The protein Ctbp2 senses cell metabolism to control heart muscle cell growth and heart repair

C57BL/6 mice were purchased from Liaoning Changsheng Biotechnology (Liaoning, China). Individually raised in a temperature-controlled environment (22 ± 1 °C), with a light/dark cycle of 12 h, food and water can be freely obtained. The weight and physiological status of animals in different experimental groups are similar. All experimental animals were raised according to the Guidelines for the Care and Use of Experimental Animals issued by China National Health Research Institute, and the experimental protocol was approved by the Ethics Committee of Harbin Medical University.

Quickly remove the hearts of neonatal mice and cut them into small pieces with scissors. Then, the cells were isolated and digested several times using 0.125% trypsin solution (Beyotime, China). Next, collect the supernatant after each round of digestion and transfer it to DMEM culture medium with 10% fetal bovine serum and 1% penicillin streptomycin solution added. Total cell suspension used 70 μM filter membrane filtration, centrifuge at 1500 rpm for 5 min. After removing the supernatant, the cells were cultured in DMEM medium with 10% fetal bovine serum and 1% penicillin streptomycin for 90 min. During this process, most non-CMs adhere to the culture bottle, while CMs remain suspended. Collect suspended cells and culture them at a certain density on a culture plate, and add Brdu (final concentration of 100 μmol/L) to the culture medium to inhibit the growth of fibroblasts.

Total RNA was extracted from cell and tissue samples using Trizol reagent (TAKARA, Japan). Total RNA was dissolved in RNase-free water (DEPC) and reverse-transcribed to cDNA. Then, Hieff qPCR SYBR Green Master Mix (YEASEN, China) was used for quantitative RT-PCR. The primers are listed in Table 1.

The isolated mouse cardiomyocytes or heart tissue were lysed in a cold lysis buffer (Beyotime, China) containing Phenylmethanesulfonyl fluoride (PMSF), and the protein was quantified using the BCA protein analysis kit (Beyotime, China). Mice cardiomyocytes or mice heart tissue nucleus were separated using the cytoplasm/nucleus separation kit (BB-36021, Bestbio, China) and tissue cytoplasm/nucleus separation kit (BB-36022, Bestbio, China). The obtained nucleus were lysed using the above method and the proteins were quantified. Protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membrane. After blocking, incubate the membrane overnight with the following primary antibodies at 4 °C: anti-Ctbp2 (1:1000; ab128871, Abcam, UK), anti-FoxO1 (1:1000; 2880T, Cell Signaling Technology, USA), anti-p21 (1:1000; ab188224, Abcam, UK), GAPDH (1:1000, TA802519, OriGene, USA). Wash the membrane with Tris Buffered saline/0.1% Tween 20 (TBST) and incubate with secondary antibody at room temperature for 1 h.

Using transfection reagents, adenovirus was transfected into cardiomyocytes to silence and overexpress Ctbp2. Adenovirus and transfection reagents were designed and synthesized by Genechem (Shanghai, China), and the transfection methods were carried out according to the manufacturer’s instructions. The transfected cells were analyzed after 48 or 72 h.

Wash the culture medium with PBS for cells cultured in vitro. Then, fix the cells with 3% frozen paraformaldehyde. Frozen section fixes with cold acetone. Cell or tissue sections were permeabilized and blocked with 10% horse serum containing 0.3% Triton X-100 at room temperature for 1 h. Then, the cells or tissue sections were incubated overnight with the following primary antibodies at 4 °C: anti-cTnT (1:400; ab8295, Abcam, UK), anti-Ctbp2 (1:50; ab128871, Abcam, UK), anti-Ki67 (1:100; 14–5698-82, Invitrogen, USA), anti-pH3 (1:1000; ab303567, Abcam, UK). After washing the cell or tissue sections with PBS, incubate them with the following secondary antibodies at room temperature for 1 h: goat anti-mouse IgG/Alexa Fluid 488 (1:500; ab150113, Abcam, UK), donkey anti-rat IgG/Alexa Fluid 647 (1:500; ab150155, Abcam, UK), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody/Alexa Fluor Plus 647 (1:500; A32733TR, Invitrogen, USA). After washing the cell or tissue sections with PBS, incubate them with DAPI at room temperature. Use a fluorescence microscope for image acquisition.

