Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells from Type2 Diabetes Patients

Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation

Francesco Spallotta, Chiara Cencioni, Sandra Atlante, Davide Garella, Mattia Cocco, Mattia Mori, Raffaella Mastrocola, Carsten Kuenne, Stefan Guenther, Simona Nanni, Valerio Azzimato, Sven Zukunft, Angela Kornberger, Duran Sürün, Frank Schnütgen, Harald Von Melchner, Antonella Di Stilo, Manuela Aragno, Maarten Braspenning, Wim Van Criekinge & 13 others Miles J. De Blasio, Rebecca H. Ritchie, Germana Zaccagnini, Fabio Martelli, Antonella Farsetti, Ingrid Fleming, Thomas Braun, Andres Beiras-Fernandez, Bruno Botta, Massimo Collino, Massimo Bertinaria, Andreas M. Zeiher, Carlo Gaetano

Research output: Contribution to journalArticleResearchpeer-review

12 Citations (Scopus)

Abstract

Rationale: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. 

Objective: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. 

Methods and Results: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. 

Conclusions: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.

Original languageEnglish
Pages (from-to)31-46
Number of pages16
JournalCirculation Research
Volume122
Issue number1
DOIs
Publication statusPublished - 5 Jan 2018
Externally publishedYes

Keywords

  • DNA methylation
  • epigenomics
  • fibroblasts
  • heart
  • hyperglycemia
  • metabolism

Cite this

Spallotta, Francesco ; Cencioni, Chiara ; Atlante, Sandra ; Garella, Davide ; Cocco, Mattia ; Mori, Mattia ; Mastrocola, Raffaella ; Kuenne, Carsten ; Guenther, Stefan ; Nanni, Simona ; Azzimato, Valerio ; Zukunft, Sven ; Kornberger, Angela ; Sürün, Duran ; Schnütgen, Frank ; Von Melchner, Harald ; Di Stilo, Antonella ; Aragno, Manuela ; Braspenning, Maarten ; Van Criekinge, Wim ; De Blasio, Miles J. ; Ritchie, Rebecca H. ; Zaccagnini, Germana ; Martelli, Fabio ; Farsetti, Antonella ; Fleming, Ingrid ; Braun, Thomas ; Beiras-Fernandez, Andres ; Botta, Bruno ; Collino, Massimo ; Bertinaria, Massimo ; Zeiher, Andreas M. ; Gaetano, Carlo. / Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells from Type2 Diabetes Patients : Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation. In: Circulation Research. 2018 ; Vol. 122, No. 1. pp. 31-46.
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title = "Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells from Type2 Diabetes Patients: Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation",
abstract = "Rationale: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. Objective: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. Methods and Results: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. Conclusions: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.",
keywords = "DNA methylation, epigenomics, fibroblasts, heart, hyperglycemia, metabolism",
author = "Francesco Spallotta and Chiara Cencioni and Sandra Atlante and Davide Garella and Mattia Cocco and Mattia Mori and Raffaella Mastrocola and Carsten Kuenne and Stefan Guenther and Simona Nanni and Valerio Azzimato and Sven Zukunft and Angela Kornberger and Duran S{\"u}r{\"u}n and Frank Schn{\"u}tgen and {Von Melchner}, Harald and {Di Stilo}, Antonella and Manuela Aragno and Maarten Braspenning and {Van Criekinge}, Wim and {De Blasio}, {Miles J.} and Ritchie, {Rebecca H.} and Germana Zaccagnini and Fabio Martelli and Antonella Farsetti and Ingrid Fleming and Thomas Braun and Andres Beiras-Fernandez and Bruno Botta and Massimo Collino and Massimo Bertinaria and Zeiher, {Andreas M.} and Carlo Gaetano",
year = "2018",
month = "1",
day = "5",
doi = "10.1161/CIRCRESAHA.117.311300",
language = "English",
volume = "122",
pages = "31--46",
journal = "Circulation Research",
issn = "0009-7330",
publisher = "Lippincott Williams & Wilkins",
number = "1",

