Mitochondrial pyruvate carriers are required for myocardial stress adaptation - PubMed (original) (raw)

. 2020 Nov;2(11):1248-1264.

doi: 10.1038/s42255-020-00288-1. Epub 2020 Oct 26.

Paul V Taufalele 1, Jesse D Cochran 1, Isabelle Robillard-Frayne 3, Jonas Maximilian Marx 4 5, Jamie Soto 1 6, Adam J Rauckhorst 1, Fariba Tayyari 7, Alvin D Pewa 7, Lawrence R Gray 1, Lynn M Teesch 7, Patrycja Puchalska 4 8, Trevor R Funari 1, Rose McGlauflin 1, Kathy Zimmerman 9, William J Kutschke 9, Thomas Cassier 1, Shannon Hitchcock 1, Kevin Lin 1, Kevin M Kato 1, Jennifer L Stueve 1, Lauren Haff 1, Robert M Weiss 9, James E Cox 10 11, Jared Rutter 10 12, Eric B Taylor 1 7 13, Peter A Crawford 4 8, E Douglas Lewandowski 4 14, Christine Des Rosiers 3, E Dale Abel 15 16 17

Affiliations

Mitochondrial pyruvate carriers are required for myocardial stress adaptation

Yuan Zhang et al. Nat Metab. 2020 Nov.

Erratum in

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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Conflict of interest statement

Conflict of interest statement

The authors have declared that no conflict of interest exists.

Figures

Extended Data Fig. 1

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 (Complexes 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.

Extended Data Fig. 2

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.

Extended Data Fig. 3

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

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

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

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

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

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

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

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 0.5mM [U-13C5]-glutamine and unlabeled substrates (10mM glucose, 0.4mM palmitate, 0.5mM lactate, and 0.1mM 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.

Figure 1.

Figure 1.. Generation and phenotype of cMPC1−/− mice

(a-c) Loss of MPC1 was confirmed by qPCR (a) in heart lysates of 4-week-old cMPC1−/− (Sample size (n): Control, 5; cMPC1−/−, 4) and Western blot (b-c) in isolated cardiomyocytes from 8-week-old cMPC1−/− mice (n: Control, 4; cMPC1−/−, 5). (d) Mitochondrial pyruvate uptake was performed in mitochondria isolated from 8-week-old cMPC1−/− hearts (n: Control, 5; cMPC1−/−, 4). (e-f) Oxygen consumption rates (OCR) were determined by Seahorse respirometry in isolated mitochondria from 8-week-old cMPC1−/− hearts in the presence of pyruvate (1 mmol/L)/malate (0.25 mmol/L) (e) (n: Control, 4; cMPC1−/−, 4) and glutamate (1mmol/L)/malate (0.25 mmol/L) (f) (n: Control, 10; cMPC1−/−, 9). Concentration of the MPC inhibitor, CHC was 1mM. See methods for concentrations of other inhibitors. (g) Midventricular cross sections of cMPC1 and control hearts at the ages as shown. Scale bar, 500 μm. Images are representative of n= 3 per group. (h-i) LV Mass and Ejection Fraction by echocardiography (j) Heart weight to tibia length ratio (HW/TL) at the ages as shown. Sample size (n) is represented as Control/cMPC1−/−. For h-i, 4-week-old and 8-week-old: 17/21; 18-week-old: 12/15; 34-week-old: 12/12. For j, 4-week-old: 11/8; 8-week-old: 10/8; 18-week-old: 7/11; 34-week-old: 8/7.) (k) mRNA levels of hypertrophic genes, ANP (NPPA), BNP (NPPB) and ACTA1 were determined by qPCR in 4- and 8-week-old cMPC1−/− hearts. For k, 4-week-old: 4/5; 8-week-old: 5/4. (l) Survival curves of control and cMPC1−/− mice over a 1-year period (Control, 34; cMPC1−/−, 30). Data are presented as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test.

Figure 2.

Figure 2.. Substrate Metabolism and Partitioning in cMPC1−/− hearts.

