Acetyl-CoA production by specific metabolites promotes cardiac repair after myocardial infarction via histone acetylation

  1. Ienglam Lei
  2. Shuo Tian
  3. Wenbin Gao
  4. Liu Liu
  5. Yijing Guo
  6. Paul Tang
  7. Eugene Chen
  8. Zhong Wang  Is a corresponding author
  1. Faculty of Health Sciences, University of Macau, China
  2. Department of Cardiac Surgery, University of Michigan-Ann Arbor, United States
6 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
8 C administration attenuates ischemia-reperfusion injury in rats.

(A) Metabolic pathways of acetyl-CoA synthesis. (B) Schematic diagram of different carbon sources administration prior to I/R surgery. (C) Quantification of IS/AAR ratio in Figure 1—figure supplement 1A by Image J. (D) Schematic diagram of 8C administration post ischemic injury. (E) Representative figures of heart sections at 24 hr after I/R with or without 8C administration at reperfusion after 45 min ischemia. Scale bar: 2.5 mm (F) Quantification of IS/AAR ratio in (E). LV EF (G) and FS (H) at 24 hr after I/R. (I) Quantification of Acetyl-CoA levels in sham and I/R rat hearts at indicated conditions. (J–K) Western blot and quantification of H3K9ac and H3 in rat hearts 24 hr after I/R. (L) Trichrome masson staining of heart section after 4 weeks of I/R. Scale bar: 2.5 mm. (M) Quantification of infarct size in (I). LV EF (N) and FS (O) at 4 weeks after I/R. Error bars represent S.D. n = 5, #p < 0.05, ##p < 0.01 vs Sham group. * p < 0.05, **p < 0.01, vs I/R group. Data were analyzed by one-way ANOVA (C,G,H,K,N and O) or two-way ANOVA (I), followed by post-hoc Tukey test, data in F and M were analyzed by two-tailed student test.

Figure 1—figure supplement 1
Effect of metabolites on myocardial I/R injury.

(A) Representative figures of heart sections at 24 hr after I/R in presence of different metabolites. Scale bar: 2.5 mm. (B) Quantification of AAR/LV in Figure 1A by Image J. (C) Quantification of AAR/LV ratio after I/R with or without 8C administration at 45 min after ligation. (D–E) Representative echo image for measurement of heart function in Figure 1G, H, N and O. (F) Quantification of Succinate levels in rat hearts after I/R with saline, 8C or 9C treatment. n = 3, *p < 0.05, **p < 0.01 vs I/R + saline group; ##p < 0.01 vs I/R + 8C group. Data were analyzed by one-way ANOVA (B and E), followed by post-hoc Tukey test, data in C was analyzed by two-tailed student test.

Figure 2 with 1 supplement
Post ischemic administration of 8C reduces the oxidative stress and cell death after I/R injury.

(A) Representative of TUNEL (green) and cTnT (red) double staining at boarder zone at 24 hr post I/R injury. Scale bar: 100 µm (B) Quantification of cardiomyocytes cell death of 12 sections (C–D) Serum cTni and CK-MB level at 24 hr post I/R. (E–F) Western blot and quantification of Bax and Bcl2 at 24 hr after I/R. (G) GSEA analysis of Kegg apoptotic pathways after I/R with and without 8C treatment. (H) ROS levels were measured by DHE staining. Scale bar: 200 µm. (I) Relative mean DHE fluorescence intensity measured by Image J. Error bars represent S.D. n = 3. *p < 0.05, **p < 0.01 vs Sham; #p < 0.05, ##p < 0.01 vs I/R group. Data were analyzed by one-way ANOVA, followed by post-hoc Tukey test.

Figure 2—figure supplement 1
8C administration altered the gene expression after I/R injury.

(A–B) Serum CK and LDH level at 24 hr post I/R. (C) Volcano plot of differential expressed genes in 8C-treated and saline-treated hearts after I/R. Differential expressed genes with abs(Log2Foldchange) > 1, and padj < 0.05 were labelled in red. (D) Top gene set enrichment of differential expressed genes using GO biological process gene sets. (E) Heatmap of antioxidant gene expression. (F) Quantification of myocardial SOD activity after I/R with or without I/R. n = 3, *p < 0.05 vs Sham group; #p < 0.05 vs I/R group. Data were analyzed by one-way ANOVA (B and D), followed by post-hoc Tukey test.

8C attenuates NRVMs apoptosis through reducing oxidative stress.