We used the EDU cell imaging kit (40277ES60, YEASEN, China) to detect cells proliferation. Briefly, add EDU (final concentration of 10 μmol/L) to the culture medium and co-incubate with cells 24 h in advance. Firstly, fix the cells with 4% paraformaldehyde for 15 min. Then permeate with 0.3% Triton X-100. Treat cells with EDU reaction mixture for 30 min, and stain the nucleus with DAPI. Use a fluorescence microscope for image acquisition.

The AAV9 vectors with the cardiomyocyte-specific cTnT promoter carrying Ctbp2 and negative control (NC) were constructed by Genechem (Shanghai, China). The AAV9 virus (1×1012v.g per mouse) was delivered into C57BL/6 male mice (4–6 weeks old) by tail vein injection for 3 weeks before MI surgery.

C57BL/6 male mice (8 weeks old) were anesthetized intraperitoneally with pentobarbital sodium and breathed using Small Animal Ventilators. After thoracotomy, ligate the left anterior descending branch of the coronary artery with 7-0 suture. The sham group mice underwent similar surgery without blocking the coronary artery. After the surgery, warm the mice until they wake up.

Use small animal cardiac ultrasound instrument Vevo3100LT (Visualsonics, JPN) to detect left ventricular systolic function in mice. The mice were anesthetized with pentobarbital sodium throughout the entire process for non-invasive examination. Using Vevo lab software, calculate the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) based on the end diastolic and end systolic inner diameters measured by M-mode.

Mice were intraperitoneally injected with Brdu (10 mg/mL), 200 µL per mouse, for 7 consecutive days. Take heart tissue for frozen sectioning. Cold acetone fixed slicing for 15 min. After washing the tissue slices with PBS, incubate them at 37 °C for 30 min with 2 mol/L HCL. After washing the tissue slices with 0.1 mol/L boric acid buffer, 10% horse serum containing 0.3% Triton X-100 was blocked at room temperature for 1 h. Then, the tissue slices were incubated overnight with primary antibodies at 4 °C: anti-Brdu (1:200; ab6326, Abcam, UK). After washing the tissue slices with PBS, the secondary antibody was incubated at room temperature for 1 h. After washing the tissue slices with PBS, DAPI was incubated at room temperature. Use a fluorescence microscope for image acquisition.

Measure the infarct size of the heart using the TTC (T8877, sigma, USA). Remove the entire heart from the mouse body and rapidly freeze for 5–10 min. Cut the heart into 2 mm thick transverse slices and place them in a well plate containing 2% TTC solution. Incubate at 37 °C for 15–30 min. Finally, take photos of the slices and measure the infarct area of different slices using ImageJ software.

Frozen sections of mice heart tissue were fixed with cold acetone for 15 min. After washing the tissue slices with PBS, the tissue slices were incubated with 5 μg/mL WGA (L4895, sigma, USA) at room temperature for 10 min. After washing the tissue slices with PBS, incubate them with DAPI at room temperature for 8 min. Use a fluorescence microscope for image acquisition. ImageJ software measures the cross-sectional area of myocardial cells.

For paraffin sections, heart tissue was fixed with 4% PFA and subsequently sent to AOPENG Biotechnology (Shanghai, China) for Masson’s trichrome staining. Briefly, the tissue was embedded in paraffin, sliced, and dehydrated. The paraffin sections were then stained using Masson’s staining solution (AOPENG, China) according to the protocol provided by the company’s technical personnel. Microscopic examination, image acquisition, and analysis were performed. For frozen sections, tissue sections were fixed with cold acetone for 15 min, washed with distilled water, and stained using Masson’s trichrome stain kit (G1340, Solarbio, China). Specific methods were followed as described in the reagent kit instructions. Microscopic examination, image acquisition, and analysis were conducted.

