: Control, 6; cMPC1-/-, 12). Data are presented as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 2 Glycolysis enzymes and intermediates in cMPC1-/- hearts.

a, Glycolysis-derived metabolic intermediates in 8-week-old control and cMPC1-/- hearts were determined by GC-MS. Mice were random fed before sacrifice. (Control, 15; cMPC1-/-, 11). b, hexokinase I (HK I), GAPDH, O-GlcNAc transferase (OGT) and glycogen synthase (GS) blots were performed in lysates of cardiomyocytes isolated from 8-week-old control and cMPC1-/- mice. Protein quantification was normalized to total Coomassie blue staining. (n = 5 both groups). Data are presented as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 3 Flux scheme of 13C-labeled substrate utilization in cMPC1-/- hearts.

Schematic depicting metabolic fate of uniformly labeled glucose ([U-13C6]-glucose) into glycolysis, the pentose phosphate pathway (PPP), serine biosynthesis pathway (SBP) and the Citric Acid Cycle (CAC). Closed and open circles represent 13C-labeled and 12C-labeled carbons respectively.

Extended Data Fig. 4 13C-MPE for pyruvate, lactate, alanine and serine from [U-13C6]-glucose perfusion.

13C-isotopomer labeling pattern of pyruvate (a), lactate (b), alanine (c) and serine (d) following [U-13C6]-glucose perfusion (Control, 7; cMPC1-/-, 9). Data are presented as mean ± SEM and P values were determined by two-tailed unpaired Student’s t-test.

Extended Data Fig. 5 13C-isotopomer labeling pattern of CAC intermediates from 13C-β-HB perfusion.

13C-labeled CAC intermediates analysis of Langendorff-perfused hearts perfused with [2,4-13C2]-β-HB and unlabeled glucose, palmitate and lactate. Fractional enrichment 13C-labeled isotopomers of citrate (a), glutamate (b), succinate (c), α-ketoglutarate (KG) (d), malate (e), aspartate (f) and β-hydroxybutyrate (HB) (g) were determined by LC-MS (Control, 3; cMPC1-/-, 5). Data are presented as mean ± SEM and P values were determined by two-tailed unpaired Student’s t-test.

Extended Data Fig. 6 Expression levels of hypertrophic markers and selected transcripts encoding metabolic genes in the cMPC1-/- hearts on 2920X and ketogenic diet.

The cMPC1-/- mice under protocol 2 were analyzed for gene expression after 8-week-feeding on Ketogenic (Keto) Diet or control diet (2920X). Sample sizes: n = 4 (Control-2920X), n = 4 (cMPC1-/–2920X), n = 5 (Control-Keto), n = 6 (cMPC1-/–Keto). Data are presented as mean ± SEM and P values were determined by two-way ANOVA followed by Tukey multiple comparison test.

Extended Data Fig. 7 Cardiac function of cMPC1-/- after switching ketogenic diet to regular chow.

10-week-old control and cMPC1-/- mice were fed a Keto Diet for 8 weeks and then 50% of mice of each genotype were switched to regular chow for 6 weeks. The feeding scheme is shown in panel (a). LV mass (b) and ejection fraction (c) were determined via echocardiography at the age of 22 weeks (4 weeks after chow switch). Heart weight and tibia length (d) was determined at the age of 24 weeks. Sample sizes: n = 10 (Control-2920X), n = 8 (cMPC1-/–2920X), n = 9 (Control-Keto), n = 8 (cMPC1-/–Keto). Data are presented as mean ± SEM and P values were determined by two-way ANOVA followed by Tukey multiple comparison test.

Extended Data Fig. 8 Effects of ketogenic diet feeding on pressure overload-induced cardiac remodeling in WT mice.

a–c, 8-week-old WT C57Bl6/J mice were fed with chow and Keto Diet 1 day before TAC surgery. LV mass (a) and ejection fraction (b) were measured by echocardiography prior to surgery and 3 weeks post TAC. Heart weight normalized to tibia length (c) was determined at the time of sacrifice. (n = 5 for both groups). d–f, 12-week-old WT C57Bl6/J mice were fed with chow and Keto Diet 1 day before sham/TAC surgery. LV mass (d) and ejection fraction (e) were measured by echocardiography prior to surgery and 3 weeks post TAC. Heart weight normalized to tibia length (f) was determined at the time of sacrifice. (n = 10 for Keto Diet-TAC group and n = 5 for other groups). Data are presented as mean ± SEM and P value was determined by two-way ANOVA followed by Tukey multiple comparison test.

Extended Data Fig. 9 mRNA level of ME isoforms and ALT activity in cMPC1-/- hearts.

a, mRNA level of three malic enzyme isoforms were determined by qPCR in the hearts from control and cMPC1-/- mice (Control, 6; cMPC1-/-, 5). b, c, ALT activity (Cayman 700260) were determined in the hearts from 8-week-old and 18-week-old control and cMPC1-/- mice (8-week-old group: Control, 6; cMPC1-/-, 4. 18-week-old group: Control, 7; cMPC1-/-,7). Data are presented as mean ± SEM and P values were determined by two-tailed unpaired Student’s t-test.

Extended Data Fig. 10 13C-labeled CAC intermediates analysis from [U-13C5]-glutamine perfusion.

13C-labeled CAC intermediates analysis of Langendorff-perfused hearts perfused with 0.5 mM [U-13C5]-glutamine and unlabeled substrates (10 mM glucose, 0.4 mM palmitate, 0.5 mM lactate, and 0.1 mM β-HB). 13C-MPE of glutamine (a), glutamate (b), alpha ketoglutarate (a-KG) (c), citrate (d), malate (e), succinate (f) and pyruvate (g) were determined by GC-MS (Control, 6; cMPC1-/-, 6). Data are presented as mean ± SEM and P values were determined by two-tailed unpaired Student’s t-test.

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Unprocessed western blots.

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Unprocessed western blots.

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Zhang, Y., Taufalele, P.V., Cochran, J.D. et al. Mitochondrial pyruvate carriers are required for myocardial stress adaptation. Nat Metab 2, 1248–1264 (2020). https://doi.org/10.1038/s42255-020-00288-1

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