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CRAT links cholesterol metabolism to innate immune responses in the heart

Nature Metabolism volume 5, pages 1382–1394 (2023)Cite this article

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Abstract

Chronic inflammation is associated with increased risk and poor prognosis of heart failure; however, the precise mechanism that provokes sustained inflammation in the failing heart remains elusive. Here we report that depletion of carnitine acetyltransferase (CRAT) promotes cholesterol catabolism through bile acid synthesis pathway in cardiomyocytes. Intracellular accumulation of bile acid or intermediate, 7α-hydroxyl-3-oxo-4-cholestenoic acid, induces mitochondrial DNA stress and triggers cGAS–STING-dependent type I interferon responses. Furthermore, type I interferon responses elicited by CRAT deficiency substantially increase AIM2 expression and AIM2-dependent inflammasome activation. Genetic deletion of cardiomyocyte CRAT in mice of both sexes results in myocardial inflammation and dilated cardiomyopathy, which can be reversed by combined depletion of caspase-1, cGAS or AIM2. Collectively, we identify a mechanism by which cardiac energy metabolism, cholesterol homeostasis and cardiomyocyte-intrinsic innate immune responses are interconnected via a CRAT-mediated bile acid synthesis pathway, which contributes to chronic myocardial inflammation and heart failure progression.

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Fig. 1: CRAT is downregulated in patients with HF and its activity is decreased in a mouse model of HF induced by transaortic constriction.
Fig. 2: Cardiomyocyte-specific deletion of CRAT leads to dilated cardiomyopathy in mice.
Fig. 3: Depletion of CRAT triggers type I interferon responses in primary cardiomyocytes.
Fig. 4: Bile acid synthesis intermediate 7-HOCA promotes mtDNA stress and induces ISG expression.
Fig. 5: Depletion of CRAT activates DNA-sensing AIM2 inflammasome.
Fig. 6: Depletion of caspase-1, cGAS or AIM2 reverses myocardial inflammation and dilated cardiomyopathy induced by CRAT knockout.
Fig. 7: Schematic model of the role of CRAT in cholesterol metabolism and innate immune responses.

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Data availability

The data that support the findings of this study are available within the paper in Supplementary Information files. Detailed information of key reagents is provided in Supplementary Table 1. The raw sequence data reported in this paper have been deposited in GEO (accession no. GSE200057). This RNA-seq dataset, together with the other single-cell RNA-seq datasets GSE109816 and GSE121893 (ref. 6), is publicly accessible at GEO. Source data are provided with this paper.

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Acknowledgements

This work is supported by National Institutes of Health grants R01 HL122736 (to L.X.), R01 HL166549 (to L.X.) and R01 DK123186 (to X.P.).

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Author notes

  1. These authors contributed equally: Hua Mao, Aude Angelini, Shengyu Li.

Authors and Affiliations

  1. Department of Medicine, Section of Cardiovascular Research, Baylor College of Medicine, Houston, TX, USA

    Hua Mao, Aude Angelini, Luge Li, Xinchun Pi & Liang Xie

  2. Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX, USA

    Hua Mao, Aude Angelini, Luge Li, Xinchun Pi & Liang Xie

  3. Center for Cardiovascular Regeneration, Houston Methodist Research Institute, Houston, TX, USA

    Shengyu Li & Guangyu Wang

  4. Department of Cardiothoracic Surgery, Weill Cornell Medicine, Cornell University, New York, NY, USA

    Shengyu Li & Guangyu Wang

  5. University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Cam Patterson

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Contributions

L.X. and X.P. conceived and designed the studies. H.M., A.A., L.L., C.P., X.P. and L.X. performed the experiments and interpreted data. G.W. and S.L. performed the RNA-seq analysis. L.X. and X.P. co-wrote the manuscript. L.X. carried out experimental design and interpretation and supervised the project. All authors approved the manuscript.

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Correspondence to Liang Xie.

