The transcriptional repressor Ctbp2 as a metabolite sensor regulating cardiomyocytes proliferation and heart regeneration

Figures

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  Fig. 1. Ctbp2 participates in the regulation of cardiomyocytes proliferation. A Ki67 and cTnT immunofluorescence staining of heart from P1, P7, P21, and P42 mice. Scale bar = 100 µm. B Statistical analysis of the proportion of Ki67-positive cardiomyocytes (n = 3). Data were expressed as mean ± SD. *p vs. the P1 group, #p vs. the P7 group. **P < 0.01, ***P < 0.001, ##P < 0.01, One-way ANOVA. C qPCR analysis of Ctbp2 in hearts from P1, P21, and P42 mice (n ≥ 4). Data were expressed as mean ± SD. **P < 0.01, One-way ANOVA. D, E Western-blot analysis of Ctbp2 in hearts from P1, P21, and P42 mice (n = 4). Data were expressed as mean ± SD. **P < 0.01, ***P < 0.001, One-way ANOVA. F qPCR analysis of Ctbp2 in cardiomyocytes from P1 and P7 mice (n = 3). Data were expressed as mean ± SD. *P < 0.05, t test. G Ctbp2 and WGA immunofluorescence staining of heart from P1 and Adult mice. Scale bar = 50 µm

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  Fig. 2. Silencing Ctbp2 inhibits the proliferation of primary neonatal mice cardiomyocytes. A qPCR analysis of Ctbp2 in cardiomyocytes from P1 mice after 72 h of transfection with Ad-sh-NC and Ad-sh-Ctbp2 (n ≥ 3). Data were expressed as mean ± SD. ***P < 0.001, One-way ANOVA. B, C Western-blot analysis of Ctbp2 in cardiomyocytes from P1 mice after 72 h of transfection with Ad-sh-NC and Ad-sh-Ctbp2 (n ≥ 3). Data were expressed as mean ± SD. ***P < 0.001, One-way ANOVA. D Ki67 and cTnT immunofluorescence staining of cardiomyocytes from P1 mice after 72 h of transfection with Ad-sh-NC and Ad-sh-Ctbp2. Scale bar = 100 µm. E, F Statistical analysis of the number of Ki67-positive cardiomyocytes and the total number of cardiomyocytes (n = 3). Data were expressed as mean ± SD. **P < 0.01, ***P < 0.001, One-way ANOVA. G EDU and cTnT immunofluorescence staining of cardiomyocytes from P1 mice after 72 h of transfection with Ad-sh-NC and Ad-sh-Ctbp2. Scale bar = 200 µm. H, I Statistical analysis of the number of EDU-positive cardiomyocytes and the total number of cardiomyocytes. Data were expressed as mean ± SD (n ≥ 3). ***P < 0.001, One-way ANOVA. J–M qPCR analysis of cell cycle activators (CDK1,…

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  Fig. 3. Overexpression of Ctbp2 has no effect on the heart of normal adult mice. A Schematic diagram for experimental procedure. B cTnT immunofluorescence staining of heart from 4 to 6-week-old mice after 21 days of tail vein injection of AAV9-cTnT-Ctbp2-RFP. Scale bar = 100 µm. C, D Western-blot analysis of Ctbp2 in hearts from AAV9-NC mice and AAV9-Ctbp2 mice (n = 3). Data were expressed as mean ± SD. ***P < 0.001, t test. E Representative images of heart from AAV9-NC mice and AAV9-Ctbp2 mice. Scale bar = 2 mm. F The ration of heart weight to body weight from AAV9-NC mice and AAV9-Ctbp2 mice (n = 5). Data were expressed as mean ± SD. t test. G Masson’s trichrome staining of heart from AAV9-NC mice and AAV9-Ctbp2 mice. Scale bar = 2 mm. H, I WGA staining of heart from AAV9-NC mice and AAV9-Ctbp2 mice, ImageJ measurement of cardiomyocyte size (n = 4). Scale bar = 40 µm. Data were expressed as mean ± SD. t test. J–L Cardiac function (PSAX) of wild-type mice, AAV9-NC mice and AAV9-Ctbp2 mice (n ≥ 3). Data were expressed as mean ± SD. One-way ANOVA. M–O Cardiac function (PSLAX) of wild-type mice, AAV9-NC mice and AAV9-Ctbp2 mice (n ≥ 3). Data were expressed as mean ± SD. One-way ANOVA. P…