Frozen tissue sections were fixed with cold acetone for 15 min, washed with distilled water, and stained using the Sirius red stain kit (G1472, Solarbio, China). The staining procedure was performed according to the instructions provided with the reagent kit. Microscopic examination, image acquisition, and analysis were carried out.

After overexpression of Ctbp2 in primary neonatal mice cardiomyocytes, different concentrations of KA, different ratios of lactate, and different concentrations of sodium palmitate were added to the cell culture medium. At the same time, 1/10 volume of CCK8 solution (C0005, TargetMol, USA) was added, thoroughly mixed, and then placed in a cell culture incubator for cultivation. The absorbance (450 nm) was measured every hour using microplate reader.

After euthanizing SHAM mice and myocardial infarction mice (3 days after myocardial infarction), the mouse heart tissue was isolated, excess tissues such as large blood vessels were removed, and the heart tissue was washed in cold physiological saline. After washing, it was dipped to dry on filter paper. Place the mouse heart in a pre-cooled mouse heart slicing mold, observe the heart infarction under a stereomicroscope, divide the heart into zones (white for infarction area, pink for border, red for remote), and use a sharp blade for segmentation. After cutting the heart tissue into pieces, the tissue cytoplasm/nucleus separation kit (BB-36022, Bestbio, China) was used to extract the nucleus. NAD+/NADH assay kit (S0175, Beyotime, China) was used to detect the levels of NADH and NAD+ in the nucleus through the WST-8 colorimetric method. The extraction and detection of fatty acyl-CoA were based on the method described in the previous research (Sekiya et al. 2021; Mangino et al. 1992). The specific method is to separate lipids from the nucleus using the Bligh Dyer method, while collecting fatty acyl-CoA distributed in methanol/aqueous phase. Fatty acyl-CoA is oxidized by fatty acyl-CoA oxidase (A0270, Solarbio, China) to produce 2,3-trans-enyl-CoA and H₂O₂. Indirect quantification of fatty acyl-CoA by detecting H₂O₂ content using the H₂O₂ content assay kit (BC3595, Solarbio, China).

All results were analyzed using GraphPad Prism 8. All data were expressed as the mean ± SD. Unpaired Student’s t-test was used for two-group comparisons and one-way analysis of variance (ANOVA) was used for multigroup comparisons. P < 0.05 was considered statistically significant:*P < 0.05; **P < 0.01; ***P < 0.001.

First, we examined the changes in cardiomyocyte proliferative capacity at different developmental stages in mice. Using Ki67 and cardiac troponin T (cTnT) immunofluorescence staining, we assessed the proliferative capacity of cardiomyocytes in P1, P7, P21, and P42 mice heart tissues. The results showed a rapid decline in the proliferative capacity of cardiomyocytes over time after birth (Fig. 1A, B). Next, we analyzed the expression levels of Ctbp2 in P1, P21, and P42 mice heart tissues at both the mRNA and protein levels. The results indicated that the expression level of Ctbp2 in adult mice heart decreased compared to that in P1 mice (Fig. 1C–E). Then, using trypsin digestion, we extracted cardiomyocytes from P1 and P7 mice heart tissues and analyzed the expression levels of Ctbp2 in these cells at the mRNA level. The results showed that the expression level of Ctbp2 in P7 cardiomyocytes was lower than that in P1 cardiomyocytes (Fig. 1F). Subsequently, we used Ctbp2 and WGA/cTnT immunofluorescence staining to detect the expression levels of Ctbp2 in cardiomyocytes in P1 and adult mice heart tissues. The results indicated that the expression level of Ctbp2 in adult cardiomyocytes was lower than that in P1 cardiomyocytes (Figs. 1G, S1). In summary, the expression level of Ctbp2 in adult cardiomyocytes decreased compared to P1 cardiomyocytes, consistent with the decline in the proliferative capacity of cardiomyocytes. This suggests that Ctbp2 may be involved in the regulation of cardiomyocyte proliferation in mice.

