Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure
- Faculty of Biology, Medicine, and Health, the University of Manchester, United Kingdom
- Department of Internal Medicine III, University of Kiel, Germany
- DZHK, German Centre for Cardiovascular Research, Partner Site Hamburg/Kiel/Lübeck, Germany
- Institute of Pharmacology and Toxicology, Faculty of Medicine Carl Gustav Carus, Technische Universitaet Dresden, Germany
- Edinburgh University Medical School, United Kingdom
- Institute of Vascular Medicine, Peking University, China
Figures
Figure 1
IRS1 is downregulated in failing hearts and prolonged hypoxia-stressed cardiomyocytes.
(A) Experimental design of male C57BL/6N mice subject to 4w-MI. (B) M-mode images captured by echocardiography determined cardiac dysfunction after 4w-MI, measured by fractional shortening (FS%) and …
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Figure 1—source data 1
Insulin sensitivity and Irs1 downregulation in MI-induced heart failure and rat cardiomyocytes under prolonged hypoxic stress.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig1-data1-v2.xlsx
Figure 2 with 5 supplements
Hypoxia-upregulates mir128-3p and degrades Irs1.
(A) Mature mir128-3p was increased in 4w-MI myocardium (N = 6–8 mice). Data were normalized against mature mir191-5p expression. (B) In situ hybridization with digoxigenin-labeled mir128-3p probes …
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Figure 2—source data 1
Study of mir128-3p expression under hypoxia and its function in regulating Irs1.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
MiRNA analysis in 4w-MI myocardium.
(A) Mature mir144-3p and mir145-5p in 4w-MI myocardium were measured using TaqMan Advanced miRNA qPCR (N = 6–8 mice). Data were normalized against mature mir191-5p expression. (B) In situ …
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Figure 2—figure supplement 1—source data 1
MiRNA analysis in 4w-MI myocardium.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Mir128-3p level in rat cardiomyocytes after hypoxia.
Mature mir128-3p was measured using TaqMan Advanced miRNA qPCR (N = 6 experiments). Data were normalized against mature mir191-5p expression. Data presented as mean ± SEM. Groups were compared using …
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Figure 2—figure supplement 2—source data 1
Mir128-3p level in rat cardiomyocytes after hypoxia.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig2-figsupp2-data1-v2.xlsx
Figure 2—figure supplement 3
Pri-mir128-2 location and structure.
Location of Pri-miR-128-2 hosted by human ARPP21 gene (GRCh38:3:35638945:35794496:1, Ensembl ENSG00000172995) and the stem-loop structure of Pre-miR-128-2.
Figure 2—figure supplement 4
Pri-mir128-1 level in rat cardiomyocytes after hypoxia.
Pri-mir128-1 in cardiomyocytes was performed using TaqMan Gene expression (N = 5 experiments). Data were normalised against 18S expression. Data presented as mean ± SEM. Groups were compared using …
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Figure 2—figure supplement 4—source data 1
Pri-mir128-1 level in rat cardiomyocytes after hypoxia.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig2-figsupp4-data1-v2.xlsx
Figure 2—figure supplement 5
Mir128-3p overexpression and inhibition optimization.
Validation of mir128-3p mimic and antagomir. (A) mir128-3p mimic and (B) antagomir validation was carried out on H9C2 cells at different concentrations. Mature miRNA was measured by TaqMan Advanced …
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Figure 2—figure supplement 5—source data 1
Mir128-3p overexpression and inhibition optimization.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig2-figsupp5-data1-v2.xlsx
Figure 3 with 7 supplements
MAPK7 regulation of CEBPβ transcriptionally represses mir128-3p.
(A) Pri-mir128-1 promotor luciferase activity in H9C2 was increased by long-term hypoxia (N = 4 experiments). (B) Schematic figure showing CEBP consensus binding sites in the intronic promoter …
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Figure 3—source data 1
Role of MAPK7 and CEBPβ in mir128-3p transcriptional regulation.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Hypoxia effect on R3hdm1 expression.
Luciferase reporter assay performed on H9C2 cells transfected with pGL3 or pGL3-R3hdm1 promoter (−1.5 kbp of transcription start site TSS downstream of R3hdm1 exon1) demonstrates that R3hdm1 …
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Figure 3—figure supplement 1—source data 1
Hypoxia effect on R3hdm1 expression.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Mapk7 and Cebpa expression in 4w-MI myocardium.
(A) Cebpa transcript expression was not changed in 4w-MI myocardium, while (B) Mapk7 was decreased measured by qPCR (N = 6–8 mice). Data were normalized against 18S expression. Data presented as …
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Figure 3—figure supplement 2—source data 1
Mapk7 and Cebpa expression in 4w-MI myocardium.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig3-figsupp2-data1-v2.xlsx
Figure 3—figure supplement 3
Role of CEBPβ in miR128-3p and Irs1 regulation in H9C2 cells.