(A) FACS analysis of Annexin V and PI staining in NRVMs exposed to sI/R with and without 8C treatment. (B) Quantification of percentage of Annexin V + and PI+ cells. Cell viability and cell death measurement in NRVMs with sI/R using CCK8 detection kit (C) and LDH assay kit (D). (E) Western blot of cleaved PARP in NRVMs after sI/R treatment. (F) NRVM cellular ROS levels are indicated by DHE staining after sI/R treatment. Scale bar: 200 µm (G) FACS analysis of NRVM DHE staining after sI/R. (H) Relative mean fluorescence intensity of DHE staining measured by Flowjo. n = 3, **p < 0.01, ***p < 0.001, vs Normoxia + PBS; ## p < 0.01, ###p < 0.001 vs sI/R + PBS. Data were analyzed by two-way ANOVA, followed by post-hoc Tukey test.

Figure 4 with 1 supplement
8C stimulates histone acetylation and promotes antioxidant gene expression.

(A) Quantification of Acetyl-CoA concentrations in NRVMs subjected to sI/R. (B–E) 8 C rescues sI/R reduced H3K9ac, H3K27ac, H3K14ac and total acH3 levels. NRVMs were treated with or without 0.5 mM 8 C under sI/R. The histone acetylation levels were determined by western blot. Total H3 in the same blot was used as loading control. (F) Representative MS spectra from NRVMs treated with [U-13C] sodium octanoate indicating isotope distribution on acetyl Histone H3. The mass shifts indicate the incorporation of heavy carbon to the acetyl histone lysines. (G) Enrichment of H3K9ac over H3 at promoters of HO1, NQO1, and SOD2 in NRVMs after sI/R. (H) mRNA expression of HO1, NQO1, and SOD2 in NRVMs after sI/R. *p < 0.05, **p < 0.01, ***p < 0.001, vs Normoxia + PBS; #p < 0.05, ##p < 0.01, ###p < 0.001 vs sI/R + PBS. Data were analyzed by two-way ANOVA, followed by post-hoc Tukey test.

Figure 4—figure supplement 1
8C administration altered the gene expression after I/R injury.

(A-B) Serum CK and LDH level at 24 hr post I/R. (C) Volcano plot of differential expressed genes in 8C-treated and saline-treated hearts after I/R. Differential expressed genes with abs(Log2Foldchange) >1, and padj < 0.05 were labeled in red. (D) Top gene set enrichment of differential expressed genes using GO biological process gene sets. (E) Heatmap of antioxidant gene expression. (F) Quantification of myocardial SOD activity after I/R with orwithout I/R. n=3, *p < 0.05 vs Sham group; # p < 0.05 vs I/R group. Data were analyzed by oneway ANOVA (B and D), followed by post-hoc Tukey test.

Figure 5 with 1 supplement
MCAD was required for the conversion of 8 C into acetyl-CoA and subsequent histone acetylation increase and heart protection.

(A) Western blot of H3K9ac level showed MCAD knockdown reduced 8C-induced H3K9ac increase in NRVMs under both normoxia and sI/R. (B) Measurement of medium LDH levels in NRVMs at indicated condition using LDH assay kit. (C) FACS analysis of DHE staining NRVMs after sI/R. (D) Relative mean fluorescence intensity of DHE staining. (E) Enrichment of H3K9ac over H3 at promoters of HO1 and NQO1 after sI/R at indicated conditions. (F) mRNA expression of HO1, NQO1 and MCAD in NRVMs after sI/R. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, vs Normoxia + PBS + shCTL; #p < 0.05, ##p < 0.01, ###p < 0.001 vs sI/R + PBS + shCTL. Data were analyzed by two-way ANOVA, followed by post-hoc Tukey test.

Figure 5—figure supplement 1
Knockdown of MCAD is required for alleviating ROS accumulation.

(A) Western blot showed knockdown of MCAD by adenovirus shRNA. (B) Cell viability measurement in NRVMs at shown condition using CCK8 detection kit. (C) NRVM cellular ROS levels are indicated by DHE staining after sI/R treatment. Scale bar: 200 µm. (D–F) Western blot and quantifications of HO1 and NQO1 in NRVMs after sI/R. n = 3, *p < 0.05, **p < 0.01, vs Normoxia + PBS + shCTL; #p < 0.05, ##p < 0.01 vs sI/R + PBS + shCTL. Data were analyzed by two-way ANOVA, followed by post-hoc Tukey test.

Figure 6 with 1 supplement
HAT enzyme Kat2a was required for 8 C mediated histone acetylation to inhibit oxidative stress in heart protection.