}

Spallotta, F, Cencioni, C, Atlante, S, Garella, D, Cocco, M, Mori, M, Mastrocola, R, Kuenne, C, Guenther, S, Nanni, S, Azzimato, V, Zukunft, S, Kornberger, A, Sürün, D, Schnütgen, F, Von Melchner, H, Di Stilo, A, Aragno, M, Braspenning, M, Van Criekinge, W, De Blasio, MJ, Ritchie, RH, Zaccagnini, G, Martelli, F, Farsetti, A, Fleming, I, Braun, T, Beiras-Fernandez, A, Botta, B, Collino, M, Bertinaria, M, Zeiher, AM & Gaetano, C 2018, 'Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells from Type2 Diabetes Patients: Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation', Circulation Research, vol. 122, no. 1, pp. 31-46. https://doi.org/10.1161/CIRCRESAHA.117.311300

Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells from Type2 Diabetes Patients : Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation. / Spallotta, Francesco; Cencioni, Chiara; Atlante, Sandra; Garella, Davide; Cocco, Mattia; Mori, Mattia; Mastrocola, Raffaella; Kuenne, Carsten; Guenther, Stefan; Nanni, Simona; Azzimato, Valerio; Zukunft, Sven; Kornberger, Angela; Sürün, Duran; Schnütgen, Frank; Von Melchner, Harald; Di Stilo, Antonella; Aragno, Manuela; Braspenning, Maarten; Van Criekinge, Wim; De Blasio, Miles J.; Ritchie, Rebecca H.; Zaccagnini, Germana; Martelli, Fabio; Farsetti, Antonella; Fleming, Ingrid; Braun, Thomas; Beiras-Fernandez, Andres; Botta, Bruno; Collino, Massimo; Bertinaria, Massimo; Zeiher, Andreas M.; Gaetano, Carlo.

In: Circulation Research, Vol. 122, No. 1, 05.01.2018, p. 31-46.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells from Type2 Diabetes Patients

T2 - Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation

AU - Spallotta, Francesco

AU - Cencioni, Chiara

AU - Atlante, Sandra

AU - Garella, Davide

AU - Cocco, Mattia

AU - Mori, Mattia

AU - Mastrocola, Raffaella

AU - Kuenne, Carsten

AU - Guenther, Stefan

AU - Nanni, Simona

AU - Azzimato, Valerio

AU - Zukunft, Sven

AU - Kornberger, Angela

AU - Sürün, Duran

AU - Schnütgen, Frank

AU - Von Melchner, Harald

AU - Di Stilo, Antonella

AU - Aragno, Manuela

AU - Braspenning, Maarten

AU - Van Criekinge, Wim

AU - De Blasio, Miles J.

AU - Ritchie, Rebecca H.

AU - Zaccagnini, Germana

AU - Martelli, Fabio

AU - Farsetti, Antonella

AU - Fleming, Ingrid

AU - Braun, Thomas

AU - Beiras-Fernandez, Andres

AU - Botta, Bruno

AU - Collino, Massimo

AU - Bertinaria, Massimo

AU - Zeiher, Andreas M.

AU - Gaetano, Carlo

PY - 2018/1/5

Y1 - 2018/1/5

N2 - Rationale: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. Objective: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. Methods and Results: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. Conclusions: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.

AB - Rationale: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. Objective: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. Methods and Results: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. Conclusions: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.

KW - DNA methylation

KW - epigenomics

KW - fibroblasts

KW - heart

KW - hyperglycemia

KW - metabolism

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U2 - 10.1161/CIRCRESAHA.117.311300

DO - 10.1161/CIRCRESAHA.117.311300

M3 - Article

VL - 122

SP - 31

EP - 46

JO - Circulation Research

JF - Circulation Research

SN - 0009-7330

IS - 1

ER -