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  Fig. 4. Overexpression of Ctbp2 has a protective effect on adult mice with heart injury. A Schematic diagram for experimental procedure. B–D Cardiac function (PSAX) of SHAM mice, MI+AAV9-NC mice and AAV9-Ctbp2 mice (n ≥ 5). Data were expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA. E–G Cardiac function (PSLAX) of SHAM mice, MI+AAV9-NC mice and AAV9-Ctbp2 mice (n ≥ 4). Data were expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA. H Representative images of heart from MI+AAV9-NC mice and AAV9-Ctbp2 mice. Scale bar = 4 mm (left), Scale bar = 2 mm (right). I, J WGA staining of heart from SHAM mice, MI+AAV9-NC mice and MI+AAV9-Ctbp2 mice, ImageJ measurement of cardiomyocyte size (n = 4). Scale bar = 40 µm. Data were expressed as mean ± SD. *P < 0.05, ***P < 0.001, One-way ANOVA. K, L TTC staining of heart from SHAM mice, MI+AAV9-NC mice and MI+AAV9-Ctbp2 mice (n = 4). Data were expressed as mean ± SD. **P < 0.01, t test. M Masson’s trichrome staining of heart from MI+AAV9-NC mice and MI+AAV9-Ctbp2 mice. Scale bar = 1 mm. N Brdu and WGA immunofluorescence staining of heart from MI+AAV9-NC mice and MI+AAV9-Ctbp2 mice. Scale bar = 100 µm. O, P…

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  Fig. 5. Content of nuclear metabolites NADH/NAD+ and fatty acyl-CoAs in heart tissue of mice. A–E NADH/NAD+ content and fatty acyl-CoAs content in heart tissue nucleus of SHAM mice and MI mice (n ≥ 3). Scale bar = 2 mm. Data were expressed as mean ± SD. *P < 0.05, **P < 0.01, t test. F–J NADH/NAD+ content and fatty acyl-CoAs content in heart tissue (infarction area, Border, Remote) nucleus of MI mice (n ≥ 3). Scale bar = 2 mm. Data were expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA

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  Fig. 6. Ctbp2 regulation of cardiomyocyte proliferation is influenced by the metabolites NADH/NAD+ and fatty acyl-CoAs. A Schematic diagram for experimental procedure. B CCK8 analysis of control, Ad-NC, Ad-Ctbp2, Ad-Ctbp2+1 μmol/L KA, Ad-Ctbp2+5 μmol/L KA, Ad-Ctbp2+10 μmol/L KA cardiomyocytes (n = 5). Data were expressed as mean ± SD. *P < 0.05, ***P < 0.001, One-way ANOVA. C CCK8 analysis of control, Ad-NC, Ad-Ctbp2, Ad-Ctbp2+1:1 (Pyruvate:Lactate), Ad-Ctbp2+1:10 (Pyruvate:Lactate), Ad-Ctbp2+1:100 (Pyruvate:Lactate) cardiomyocytes (n = 5). Data were expressed as mean ± SD. *P < 0.05, ***P < 0.001, One-way ANOVA. D CCK8 analysis of control, Ad-NC, Ad-Ctbp2, Ad-Ctbp2+100 μmol/L Sodium Palmitate, Ad-Ctbp2+300 μmol/L Sodium Palmitate, Ad-Ctbp2+500 μmol/L Sodium Palmitate cardiomyocytes (n = 4). Data were expressed as mean ± SD. *P < 0.05, ***P < 0.001, One-way ANOVA. E EDU and cTnT immunofluorescence staining of control, Ad-NC, Ad-Ctbp2, Ad-Ctbp2+KA, Ad-Ctbp2+Lactate, Ad-Ctbp2+Sodium palmitate cardiomyocytes. Scale bar = 40 µm. F Statistical analysis of the number of EDU-positive cardiomyocytes (n ≥ 4). Data were expressed as mean ± SD. *P < 0.05, **P < 0.01, One-way ANOVA. G EDU and cTnT…

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  Fig. 7. Metabolites NADH/NAD+ and fatty acyl-CoAs affects the regulation of Ctbp2 on the cardiomyocytes proliferation may be achieved through the Ctbp2-FoxO1-p21/p27 pathways. A qPCR analysis of FoxO1 in hearts from AAV9-NC and AAV9-Ctbp2 mice (n = 3). The data is represented as mean ± SD. t test. B, C Western-blot analysis of FoxO1 in heart tissue nucleus of AAV9-NC mice and AAV9-Ctbp2 mice (n = 3). The data is represented as mean ± SD. t test. D, E qPCR analysis of p21 and p27 in hearts from AAV9-NC and AAV9-Ctbp2 mice (n = 4). The data is represented as mean ± SD. t test. F qPCR analysis of FoxO1 in hearts from MI+AAV9-NC and MI+AAV9-Ctbp2 mice (n ≥ 3). Data were expressed as mean ± SD. One-way ANOVA. G, H Western-blot analysis of FoxO1 in heart tissue nucleus of MI+AAV9-NC mice and MI+AAV9-Ctbp2 mice (n ≥ 3). The data is represented as mean ± SD. *P < 0.05, **P < 0.01, One-way ANOVA. I, J qPCR analysis p21 and p27 in hearts from MI+AAV9-NC and MI+AAV9-Ctbp2 mice (n ≥ 3). Data were expressed as mean ± SD. *P < 0.05, ***P < 0.001, One-way ANOVA. K qPCR analysis of FoxO1 in control, Ad-NC, Ad-Ctbp2, Ad-Ctbp2+KA, Ad-Ctbp2+Lactate and Ad-Ctbp2+Sodium Palmitate cardiomyocytes (n = 4). Data…

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  Fig. 8. Schematic illustration on the signaling pathway of the transcriptional repressor Ctbp2 as a metabolite sensor regulating cardiomyocyte proliferation and heart regeneration