To investigate the role of Ctbp2 in cardiomyocyte proliferation, we silenced or overexpressed Ctbp2 in P1 mice cardiomyocytes using adenoviral transfection. We validated the effects of adenoviral silencing or overexpression of Ctbp2 at the mRNA and protein levels (Figs. 2A–C, S2A–C). After silencing or overexpressing Ctbp2 in P1 cardiomyocytes, we assessed cardiomyocyte proliferation using Ki67 and 5-ethynyl-2′-deoxyuridine (EDU) immunofluorescence staining. The results showed that silencing Ctbp2 reduced the number of proliferating cardiomyocytes and total cardiomyocytes (Fig. 2D–I), while overexpressing Ctbp2 increased both populations (Fig. S2D–I). Subsequently, we performed qPCR to detect the expression of cell cycle activators (CDK1, CDK4, Cyclin B1, and Cyclin D1) in cardiomyocytes. Silencing Ctbp2 reduced the expression of CDK1, CDK4, Cyclin B1, and Cyclin D1 compared to the control (Fig. 2J–M), whereas overexpressing Ctbp2 increased their expression (Fig. S2J–M). Additionally, qPCR and Western blot were used to detect the expression of the cell cycle negative regulator p21 at the mRNA and protein levels. Silencing Ctbp2 increased the expression of p21 compared to the control (Fig. 2N–P), while overexpressing Ctbp2 decreased its expression (Fig. S2N–P). These results suggest that Ctbp2 promotes the proliferation of primary neonatal mice cardiomyocytes.

To investigate whether Ctbp2 promotes adult cardiomyocyte proliferation, we performed the following experiment (Fig. 3A). First, we used 4–6 weeks old C57BL/6 mice and administered AAV9 carrying a cardiomyocyte-specific promoter cTnT via tail vein injection to achieve cardiomyocyte specific overexpression of Ctbp2. After 21 days of injection, immunofluorescence and Western blot confirmed that the injection of AAV9-cTnT-Ctbp2-RFP successfully overexpressed Ctbp2 in adult mice cardiomyocytes (Fig. 3B–D). Subsequent analysis of heart shape and the ratio of heart weight to body weight revealed that, compared to the control group, overexpression of Ctbp2 did not alter heart volume or weight in mice (Fig. 3E, F). Masson’s trichrome staining of mice heart tissue paraffin sections showed no fibrosis compared to the control group, and panoramic scanning of heart tissue sections indicated no structural changes following Ctbp2 overexpression (Fig. 3G). Wheat germ agglutinin (WGA) staining of frozen sections of mice heart tissue showed that the cross-sectional area of cardiomyocytes did not change, indicating no occurrence of cardiomyocyte hypertrophy compared to the control group (Fig. 3H, I). Echocardiography results indicated no significant changes in cardiac cavity size or structure on B-mode in both short-axis (PSAX) and long-axis (PSLAX) views after overexpression of Ctbp2. Additionally, M-mode showed no significant difference in left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) among the three groups, suggesting no significant differences in left ventricular systolic function (Fig. 3J–O). Mice were intraperitoneally injected with 5-bromodeoxyuridine (Brdu) for 7 consecutive days, and heart tissue was then collected for frozen sectioning. Brdu immunofluorescence staining revealed no difference in cardiomyocyte proliferation compared to the control group, indicating that overexpression of Ctbp2 did not affect cardiomyocyte proliferation (Fig. 3P, Q). Overall, these results suggest that under normal conditions, cardiomyocyte specific overexpression of Ctbp2 in adult mice has no impact on heart volume, weight, structure, function, cardiomyocyte cross-sectional area, or proliferative capacity.