(A) Western blot of H3K9ac level showed Kat2a knockdown reduced 8C-induced H3K9ac increase in NRVMs under both normoxia and sI/R. (B) Measurement of medium LDH levels in NRVMs at indicated condition using LDH assay kit. (C) FACS analysis of DHE staining NRVMs after sI/R. (D) Relative mean fluorescence intensity of DHE staining. (E) Enrichment of H3K9ac over H3 at promoters of HO1 and NQO1 after sI/R at indicated conditions. (F) mRNA expression of HO1,NQO1 and Kat2a in NRVMs after sI/R. (G) Schematic diagram of 8 C metabolism for cardiac repair. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, vs Normoxia + PBS + shCTL; #p < 0.05, ##p < 0.01, ###p < 0.001 vs sI/R + PBS + shCTL. Data were analyzed by two-way ANOVA, followed by post-hoc Tukey test.

Figure 6—figure supplement 1
Knockdown of Kat2a is required for alleviating ROS accumulation.

(A) Kat2a was upregulated after I/R based on RNA-seq results. (B) Western blot showed knockdown of Kat2a by adenovirus sh-RNA. (C) Cell viability measurement in NRVMs at indicated condition using CCK8 detection kit. (D) NRVM cellular ROS levels are indicated by DHE staining after sI/R treatment. Scale bar: 200 µm. (E–G) Western blot and quantifications of HO1 and NQO1 in NRVMs after sI/R. n = 3, *p < 0.05, **p < 0.01, vs Normoxia + PBS + shCTL; # p < 0.05 vs sI/R + PBS + shCTL. Data were analyzed by two-way ANOVA, followed by post-hoc Tukey test.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyBcl2 (Rabbit monoclonal antibody)AbcamCat#: ab182858; RRID: AB_2715467Western blot (1:1000)
AntibodyBax (Rabbit polyclonal antibody)Cell Signaling TechnologyCat#: 2,772 S; RRID: AB_10695870Western blot (1:1000)
Antibodyα-Tubulin (mouse monoclonal antibody)Cell Signaling TechnologyCat#: 3,873 S; RRID: AB_1904178Western blot (1:1000)
AntibodyPARP (Rabbit polyclonal antibody)Cell Signaling TechnologyCat#: 9542; RRID: AB_2160739Western blot (1:1000)
AntibodyH3K9ac (Rabbit polyclonal antibody)SigmaCat#: 06–942; RRID: AB_310308Western blot (1:3000), 1:100 for ChIP (1:100)
AntibodyH3K14ac (Rabbit polyclonal antibody)SigmaCat#: 07–353; RRID: AB_310545Western blot (1:3000)
AntibodyH3K27ac (Rabbit polyclonal antibody)MilliporeCat#: 07–360; RRID: AB_310550Western blot (1:3000)
AntibodyAcH3 (Rabbit polyclonal antibody)MilliporeCat#: 06–599; RRID: AB_2115283Western blot (1:3000)
AntibodyHistone H3 (mouse monoclonal antibody)SigmaCat#: 14,269 S; RRID: AB_2756816Western blot (1:2000)
AntibodyHO1 (Rabbit polyclonal antibody)Proteintech Group IncCat#: 10701–1-AP; RRID: AB_2118685Western blot (1:1000)
AntibodyNQO1 (Rabbit polyclonal antibody)Proteintech Group IncCat#: 11451–1-AP; RRID: AB_2298729Western blot (1:1000)
AntibodySOD2 (Rabbit polyclonal antibody)Proteintech Group IncCat#: 24127–1-AP; RRID:AB_2879437Western blot (1:1000)
AntibodyMCAD (Rabbit polyclonal antibody)Proteintech Group IncCat#: 55210–1-AP; RRID: AB_10837361Western blot (1:1000)
AntibodyKat2a (Rabbit recombinant antibody)AbcamCat#: Ab217876; RRID:AB_2811191Western blot (1:1000)
AntibodycTnT (Rabbit polyclonal antibody)AbcamCat#: ab45932; RRID: AB_956386IF (1:200), FACS (1:200)
Antibody680RD Donkey anti-Mouse IgG (H + L)LI-CORCat# 926–68072;RRID:AB_10953628Western blot (1:5000)
Antibody800CW Donkey anti-Rabbit IgG (H+ L)LI-CORCat# 926–32213;
RRID:AB_621848
Western blot (1:5000)
Chemical compound, drugSodium acetateSigmaCat#:S2889-250G