CEBPβ repression of mir128-3p retains Irs1 in H9C2 cardiomyocytes. (A) Cebpb was knocked down more than 70% by siRNA transfection with Lipofectamine LTX (N = 6 experiments). (B) Human Cebpb …
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Figure 3—figure supplement 3—source data 1
Role of CEBPβ in miR128-3p and Irs1 regulation in H9C2 cells.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig3-figsupp3-data1-v2.xlsx
Figure 3—figure supplement 4
MAPK7 activation and expression in short and long-term hypoxia.
MAPK7 activation was observed in neonatal rat cardiomyocytes (NRCMs) in response to short-term hypoxia, whereas MAPK7 was decreased by prolonged stress. Quantification is provided (N = 3–6 …
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Figure 3—figure supplement 4—source data 1
MAPK7 activation and expression in short and long-term hypoxia.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig3-figsupp4-data1-v2.xlsx
Figure 3—figure supplement 5
CEBPβ binding analysis to Pri-mir128-1 intronic promoter.
Chromatin immunoprecipitation (ChIP) assay was performed on ARCMs DNA using anti-CEBPβ antibody (anti-IgG antibody as the negative control). The enrichment of CEBPβ binding to different regions in Pr…
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Figure 3—figure supplement 5—source data 1
CEBPβ binding analysis to Pri-mir128-1 intronic promoter.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig3-figsupp5-data1-v2.xlsx
Figure 3—figure supplement 6
Analysis of MAPK7 downstream effectors after hypoxia.
Potential MAPK7 downstream effectors were analysed in ARCMs by immunoblots and quantification. CREB1 phosphorylation under short-term hypoxia was blocked by Erk5 deficiency (N = 4 experiments). Data …
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Figure 3—figure supplement 6—source data 1
Analysis of MAPK7 downstream effectors after hypoxia.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig3-figsupp6-data1-v2.xlsx
Figure 3—figure supplement 7
Hypoxia-induced CREB1 and CEBPβ nuclear translocation in NRCMs.
The impacts of Mapk7 knockdown on CREB1 activation and CEBPβ translocation to the nucleus in NRCMs in response to short-term hypoxia, which was determined by (A) immunoblots and (B) …
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Figure 3—figure supplement 7—source data 1
Hypoxia-induced CREB1 and CEBPβ nuclear translocation in NRCMs.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig3-figsupp7-data1-v2.xlsx
Figure 4 with 5 supplements
MAPK7 deficiency promotes cardiac insulin resistance and injury under hypoxic condition.
(A) Experimental design of male cardiac-specific Mapk7-deleted mice subject to 1w-MI. (B) Echocardiography determined detrimental cardiac function in Mapk7-cko mice post-MI, indicated by fractional …
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Figure 4—source data 1
Effect of MAPK7 deficiency on cardiac insulin resistance and in vivo and in vitro.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig4-data1-v2.xlsx
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Figure 4—source data 2
Echocardiographic parameters of Mapk7-cko and control mice one week post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
MAPK7 activation and hypertrophy in mouse myocardium one week post-MI.
(A) Contrary to the analysis after 4w-MI, MAPK7 activation was observed after 1w-MI in Mapk7flox mice (N = 4–6 mice). Cardiac hypertrophic growth in Mapk7-cko and Mapk7flox mice was evaluated by (B) …
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Figure 4—figure supplement 1—source data 1
MAPK7 activation and hypertrophy in mouse myocardium one week post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Gene expression comparison between Mapk7flox and Mapk7-cko mice one week post-MI.
Real-time qPCR detection of (A) Pri-mir128-1 and Pri-mir128-2, (B) mir144-3p and mir145-5p and (C) Cebpa in Mapk7flox and Mapk7-cko subject to 1 week MI (N = 5–8 mice). (D) qPCR determined that Insr,…
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Figure 4—figure supplement 2—source data 1
Gene expression comparison between Mapk7flox and Mapk7-cko mice one week post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
Activation of the insulin cascade in sham mice and cardiac glycogen detection one week post-MI.
(A) Immunoblots illustrated that insulin-stimulated (insulin IP injection, 1 U/kg for 30 mins) activation of IRS1 and AKT was at a comparable level in the myocardium from Mapk7flox and Mapk7-cko …
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Figure 4—figure supplement 3—source data 1
Activation of the insulin cascade in sham mice and cardiac glycogen detection one week post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig4-figsupp3-data1-v2.xlsx
Figure 4—figure supplement 4
Insulin-related gene expression in MAPK7-deficient rat cardiomyocytes under hypoxic stress.
(A) Insr, Slc2a1 and Slc2a4 levels were comparable in adult rat cardiomyocytes (ARCMs) with MAPK7 deficiency despite hypoxic stress (N = 6 experiments). (B) Irs1 was reduced in neonatal …
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Figure 4—figure supplement 4—source data 1
Insulin-related gene expression in MAPK7-deficient rat cardiomyocytes under hypoxic stress.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig4-figsupp4-data1-v2.xlsx
Figure 4—figure supplement 5
Effect of Mapk7 and Irs1 knockdown in ARCMs cell death after hypoxia.
