Mitochondrial pyruvate carrier abundance mediates pathological cardiac hypertrophy

Abstract

Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodelling during stress. Here we show that mitochondrial pyruvate carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as in the myocardium of mice induced to fail by angiotensin II or through transverse aortic constriction. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-[13C2]glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse aortic constriction protected against cardiac hypertrophy and failure. Collectively, our findings demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling.

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Fig. 1: MPC1 and MPC2 abundance is lower in hearts of patients with hypertrophic cardiomyopathy and mice induced to undergo pathological hypertrophy.
Fig. 2: MPC1 or MPC2 knockout from birth induces heart hypertrophy and increases mortality.
Fig. 3: Inducibly decreasing MPC expression in adult mice switches metabolism to an anabolic programme that causes hypertrophic growth.
Fig. 4: Conserved MPC expression in MPC1 overexpressers in a TAC model of cardiac hypertrophy is cardioprotective and limits aberrant growth.

Data availability

The data that support the findings of this study are available from the corresponding author on request. Human Metabolome Technologies uses the Human Metabolome Database (HMDB) (https://hmdb.ca/). The metabolomic data and the HMDB link for each metabolite are available online as Supplementary Table 1 and Supplementary Table 2. Source data for Figs. 1–4 and Extended Data Figs. 1–3, 5 and 6 are available with the paper.

The Ensembl Gene entry used for the generation of MPC1 conditional knockout with tetO knock-in was ENSMUSG00000023861.

The Ensembl Gene entry used for the generation of MPC2 conditional knockout with tetO knock-in was ENSMUSG00000026568.

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Acknowledgements

This work was supported by the British Heart Foundation, the European Research Council (ERC Advanced award) and the Medical Research Council. P.E. is supported by The Barts Charity Cardiovascular Programme Award G00913. We also thank K. Hartmann for her technical assistance and Biobank of ‘A Coruña’ (XXIAC-Instituto de Investigación Biomédica de A Coruña) for providing healthy heart tissue samples. T.E. acknowledges support from NIHR Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and KCL; the Centre of Excellence in Medical Engineering funded by the Welcome Trust and EPSRC (WT 088641/Z/09/Z) and the KCL Comprehensive Cancer Imaging Centre funded by the Cancer Research UK (CRUK) and EPSRC in association with MRC and DoH. We acknowledge the metabolic flux analysis facility of the Barts School of Medicine and Dentistry created with the support of the Barts and the London Charity, grant MGU0401.

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Contributions

P.E. and M.F.-C. conceived the project. M.F.-C., A.K., A.A.F. and O.P. conducted experiments. S.K. performed histology. K.B. and V.M. conducted the C13 flux metabolomics using LC–MS analysis. N.D., M.G.C.-L. and M.G.V. collected human tissue samples and clinical data. M.F.-C. and T.R.E. analysed the metabolomic data. P.E. and M.F.-C. wrote the manuscript. All authors discussed the results and reviewed the manuscript.

Corresponding authors

Correspondence to Mariana Fernandez-Caggiano or Philip Eaton.

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Peer review information Primary Handling Editor: George Caputa.

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Extended data

Extended Data Fig. 1

a, Cardiac function of wild type mice injected with saline or angiotensin presented as ejection fraction, fractional shortening, cardiac output and stroke volume. Development of cardiac hypertrophy was indexed by the heart weight/tibia length ratio (n = 5 mice/group). b, Cardiac function of wild type mice subjected to a sham or thoracic aortic constriction operation presented as ejection fraction, fractional shortening, cardiac output and stroke volume. Development of cardiac hypertrophy was indexed by the heart weight/tibia length ratio (n = 5 mice/group). All statistical significances (P < 0.05) were calculated using unpaired, two-tailed, Student’s t-tests. Data are presented as mean ± S.E.M. Source data

Extended Data Fig. 2 Cardiac function of partial MPC1 KO (-/ + CreMPC1), Mhy6Cre control and wild type mice was indexed using echocardiography.

Cardiac function is presented as interventricular septum thickness at end-diastole and end systole (IVSd and IVSs), left ventricular internal-diastolic (LVIDd), internal-systolic dimension (LVIDs), left ventricular posterior wall in diastole and systole (LVPWd and LVPWs), end diastolic and end systolic volume (Vol d and Vol s), stroke volume, ejection fraction, and fractional shortening (n = 9–10 mice/group). Statistical significances (P < 0.05) were calculated using one-way ANOVA adjusted using Dunnett’s test for multiple comparisons. Data are presented as mean ± S.E.M. Source data

Extended Data Fig. 3 Western immunoblotting analysis of homozygous (TAXMPC1 KO) or heterozygous (TAXMPC1+/-) mouse hearts showed that tamoxifen injection to 8-week-old transgenic TAXMPC1 adults promoted a significant reduction in MPC1 and MPC2 protein abundance (n = 4–6 biologically independent heart samples/group).

All statistical significances (P < 0.05) were calculated using unpaired, two-tailed, Student’s t-tests. Data are presented as mean ± S.E.M. Source data

Extended Data Fig. 4 Immunofluorescence analysis of dystrophin (green), MPC1 (red) and DAPI enabled measurement of cardiomyocyte cross-sectional area.

TAXMPC1+/-, which are characterised by partial knockout of MPC expression, were hypertrophic as evidenced by the presence of larger cells. (Scale bar represents 25 μm). Representative image for n = 3 biologically independent hearts/group.

Extended Data Fig. 5 Diagram adapted from Carpenter et al36.

to show measurement of flux towards the PPP. TAXMPC1 + /- hearts presented an increase in doubly and singly labelled 13C lactate and pyruvate (n = 5–6 biologically independent heart samples/group). Peak area was normalized by HEPES. All statistical significances (P < 0.05) were calculated using unpaired, two-tailed, Student’s t-tests. Data are presented as mean ± S.E.M. Source data

Extended Data Fig. 6

a, Proliferation and growth of H9C2 cells was increased in response to the mitochondrial pyruvate carrier inhibitor UK5099 (10 µM or 20 µM). b, H9C2 cells treated with 20 µM UK5099 showed a significant reduction in NADP/NADPH and NAD/NADH ratios, whereas only the NAD/NADH ratio was significant in H9C2 cells treated with 10 µM UK5099 (n = 4 biologically independent cell samples/group). Statistical significances (P < 0.05) were calculated using one-way ANOVA adjusted using Dunnett’s test for multiple comparisons. Data are presented as mean ± S.E.M. Source data

Supplementary information

Reporting Summary

Supplementary Tables 1 and 2

Supplementary Table 1: Quantitative estimation of target metabolites Control-Cre and TAXMPC1; Supplementary Table 2: Quantitative estimation of target metabolites Control-tTA and tTAMPC1 hearts.

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Fernandez-Caggiano, M., Kamynina, A., Francois, A.A. et al. Mitochondrial pyruvate carrier abundance mediates pathological cardiac hypertrophy. Nat Metab 2, 1223–1231 (2020). https://doi.org/10.1038/s42255-020-00276-5

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