(a-f) Hearts from 8-week-old control and cMPC1−/− mice were perfused with either 14C-/3H-glucose (5mM) or 3H-palmitate (0.4mM) with unlabeled pyruvate (0.5mM) and lactate (1mM), in the working heart mode to determine glucose oxidation (a), glycolysis (b) and fatty acid oxidation (c) respectively. MVO2 (d), cardiac power (e) and cardiac efficiency (f) were also determined in the 3H-palmitate perfused hearts shown in panel c. Three replicates were obtained in each heart for panels a-f (Control, 7; cMPC1−/−, 4). (g-h) HBP flux was estimated by quantifying O-linked N-Acetylglucosamine modifications by western blot in hearts from 8-week-old control and cMPC1−/− mice (Control, 6; cMPC1−/−, 10). (i) Glycogen content was measured in the hearts from 8-week-old control and cMPC1−/− mice under fed and overnight fasting conditions (Fed: Control, 12; cMPC1−/−, 9. Fasting: Control, 15; cMPC1−/−, 14). Data are presented as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test.

Figure 3.

Figure 3.. Concentrations of metabolic intermediates in perfused cMPC1−/− hearts.

Metabolite concentrations determined in Langendorff-perfused 8-week-old hearts perfused with unlabeled substrates (10mM glucose, 0.4mM palmitate, 0.5mM lactate, 0.1mM b-hydroxybutyrate (β-HB) and 0.5mM glutamine). Tissue concentration of pyruvate (a), lactate (b), alanine (c), serine (d), ketones (e-f), and citric acid cycle (CAC) intermediates (g) were determined by GC-MS (Control, 7; cMPC1−/−, 5). The ratio of lactate/pyruvate (h) and β-HB/AcAc (i) were calculated using tissue concentrations of lactate(b), pyruvate (a), β-HB (e) and AcAc (f). Data are presented as mean ± SEM and P values were determined by two-tailed unpaired Student’s t-test.

Figure 4.

Figure 4.. 13C-enrichment of metabolites in 13C-labeled substrate-perfused cMPC1−/− hearts.

(a-d) Metabolic flux analysis of Langendorff hearts from 8-week-old mice perfused with 10mM [U-13C6]-glucose and unlabeled substrates (0.4mM palmitate, 0.5mM lactate, 0.1mM β-HB and 0.5mM glutamine). (a-b) Total molar percentage enrichment (MPE) of all 13C-labeled isotopomers of tissue pyruvate, lactate, alanine, serine and citric acid cycle (CAC) intermediates were determined by GC-MS (Control, 6; cMPC1−/−, 9). (c-f) The flux ratio of pyruvate dehydrogenase/citrate synthase (PDC/CS), pyruvate carboxylation/citrate synthase (PC/CS), PC/PDC and percentage of 13C-labeled citrate recycling into CAC were calculated from tissue 13C-MPE of isotopomers of citrate, OAA moiety of citrate (OAACIT), pyruvate and succinate (Control, 6; cMPC1−/−, 9). (g) Fractional enrichment (FE) analysis of 13C-glutamate performed on neutralized acid extracts of Langendorff hearts perfused with 10mM [1,6-13C2]-glucose, 0.5mM [3-13C]-pyruvate and 1mM [3-13C]-lactate, in addition to 0.4mM unlabeled palmitate (Control, 7; cMPC1−/−, 8). (h) Ratio of anaplerosis to citrate synthase flux (y) was determined in neutralized acid extracts of Langendorff hearts perfused with 0.4mM U-13C-palmitate, in addition to unlabeled substrates (10mM glucose, 0.5mM pyruvate and 1mM lactate) (Control, 9; cMPC1−/−, 4). (i) Langendorff perfusion was performed with 0.5mM [2,4-13C2]-β-HB (β-hydroxybutyrate) and unlabeled substrates (10mM glucose, 0.4mM palmitate, 0.5mM pyruvate and 1mM lactate) (Control, 3; cMPC1−/−, 5). Neutralized acid extracts were analyzed by NMR to determine fractional 13C-enrichment of acetyl-CoA entering the CAC from oxidation of 13C-β-HB, (_Fc_β-HB→Acetyl-CoA). Data are presented as mean ± SEM and P values were determined by two-tailed unpaired Student’s t-test.