Chemical compound, drugSodium PyruvateSigmaCat#:P2265-5G
Chemical compound, drugSodium CitrateSigmaCat#: PHR1416-1G
Chemical compound, drugSodium OctanoateFisherCat#: N029125G
Chemical compound, drugSodium NonanoateTCI AmericaCat#: N0291-25G
Chemical compound, drugEvans BlueFisherCat#: AC195550250
Chemical compound, drug2,3,5-Triphenyltetrazolium chlorideSigmaCat#: T8877
Chemical compound, drugDihydroethidium (DHE)CaymanCat#: 12,013
Chemical compound, drugU-13C-Sodium octanoateCambridge Isotope LaboratoriesCat#:CLM-9617-PK
Chemical compound, drugEDTA-free Protease Inhibitor CocktailSigmaCat#:118350001
Chemical compound, drugType II CollagenaseWorthingtonBiochemical Co.Cat#: LS004174
Chemical compound, drug2-deoxy-D-GlucoseCaymanCat#: 14,325
Peptide, recombinant proteinMicrococcal NucleaseNEBCat#: M0247S
Chemical compound, drugIHC Zinc FixativeFisherCat#: BDB550523
Commercial assay or kitCreatine Kinase Activity Assay KitSigmaCat#: MAK116-1KT
Commercial assay or kitLactate Dehydrogenase Activity Assay KitSigmaCat#: MAK066
Commercial assay or kitSOD Assay Kit-WSTDojindoCat#: S311-10
Commercial assay or kitPowerUp SYBR Green MasterMixThermo FisherCat#: A25778
Commercial assay or kitPrecision Plus Protein All Blue PrestainedBio-radCat#:1610373
Commercial assay or kitiScript cDNA Synthesis KitBio-radCat#:1708891
Commercial assay or kitAccuPrep PCR Purification KitBioneerCat#: K-3034
Commercial assay or kitTrichrome Stain (Masson) KitSigmaCat#: HT15-1KT
Commercial assay or kitTUNEL Assay KitSigmaCat#:11684795910
Commercial assay or kitCytotoxicity LDH Assay Kit-WSTDojindoCat#: CK12-05
Commercial assay or kitPicoProbeAcetyl-CoAFluorometric Assay KitFisherCat#: NC9976028
Commercial assay or kitCell Counting Kit-8DojindoCat#: CK04-13
Commercial assay or kitBD Annexin V-FITCFisherCat#: BDB556420
Commercial assay or kitSuccinate Colorimetric Assay KitFisherCat#: NC0541966
Commercial assay or kitTRIzol ReagentSigmaCat#: 15596018
Commercial assay or kitTURBO DNaseThermo FisherCat#: AM2238
Commercial assay or kitNEBNext Ultra II Directional RNA Library Prep Kit for IlluminaNEBCat#: E7760L
Commercial assay or kitCreatine kinase (CK) MB isolenzyme Elisa kitABclonalRK03571
Commercial assay or kitTroponin I (TnI) Elisa kitAbclonalRK03995
Strain, strain background (Escherichia coli)NEB stable E-coliNEBCat#: C3040H
Strain, strain background (Escherichia coli)NEB 10-beta competent E. coliNEBCat#:C3019H
Recombinant DNA reagentBLOCK-iT U6 RNAi Entry VectorKitInvitrogenCat#: K494500
Recombinant DNA reagentGateway LR Clonase II Enzyme MixThermo FisherCat#: 11791–020
Recombinant DNA reagentpAd/PL-DEST Gateway VectorKitThermo FisherCat#: V49420
Cell line (Homo-sapiens)293 A Cell LineThermo FisherR70507For package of Adenovirus
Software, algorithmImageJNIHhttps://imagej.nih.gov/ij/
Software, algorithmGraphPad Prism (version 7)Graphpad Softwarehttps://www.graphpad.com/scientific-software/prism/
Software, algorithmOdyssey CLx Imaging SystemLI-COR Bioscienceshttps://www.licor.com/
Software, algorithmFlowJoFlowjohttps://www.flowjo.com/

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Ienglam Lei
  2. Shuo Tian
  3. Wenbin Gao
  4. Liu Liu
  5. Yijing Guo
  6. Paul Tang
  7. Eugene Chen
  8. Zhong Wang
(2021)
Acetyl-CoA production by specific metabolites promotes cardiac repair after myocardial infarction via histone acetylation
eLife 10:e60311.
https://doi.org/10.7554/eLife.60311