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Nature Metabolism thanks Joseph Hill, A. Phillip West and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Activation of Myh6-MerCreMer with low doses of tamoxifen has no deleterious effects on cardiac function.

Adult Myh6-MerCreMer− or Myh6-MerCreMer + male and female mice were injected with tamoxifen for 4 consecutive days at a dose of 20 mg/kg/day. a, b, Echocardiographic analyses were performed to estimate cardiac contractile function after 2 months. c-f, RT-PCR analysis of the expression of the hypertrophic marker genes and the pro-fibrotic genes in the hearts. No significant changes were observed between these two groups. n = 5 mice per group. Data are shown as the mean ± SEM. Analysis was unpaired two-tailed Student’s t-test (a-f). NS, not significant.

Extended Data Fig. 2 Cardiomyocyte-specific deletion of CRAT increases acetyl-CoA level in the hearts.

a, Western-blots showed that CRAT was specifically depleted in CRAT-mKO male and female hearts but not in lungs or gastrocnemius muscles. b, CRAT activity was dramatically decreased in CRAT-mKO male and female hearts. c, Acetyl-CoA level was significantly increased in CRAT-mKO male and female hearts. n = 4 (a), 9 (b, WT), 7 (b, mKO), 12 (c, WT), 7 (c, mKO) mice per group. Data are shown as the mean ± SEM. Analysis was unpaired two-tailed Student’s t-test (a-c).

Source data

Extended Data Fig. 3 Knockdown of CRAT in NRVMs increases the expression of pro-inflammatory cytokines.

NRVMs were transduced with lentivirus expressing Crat shRNA or control shRNA for 5 days. RNAs were extracted from the cells and RT-PCRs were then performed to determine the expression of hypertrophy, pro-fibrotic or pro-inflammatory genes. a, knockdown of CRAT expression was confirmed by RT-PCR. b-f, the expression of hypertrophic and pro-fibrotic genes was not increased by CRAT knockdown. g-i, the expression of Il-1β, Il-6 and Tnf-α was substantially increased in CRAT-deficient NRVMs. n = 6-7 (a-i, Sh-Control), 6 (a-i, Sh-Crat) biologically independent samples per group. Data are shown as the mean ± SEM. Analysis was unpaired two-tailed Student’s t-test (a-i). NS, not significant.

Extended Data Fig. 4 CRAT deficiency induces type I interferon responses in NRVMs.

a-e, NRVMs were transduced with lentivirus expressing Crat shRNA, control shRNA or no virus for 3 or 5 days. RNAs were extracted from the cells and RT-PCRs were then performed to determine the expression of Aim2 (a), Ddx58 (b), Ifih1(c), Ifit3 (d) and Irf7 (e). n = 4 (a-e) biologically independent samples per group. Data are shown as the mean ± SEM. Analysis was two-way ANOVA followed by Bonferroni’s multiple comparison test (a-e).

Extended Data Fig. 5 CRAT deficiency induces type I interferon responses in cardiac fibroblasts.

a-d, Cardiac fibroblasts were transduced with lentivirus expressing Crat shRNA, control shRNA or no virus for 3 or 5 days. RNAs were extracted from the cells and RT-PCRs were then performed to determine the expression of Aim2 (a), Ddx58 (b), Irf7 (c) and Ifit3 (d). n = 4 (a-d, no virus, Sh-Control), 3-4 (a-d, Sh-Crat) biologically independent samples per group. Data are shown as the mean ± SEM. Analysis was two-way ANOVA followed by Bonferroni’s multiple comparison test (a-d).

Extended Data Fig. 6 Depletion of CRAT has no effect on total mitochondrial DNA level in NRVMs and adult CMs.

a, b, NRVMs were transduced with lentivirus expressing control shRNA or Crat shRNA. Cells were harvested and mitochondria and cytosol fractionation was then performed after 5 days. Western blots indicated that there was no contamination between these two fractions (a). RT-PCRs were then performed with mitochondrial fraction to quantify the total mitochondrial DNA level (b). c, Adult CRAT-WT and CRAT-mKO male and female mice were injected with tamoxifen (20 mg/kg/day) for four consecutive days. Three weeks later, CMs were isolated from hearts for mitochondria and cytosol fractionation. RT-PCRs were then performed with mitochondrial fraction to quantify the total mitochondria DNA level. n = 5 biologically independent samples (b), 6 mice (c) per group. Data are shown as the mean ± SEM. Analysis was unpaired two-tailed Student’s t-test (b-c). NS, not significant.