To further investigate the impact of Ctbp2 overexpression on the proliferative and regenerative capacity of adult cardiomyocytes, we constructed a mouse myocardial infarction model by ligating the left anterior descending coronary artery in mice with Ctbp2-overexpressing cardiomyocytes. We then evaluated cardiac function, cardiac hypertrophy, infarct size, fibrotic area, and cardiomyocyte proliferation following infarction (Fig. 4A). First, echocardiography showed no significant difference in initial cardiac function among the wild-type mice, AAV9-NC mice and AAV9-Ctbp2 mice before myocardial infarction(Fig. 3J–O), but overexpression of Ctbp2 significantly improved cardiac function after myocardial infarction (Fig. 4B–G). Observations of the heart before and after isolation showed that Ctbp2 overexpression significantly ameliorated cardiac deformation and hypertrophy post-myocardial infarction (Fig. 4H). Additionally, WGA staining of heart tissue frozen sections demonstrated that Ctbp2 overexpression significantly reduced the cross-sectional area of cardiomyocytes after myocardial infarction (Fig. 4I, J). 2,3,5-Triphenyltetrazolium Chloride (TTC) staining, Masson’s trichrome staining, and Sirius red staining of heart tissue further revealed that Ctbp2 overexpression significantly reduced the infarct size and fibrotic area (Figs. 4K–M, S3A). In adult mice with cardiomyocyte specific Ctbp2 overexpression, following the construction of myocardial infarction, Brdu was administered via intraperitoneal injection for 7 consecutive days. Brdu immunofluorescence staining of heart tissue was then used to assess cardiomyocyte proliferation. To investigate changes in proliferative capacity more precisely, we divided the infarcted heart tissue into infarction, border, and remote area (Fig. S3B). Meanwhile, we conducted statistical analysis on both cardiomyocytes and non-cardiomyocytes in the heart tissue (Fig. S3C). Brdu immunofluorescence staining results showed no significant difference in the percentage of Brdu positive non-cardiomyocytes and cardiomyocytes between MI+AAV9-NC and MI+AAV9-Ctbp2 mice in the remote area. However, in the border area, compared to MI+AAV9-NC mice, MI+AAV9-Ctbp2 mice showed no significant change in the percentage of Brdu positive non-cardiomyocytes but a significant increase in the percentage of Brdu positive cardiomyocytes (Figs. 4N–P, S3D–G). In addition, Ki67 and pH3 immunofluorescence staining also obtained the same results as Brdu immunofluorescence staining (Fig. 4Q). In summary, in adult mice, Ctbp2 overexpression promotes heart regeneration after myocardial infarction by stimulating cardiomyocytes to re-enter the cell cycle, thereby providing a protective effect against cardiac injury.