ARCMs cell death under hypoxia was augmented by Mapk7 or Irs1 knockdown, as evidenced by increased (A) lactate dehydrogenase (LDH) released to the culture medium (N = 3 experiments), (B) cleaved …
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Figure 4—figure supplement 5—source data 1
Effect of Mapk7 and Irs1 knockdown in ARCMs cell death after hypoxia.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig4-figsupp5-data1-v2.xlsx
Figure 5 with 2 supplements
Cardiac MAPK7 restoration preserves IRS1 pathway in the myocardium and cardiac function in response to hypoxia.
(A) Experimental design of cardiac MAPK7 restoration in Mapk7-cko mice via tail vein administration of AAV9-Mapk7 (1 × 1011 viral particles), followed by LAD ligation for one week. (B) …
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Figure 5—source data 1
Functional and transcriptional effects of MAPK7 restoration in Mapk7-cko mice after MI and rat cardiomyocytes under hypoxia.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig5-data1-v2.xlsx
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Figure 5—source data 2
Echocardiographic parameters of Mapk7-cko mice injected with AAV9-Gfp or AAV9-Mapk7 one week post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
MAPK7 expression and cardiac hypertrophy in AAV9-Mapk7-injected mice one week post-MI.
(A) MAPK7 expression in myocardium was restored to physiological level in Mapk7-cko mice using AAV9-Mapk7 injection (AAV9-Gfp as control) (N = 4–6 mice). Cardiac hypertrophic response was observed …
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Figure 5—figure supplement 1—source data 1
MAPK7 expression and cardiac hypertrophy in AAV9-Mapk7-injected mice one week post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
Infarct area comparison between Mapk7flox and Mapk7-cko injected with AAV9-Mapk7 seven days after MI.
Infarct area detected by triphenyltetrazolium chloride (TTC) staining in (N = 8 mice). Mapk7flox and Mapk7-cko mice with control injection and Mapk7-cko mice with AAV9-Mapk7 injection one week …
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Figure 5—figure supplement 2—source data 1
Infarct area comparison between Mapk7flox and Mapk7-cko injected with AAV9-Mapk7 seven days after MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig5-figsupp2-data1-v2.xlsx
Figure 6 with 1 supplement
Cardiac-specific CEBPβ overexpression ameliorates cardiac injury in Mapk7-cko post-MI.
(A) Experimental design of CEBPβ overexpression in Mapk7-cko myocardium via tail vein injection of AAV9-Cebpb (1 × 1011 viral particles), followed by MI for one week. (B) Echocardiography showing …
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Figure 6—source data 1
CEBPβ overexpression effect on mir128-3p and Irs1 expression in Mapk7-cko post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig6-data1-v2.xlsx
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Figure 6—source data 2
Echocardiographic parameters of Mapk7-cko mice injected with AAV9-Gfp or AAV9-Cebpb one week post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
Myocardial CEBPβ expression level after AAV9-Cebpb tail vein injection.
Immunoblots and quantification determined that overexpression of ectopic CEBPβ at a physiological range was achieved by AAV9-Cebpb injection (N = 5 mice). Data presented as mean ± SEM. Groups were …
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Figure 6—figure supplement 1—source data 1
Myocardial CEBPβ expression level after AAV9-Cebpb tail vein injection.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig6-figsupp1-data1-v2.xlsx
Figure 7
Silencing mir128-3p prevents Irs1 degradation and decelerates HF progression post-MI.
(A) Construction of antimir128-TuD built in the intronic region Gfp of AAV9-Gfp capsid. The sequence of antimir128-TuD is provided. (B) pmiRGlo-mir128-3p (four repeated complementary sequences of mir…
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Figure 7—source data 1
Functional effect of silencing mir128-3p post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig7-data1-v2.xlsx
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Figure 7—source data 2
Echocardiographic parameters of Mapk7-cko mice injected with AAV9-Control or AAV9-Antimir128 one week post-MI.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig7-data2-v2.xlsx
Figure 8
MAPK7-CEBPβ repression of mir128-3p maintains insulin sensitivity in human iPSC-CMs under hypoxia.
(A) Transcripts of Mapk7, Cebpb, and Irs1 were reduced in human iPSC-CMs in response to long-term hypoxia (N = 6 experiments). (B) Immunoblots and quantifications showed that phosphorylation of …
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Figure 8—source data 1
Analysis of the MAPK7-CEBPβ-mir128-3p axis in human iPSC-CMs under hypoxia.
- https://cdn.elifesciences.org/articles/54298/elife-54298-fig8-data1-v2.xlsx
Figure 9
Schematic representation of MAPK7-CEBPβ signaling cascade negative regulation of mir128-3p under ischemia/hypoxic condition.
Short-term ischemia/hypoxia activates MAPK7-CREB1, which facilitates CEBPβ expression and its nucleus translocation to act as transcriptional repressor on Pri-mir128-1, consequently maintaining IRS1 …
Additional files
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Supplementary file 1
Key resources table.
- https://cdn.elifesciences.org/articles/54298/elife-54298-supp1-v2.docx
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Transparent reporting form
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