Figure 5.

Figure 5.. Ketogenic diet feeding prevents cardiac dysfunction in cMPC1−/− mice.

Control and cMPC1−/− mice were fed with normal chow (2920X) or a ketogenic diet at the age of 3 weeks. (a-b) serum concentration of β-HB (a) and FGF21 (b) after 8 weeks of feeding. (c) Trichrome staining reveals no increase in cardiac fibrosis in cMPC1−/− mouse hearts. Scale bar, 200 μm. Images are representative of n= 3 per group. (d-e) LV mass (d) and ejection fraction (e) were determined by echocardiography after 15 weeks of feeding. (f-g) Heart weight/Tibia length (f) and gene expression analysis (g) were determined at the time of sacrifice after 18 weeks of feeding. For panels a and d-f, n=5 (Control-2920X), n=5 (cMPC1−/−−2920X), n=11 (Control-Keto), n=10 (cMPC1−/−-Keto); for panel b, n=5 (Control-2920X), n=5 (cMPC1−/−−2920X), n=6 (Control-Keto), n=6 (cMPC1−/−-Keto); for panel g, n=6 (Control-2920X), n=5 (cMPC1−/−−2920X), n=6 (Control-Keto), n=6 (cMPC1−/−-Keto). Data are presented as mean ± SEM and P value was determined by two-way ANOVA followed by Tukey multiple comparison test.

Figure 6.

Figure 6.. Alternative substrates feeding reverses cardiac dysfunction in cMPC1−/− mice.

Three different cohorts of mice were fed with ketogenic diet at the age of 10 (a-b), 18 (c-d) and 24 (e-f) weeks. (a) Heart weight/Tibia length (HW/TL) and (b) ejection fraction were determined after 8 weeks of ketogenic diet in the 10-week-old cohort. (c-f) LV mass (c, e) and ejection fraction (d, f) were measured by echocardiography after 8 weeks in the 18- and 24-week-old cohorts respectively. (g-h) Trichrome stained sections reveal mild cardiac interfibrillar fibrosis in the 24-week-old cMPC1−/− mice. Scale bar, 50 μm. Images are representative of n= 7(control)/6(cMPC1−/−). For panels a-b, n=7 (Control-2920X), n=8 (Control-Keto), n=13 (cMPC1−/−−2920X), n=14 (cMPC1−/−-Keto); for the other panels, sample sizes (n) of Control/cMPC1−/−: 7/4 (c-d); 10/3 (e-f); 7/6 (h). Control and cMPC1−/− mice were fed with ketone ester (KE) chow at the age of 10 weeks and serum concentration of β–Hydroxybutyrate (i) was determined after 2 weeks of feeding. Heart weight/Tibia length (j), LV mass (k) and ejection fraction (l) were determined after 8 weeks of KE chow feeding. Another cohort of mice were fed a normal chow diet (NCD) or a 60% high fat diet (HFD) at the age of 10 weeks and LV mass (m) and ejection fraction (n) were measured after 8 weeks of feeding by echocardiography. For panels i-j, n=8 (Control-2920X), n=5 (cMPC1−/−−2920X), n=15 (Control-KE), n=11(cMPC1−/−-KE); for panels k-l, n=4 (all groups); for panels m-n, n=6 (Control-NCD), n=4 (Control-HFD), n=4(cMPC1−/−-NCD), n=5 (cMPC1−/−-HFD). Data are presented as mean ± SEM and P value was determined by two-way ANOVA followed by Tukey multiple comparison test. Control vs cMPC1−/−: **P < 0.01. Treatment vs baseline: ##P < 0.01.

Figure 7.

