Accumulation of endogenous adenosine improves cardiomyocyte metabolism via epigenetic reprogramming in an ischemia-reperfusion model

Figures

• 

  Fig. 1. ADK was identified as a potential candidate gene involved in the metabolic changes during I/R. (A) Pathways with potential difference in the setting of I/R based on RNA-sequencing, the red column marks pathways involved with ADK. (n = 3 per group). (B) Potential candidate genes with changed expression in the setting of I/R based on RNA-sequencing, the bold black arrow marks the position of ADK. (n = 3 per group). Log2 fold change in X axis legend means the expression changes of target genes. Log10 adjust P value means the difference of target genes between groups.(C) Comparison of target genes between WT and WT + I/R group in the setting of 3-h I/R injury, Group A, wild-type mice; Group B, wild-type mice with I/R surgery, other details were demonstrated in the Supplementary Fig. 9. (n = 3 per group). (D) Logistics analysis of ADK to other genes. Red color represents positive association. Blue color represents negative association. Abbreviations in C and D represents the candidate genes with potential difference. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

• 

  Fig. 2. ADK inhibition attenuated myocardial I/R injury via cardiomyocyte adenosine accumulation in mice. (A) Evans blue/TTC staining of frozen myocardial tissue sections from wild-type mice treated with 5-iodotubercidin (ITU), ABT-702 dihydrochloride (ABT-702), or saline for 7 days prior to I/R surgery. (B) IR/AAR assessed from Evans blue/TTC staining. (n = 5 per group). (C) AAR/LV assessed from Evans blue/TTC staining. (n = 5 per group). (D) Echocardiographic analysis of ejection fraction in mice from the indicated treatment groups (n = 5 per group). (E) Echocardiographic analysis of fractional shortening in mice from the different treatment groups (n = 5 per group). (F) High-performance liquid chromatography of myocardial cellular adenosine levels (n = 5–7 per group). (G) High-performance liquid chromatography of circulating adenosine levels (n = 5–7 per group). (H) Western blot analysis of ADK expression. (I) Analysis of ADK activity. All marks indicated P < 0.05, *, † and ‡ to indicate significance versus 1st, 2nd, and 3rd group accordingly. I/R: I/R surgery alone; I/R + ITU: I/R surgery and ITU treatment; I/R + ABT-702: I/R surgery and ABT-702 treatment. (For interpretation of the…

• 

  Fig. 3. Cardiomyocyte-specific ADK knockout ameliorated myocardial I/R injury via adenosine-induced DNMT1 inhibition. (A) Detection of ADK expression in different cell types isolated from the hearts of wild-type mice at 3 and 24 h after I/R surgery. (n = 5 per group) (B) Evans blue/TTC staining of myocardial tissue sections from cardiomyocyte-specific ADK-knockout and wild-type mice after I/R surgery. (n = 6 per group).(C) Echocardiographic analysis of ejection fraction in the ADK-knockout and wild-type mice after I/R surgery. (n = 5 per group). (D) Echocardiographic analysis of fractional shortening in the ADK-knockout and wild-type mice after I/R surgery. (n = 5 per group). (E) IR/AAR assessed from the Evans blue/TTC staining. (n = 6 per group).(F) Apoptosis levels assessed by TUNEL staining of the myocardial tissues from the ADK-knockout and wild-type mice after I/R surgery. Bars are 5 μm marked with white color in the merged version. (G) Detection of DNMT1 expression and activity. (n = 5 per group). WT + I/R: wild-type mice with I/R surgery; ADK-Cre + I/R: cardiomyocyte-specific ADK-knockout mice with I/R surgery. All marks indicated P < 0.05, *, † and ‡ to indicate significance…

• 

  Fig. 4. Cardiomyocyte-specific ADK knockout induced the hypomethylation of genes involved in metabolic pathways and increased IGF-1 transcription via the hypomethylation of its promoter. (A) Global DNA methylation levels assessed by methylated DNA immunoprecipitation (MeDIP) in the hearts of wild-type and cardiac-specific ADK-knockout mice with or without I/R surgery. (n = 6 per group).(B) Relative DNMT1 activity in the hearts of the mice. (n = 5 per group). (C) Quantification of SAH in the hearts of the mice. (n = 5 per group). (D) Quantification of SAM in the hearts of the mice. (n = 5 per group). (E) KEGG analysis of the MeDIP data from the wild-type and ADK-knockout mice after I/R surgery. (n = 5 per group).(F) Pathway enrichment analysis in cardiomyocytes from the ADK-knockout mice after I/R surgery. (n = 5 per group).(G) DNA methylation detected by bisulfite sequencing PCR (BSP) in the IGF-1 promoter. (n = 5 per group). (H) PCR analysis for IGF-1 transcription. (n = 5 per group). For (A)–(D) and (H), WT: wild-type mice with sham surgery; ADK-Cre: cardiomyocyte-specific ADK-knockout mice with sham surgery; WT + I/R: wild-type mice with I/R surgery; ADK-Cre + I/R:…