Source data

Extended Data Fig. 7 RNA-sequencing analysis for NRVMs transduced with lentivirus expressing control shRNA or Crat shRNA.

a, b, Heatmap and volcano plot of the significantly differential expressed genes. c, d, GSEA analysis of hallmark pathways shows 17 upregulated and 17 downregulated pathways in CRAT knockdown (KD) NRVMs. e. Heatmap analysis of the major enzymes involved in cholesterol biosynthesis pathway.

Extended Data Fig. 8 Bile acids and 7-HOCA induce type I interferon responses in NRVMs.

a, b, NRVMs were treated with different doses of CDCA for 1 day. Cells were then harvested for RT-PCR to determine the expression of the indicated ISGs. c, d, NRVMs were treated with different doses of CDCA, 7-HOCA or MCA for 1 day. Cells were then harvested for RT-PCR to determine the expression of the indicated ISGs. n = 6 (a-d) biologically independent samples per group. Data are shown as the mean ± SEM. Analysis was one-way ANOVA (a) or two-way ANOVA (c-d) followed by Bonferroni’s multiple comparison test and unpaired two-tailed Student’s t-test (b).

Extended Data Fig. 9 PPARs inhibitor, GW9662, inhibits the type I interferon responses in CRAT-deficient NRVMs.

NRVMs were transduced with lentivirus expressing control shRNA or Crat shRNA. Cells were then treated with GW9662 (10 μM) or T0070907 (10 μM) as indicated for two days. a, b, RT-PCR indicated that GW9662 inhibited the induction of Cyp27a1 (a) and Cyp7b1(b) in CRAT-deficient NRVMs. c, d, GW9662 but not T0070907 abolished the increase in ISG expression induced by CRAT depletion. n = 6 (a-c), 4 (d) biologically independent samples per group. Data are shown as the mean ± SEM. Analysis was two-way ANOVA (a-b) or one-way ANOVA (d) followed by Bonferroni’s multiple comparison test and unpaired two-tailed Student’s t-test (c). NS, not significant.

Extended Data Fig. 10 Mitochondrial DNA activates AIM2-inflammasome in vitro.

Purified GST–AIM2 (2 μg), ASC protein (1 μg), His-Caspase-1 (2 μg) were incubated in the presence or absence of mtDNA (1 μg) or poly (dA:dT) (1 μg) in TE buffer for 30 min at 37 °C. The reaction mixtures were then loaded on SDS–PAGE gel and Western-blots were performed with the indicated antibodies. Here is the representative result from three independent experiments.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting summary

Source data

Source Data Figs. 1–6

Statistical source data for Figs. 1–6.

Source Data Fig. 1

Unprocessed western blots for Fig. 1d.

Source Data Fig. 3

Unprocessed western blots for Fig. 3i.

Source Data Fig. 5

Unprocessed western blots for Fig. 5b,g.

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Unprocessed western blots for Fig. 6i.

Source Data Extended Data Figs. 1–9

Statistical source data for Extended Data Figs. 1–9.

Source Data Extended Data Fig. 2

Unprocessed western blots for Extended Data Fig. 2a.

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Unprocessed western blots for Extended Data Fig. 6a.

Source Data Extended Data Fig. 10

Unprocessed western blots for Extended Data Fig. 10.

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Mao, H., Angelini, A., Li, S. et al. CRAT links cholesterol metabolism to innate immune responses in the heart. Nat Metab 5, 1382–1394 (2023). https://doi.org/10.1038/s42255-023-00844-5

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