Figure 7.. Metabolite Profiles in KD hearts

Control and cMPC1−/− mice were fed with ketogenic diet starting at the age of 8 weeks and hearts were freeze-clamped after 6 weeks of feeding. GC-MS and LC-MS were employed to determine levels of metabolites in the heart tissue. The fold change of all metabolites was normalized to control hearts fed on normal chow (2920X). Two-way heatmap (a) and Principal Component Analysis (PCA) (b) were analyzed and plotted by MetaboAnalyst. Acyl-carnitines(c), glycolysis intermediates (d), CAC intermediates (e), ADP and ATP (f), PPP and SBP intermediates (g) and nucleotides (h) were plotted and analyzed by two-way ANOVA followed by Tukey multiple comparison test. Sample sizes are as follows: n=7 (Control-2920X), n=5 (cMPC1−/−−2920X), n=5 (Control-Keto), n=6 (cMPC1−/−-Keto). O-GlcNAc blot (i) normalized to total Coomassie blue staining (i) and glycogen storage (k) was determined in 18-week-old control and cMPC1−/− mice after 8 weeks of KD (Protocol 2). For panel j, sample sizes are as follows: n=6 (Control-2920X), n=5 (cMPC1−/−−2920X), n=5 (Control-Keto), n=4 (cMPC1−/−-Keto). For panel k, sample sizes are as follows: n=7 (Control-2920X), n=5 (cMPC1−/−−2920X), n=5 (Control-Keto), n=6 (cMPC1−/−-Keto). Data are presented as mean ± SEM and analyzed by two-way ANOVA followed by Tukey multiple comparison test. All P values <0.05 are shown on the figure panels. If not indicated, then P values are >0.05.

Figure 8.

Figure 8.. Divergent effects of ketogenic diet feeding on pressure overload-induced cardiac remodeling in cMPC1−/− mice.

Control and cMPC1−/− mice were subjected to TAC surgery at the age of 6 weeks and heart weights or lung weights normalized to tibia length (a-b) and ejection fraction (c) were determined 3 weeks post TAC. Cardiac fibrosis was revealed by Trichrome staining (d-e) at time of sacrifice. Scale bar, 200 μm. Images are representative of n= 3 per group. Transverse aortic constriction surgery was performed on 8-week-old control and cMPC1−/− mice that were maintained on chow diet or initiated on a ketogenic diet (KD) at the time of TAC surgery. The survival curve following TAC is shown in panel f. Control and cMPC1−/− mice were fed with ketogenic diet at weaning and were subjected to TAC surgery at the age of 6 weeks. Ejection fraction (g) and heart weights or lung weights normalized to tibia length (h, i) were assessed at 3 weeks post TAC. For panels a-c and e, n=3 (all groups); for panel F, n=6 (Control-2920X), n=6 (Control-Keto), n=7 (cMPC1−/−−2920X), n=8 (cMPC1−/−-Keto); for panel g-i, n=5 (Control-2920X), n=5 (cMPC1−/−−2920X), n=7 (Control-Keto), n=6 (cMPC1−/−-Keto). Data are presented as mean ± SEM and P value was determined by two-way ANOVA followed by Tukey multiple comparison test.

References

    1. Wende AR, Brahma MK, McGinnis GR & Young ME Metabolic Origins of Heart Failure. JACC Basic Transl Sci 2, 297–310, doi: 10.1016/j.jacbts.2016.11.009 (2017). - DOI - PMC - PubMed
    1. Doenst T, Nguyen TD & Abel ED Cardiac metabolism in heart failure: implications beyond ATP production. Circ Res 113, 709–724, doi: 10.1161/CIRCRESAHA.113.300376 (2013). - DOI - PMC - PubMed
    1. Owen OE, Kalhan SC & Hanson RW The key role of anaplerosis and cataplerosis for citric acid cycle function. J Biol Chem 277, 30409–30412, doi: 10.1074/jbc.R200006200 (2002). - DOI - PubMed
    1. Des Rosiers C, Labarthe F, Lloyd SG & Chatham JC Cardiac anaplerosis in health and disease: food for thought. Cardiovasc Res 90, 210–219, doi: 10.1093/cvr/cvr055 (2011). - DOI - PMC - PubMed
    1. Pound KM et al. Substrate-enzyme competition attenuates upregulated anaplerotic flux through malic enzyme in hypertrophied rat heart and restores triacylglyceride content: attenuating upregulated anaplerosis in hypertrophy. Circ Res 104, 805–812, doi: 10.1161/CIRCRESAHA.108.189951 (2009). - DOI - PMC - PubMed

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