• 

  Fig. 5. DNMT1 was a limiting factor for the cardioprotective effects of ADK knockout-induced adenosine accumulation. (A) PCR analysis for IGF-1 transcription. (B) PCR analysis for IGF-1R transcription. (C) DNA methylation detected by bisulfite sequencing PCR (BSP) in the IGF-1 promoter. (D) DNA methylation detected by BSP in the IGF-1R promoter. (E) Quantification of myocardial adenosine levels. (F) SAH levels detected by ELISA. (G) SAM levels detected by ELISA. (H) Western blot analysis of in vitro DNMT1, SAHH, IGF-1, and IGF-1R expression (I)In vitro apoptosis levels assessed by flow cytometry. (J) SAHH activity in different cells in the presence or absence of ADO, DNMT1. For (A)–(G), ADO: adenosine supplementation, "-" signifies no adenosine supplementation; DNMT1: DNMT1 lentiviral transduction, "-" signifies null vector transduction. For (H) and (I), Con: wild-type cardiomyocytes cultured under hypoxia/reoxygenation (H/R) conditions; DNMT1: wild-type cardiomyocytes cultured under H/R conditions with DNMT1 lentiviral transduction; ADO: wild-type cardiomyocytes cultured under H/R conditions with adenosine supplementation; ADO + DNMT1: wild-type cardiomyocytes cultured under H/R…

• 

  Fig. 6. Cardiomyocyte-specific ADK knockout improved myocardial metabolism via upregulation of the IGF-1-associated pathway after I/R. (A) Western blot analysis of IGF-1, ADK, and DNMT1 expression. (B) PET/CT of cardiac glucose uptake. (n = 5 per group) (C) Evans blue/TTC staining of myocardial tissues from wild-type and ADK-knockout mice after I/R surgery and with or without IGF-1 inhibitor treatment. (D) IR/AAR assessed from the Evans blue/TTC staining (n = 8 per group). (E) AAR/LV assessed from the Evans blue/TTC staining (n = 8 per group). (F) Echocardiographic analysis of ejection fraction in the mice from the different groups (n = 5 per group). (G) Echocardiographic analysis of fractional shortening in the mice from the different groups (n = 5 per group). (H) Immunofluorescence analysis of the spatial distribution. Bars are 50 μm marked with white color in the merged version. (n = 5 per group).All marks indicated P < 0.05, *, † and ‡ to indicate significance versus 1st, 2nd, and 3rd group accordingly. For (A) and (B), WT: wild-type mice with sham surgery; ADK-Cre: cardiomyocyte-specific ADK-knockout mice with sham surgery; WT + I/R: wild-type mice with I/R surgery; ADK-Cre + I/R:…

• 

  Fig. 7. IGF-1 was identified as a downstream target gene required for the metaboloepigenetic effects of cardiomyocyte-specific ADK knockout in response to I/R (All data in Fig. 7 were under the condition of I/R setting). (A) Evans blue/TTC staining of myocardial tissues from WT, cardio-ADK KO, cardio-IGF-1 KO, cardio-ADK/IGF-1-KO mice (n = 8 per group).(B) IR/AAR assessed from the Evans blue/TTC staining. (C) AAR/LV assessed from the Evans blue/TTC staining. (D) Echocardiographic analysis of ejection fraction in the mice (n = 5 per group). (E) Echocardiographic analysis of fractional shortening in the mice (n = 5 per group). (F) Western blot analysis of IGF-1, BAX, and DNMT1 expression. (G) Detection of in vivo ROS levels by DHE staining. (H) Examination of cardiac glucose uptake (n = 5 per group). (I)Examination of cardiac lactate production (n = 5 per group). (J) Immunofluorescence analysis of the spatial distribution of IGF-1, ADK, BAX, and DNMT1 expression. Bars are 50 μm marked with white color in the merged version.All marks indicated P < 0.05, *, † and ‡ to indicate significance versus 1st, 2nd, and 3rd group accordingly. WT: wild-type mice with I/R surgery; cardio-ADK KO:…

• 

  Fig. 8. Activation of the myocardial ADK/DNMT1/IGF-1 signaling axis improved cellular energy metabolism in vitro. (A) Examination of myocardial glucose uptake in vitro. (n = 5 per group) (B) Examination of myocardial lactate production in vitro. (n = 5 per group) (C) Quantification of myocardial adenosine levels. (n = 5 per group) (D) SAH levels detected by ELISA. (n = 5 per group) (E) SAM levels detected by ELISA. (n = 5 per group) (F) Analysis of mitochondrial respiration in cardiomyocytes isolated from mice. (n = 3 per group) (G) Analysis of glycolytic function in cardiomyocytes isolated from mice. (n = 3 per group) (H) Mechanistic model of the myocardial ADK/DNMT1/IGF-1 axis and its downstream metabolic effects. All marks indicated P < 0.05, *, † and ‡ to indicate significance versus 1st, 2nd, and 3rd group accordingly. ADK: wild-type ADK, "-" signifies ADK knockout; IGF-1: wild-type IGF-1, "-" signifies IGF-1 knockout. All experiments were repeated 3 times.