These results indicate that under normal conditions, overexpression of Ctbp2 in adult mice cardiomyocytes does not affect their proliferative capacity. However, in the context of heart injury, overexpression of Ctbp2 in adult mice cardiomyocytes promotes their proliferation. Therefore, we aimed to explore why Ctbp2 overexpression in adult mice cardiomyocytes promotes proliferation only when heart injury is present. Previous studies have shown that in the liver, Ctbp2 can bind to the transcription factor FoxO1 and directly inhibit FoxO1 mediated gluconeogenesis. This inhibitory ability, however, is influenced by the metabolites NADH/NAD+ and fatty acyl-CoAs. NADH activates Ctbp2 by binding to it, enhancing its ability to inhibit FoxO1’s transcriptional activity, whereas fatty acyl-CoAs inactivates Ctbp2 by binding to it, thus inhibiting its transcriptional repression of FoxO1 (Sekiya et al. 2021, 2024). Other studies have indicated that transcription factor FoxO1, besides regulating metabolism, can also inhibit the cardiomyocyte cell cycle through various mechanisms, including the activation of its target genes, cell cycle negative regulators p21 and p27 (Evans-Anderson et al. 2008; Ho et al. 2008; Sengupta et al. 2013). It is well known that normal adult cardiomyocytes primarily rely on fatty acid oxidation for energy. However, during cardiomyocyte ischemia, the inability of mitochondria to utilize oxygen disrupts the oxidation of NADH to NAD+, leading to an accumulation of NADH within the cells. Given that Ctbp2, as a transcriptional repressor, is primarily localized in the nucleus, we first examined the changes in nuclear levels of NADH and fatty acyl-CoAs, an intermediate product of fatty acid oxidation, in cardiomyocytes under ischemic and hypoxic conditions. The results showed that in sham mice heart tissue, nuclear fatty acyl-CoAs levels were higher than NADH levels. However, in myocardial infarction mice heart tissue, nuclear NADH levels increased, NAD+ levels decreased, and the NADH/NAD+ ratio increased, with a slight increase in fatty acyl-CoAs levels. Consequently, nuclear NADH levels were higher than fatty acyl-CoAs levels in myocardial infarction mice heart tissue (Fig. 5A–E). Additionally, regional analysis of the infarcted heart tissue revealed that the closer the tissue was to the infarction area, the higher the NADH levels, the lower the NAD+ levels, and the higher the NADH/NAD+ ratio. Moreover, the closer the tissue was to the infarction area, the slightly higher the fatty acyl-CoAs levels. Therefore, in the remote area, nuclear fatty acyl-CoAs levels were higher than NADH levels, whereas in the border area and infarction area, nuclear NADH levels were higher than fatty acyl-CoAs levels (Fig. 5F–J). Based on previous research and the above experimental results, we hypothesize that in normal adult cardiomyocytes, fatty acid oxidation predominates as the primary energy source, with the intermediate product fatty acyl-CoAs inhibiting Ctbp2 activity and preventing it from exerting its effects. However, during ischemia and hypoxia, the accumulation of NADH within the cells activates Ctbp2, enabling it to promote cardiomyocyte proliferation.

To validate our hypothesis, we overexpressed Ctbp2 in primary neonatal mice cardiomyocytes and subsequently added varying concentrations of koningic acid (KA), different ratios of lactate, and varying concentrations of sodium palmitate to the cell culture medium. This was done to modulate the levels of NADH/NAD+ and fatty acyl-CoAs in the cells and assess changes in the proliferation ability of cardiomyocytes (Fig. 6A). The CCK8 assay results showed that, compared to the control, overexpression of Ctbp2 promoted cardiomyocyte proliferation. Upon the addition of KA, cardiomyocyte proliferation was inhibited as the intracellular NADH decreased, with this inhibition gradually increasing with higher concentrations of KA (Fig. 6B). Conversely, the addition of the lactate solution led to an increase in NADH, which further promoted cardiomyocyte proliferation. This proliferative effect was enhanced with higher concentrations of the lactate solution (Fig. 6C). Following the addition of sodium palmitate, an increase in intracellular fatty acyl-CoAs inhibited cardiomyocyte proliferation, with this inhibition also increasing with higher concentrations of sodium palmitate (Fig. 6D). To visualize the regulatory effects of NADH/NAD+ and fatty acyl-CoAs on Ctbp2 in cardiomyocytes, we used EDU and cTnT immunofluorescence staining to further examine cell proliferation. The results indicated that, compared to Ad-NC cardiomyocytes, the number of EDU positive cardiomyocytes increased in Ad-Ctbp2 cardiomyocytes. In comparison to Ad-Ctbp2 cardiomyocytes, the number of EDU positive cardiomyocytes decreased in Ad-Ctbp2+KA and Ad-Ctbp2+Sodium palmitate cardiomyocytes, whereas the number of EDU positive cardiomyocytes further increased in Ad-Ctbp2+Lactate cardiomyocytes (Fig. 6E, F). In order to further study, we combined the Ad-Ctbp2-RFP expression in cardiomyocytes and found that in cardiomyocytes overexpressing Ctbp2 (RFP positive cardiomyocytes), approximately 52% were EDU positive, and about 48% were EDU negative. After adding KA, approximately 26% of RFP positive cardiomyocytes were EDU positive, and about 74% were EDU negative. Following the addition of lactate, approximately 61% of RFP positive cardiomyocytes were EDU positive, and about 39% were EDU negative. After the addition of sodium palmitate, approximately 21% of RFP positive cardiomyocytes were EDU positive, and about 79% were EDU negative (Fig. 6G, H).

These results indicate that the regulation of cardiomyocyte proliferation by Ctbp2 is influenced by the metabolites NADH/NAD+ and fatty acyl-CoAs. When Ctbp2 is overexpressed in primary neonatal mice cardiomyocytes, their proliferation capacity increases. However, when NADH levels decrease or fatty acyl-CoAs levels increase, the cardiomyocytes do not exhibit enhanced proliferation despite the overexpression of Ctbp2. Conversely, when NADH levels rise, the proliferation capacity of the cardiomyocytes is further enhanced, even on the basis of Ctbp2 overexpression, confirming our hypothesis.

These results demonstrate that the metabolites NADH/NAD+ and fatty acyl-CoAs influence the regulation of cardiomyocyte proliferation by Ctbp2. To further investigate the underlying mechanisms, we examined the expression of the transcription factor FoxO1 and its target genes, the cell cycle negative regulators p21 and p27. First, in vivo experiments, we found that under normal conditions, there were no significant differences in the mRNA levels of FoxO1, p21, and p27, as well as the expression levels of the transcriptionally active FoxO1 protein in the nuclei of heart tissues between AAV9-NC and AAV9-Ctbp2 mice (Fig. 7A–E). In the context of heart injury, the mRNA levels of FoxO1, p21, and p27, along with the nuclear expression of FoxO1 protein, showed no significant differences in the remote area of heart tissue between MI+AAV9-NC and MI+AAV9-Ctbp2 mice. However, in the border area of heart tissue, compared to MI+AAV9-NC mice, MI+AAV9-Ctbp2 mice exhibited no change in FoxO1 mRNA levels, but a decrease in the nuclear expression of FoxO1 protein. Additionally, the mRNA levels of p21 and p27 were also reduced in the heart tissue (Fig. 7F–J). In vitro experiments, we observed that compared to Ad-NC cardiomyocytes, Ad-Ctbp2 cardiomyocytes showed no significant change in FoxO1 mRNA levels, but a decrease in the nuclear expression of FoxO1 protein. The mRNA levels of p21 and p27 were also reduced in these cells. Furthermore, compared to Ad-Ctbp2 cardiomyocytes, the addition of KA and sodium palmitate led to no significant change in FoxO1 mRNA levels, but an increase in the nuclear expression of FoxO1 protein, as well as an increase in the mRNA levels of p21 and p27. Conversely, the addition of lactate resulted in no significant change in FoxO1 mRNA levels, but a decrease in the nuclear expression of FoxO1 protein, and a reduction in the mRNA levels of p21 and p27 (Fig. 7K–O).

Combining the in vivo and in vitro results, the expression of FoxO1, p21, and p27 is correlated with the regulation of cardiomyocyte proliferation by Ctbp2. Specifically, when intracellular NADH levels increase, the transcriptional repressor Ctbp2 is activated in the nucleus, leading to a decrease in the expression of transcriptionally active FoxO1 protein. This suppresses the transcription of its target genes, the cell cycle negative regulators p21 and p27, thereby promoting cell proliferation. Conversely, when intracellular NADH levels decrease or fatty acyl-CoAs levels increase, Ctbp2 becomes inactivated in the nucleus, resulting in an increase in the expression of transcriptionally active FoxO1 protein. This enhances the transcription of its target genes, p21 and p27, thereby inhibiting cell proliferation. In summary, the metabolites NADH/NAD+ and acyl-CoAs affects the regulation of Ctbp2 on the cardiomyocytes proliferation may be achieved through the Ctbp2-FoxO1-p21/p27 pathways.

All experimental animals were raised according to the Guidelines for the Care and Use of Experimental Animals issued by China National Health Research Institute, and the experimental protocol was approved by the Ethics Committee of Harbin Medical University.

All of the authors have consented to the publication of this research.

The authors declare that they have no competing interests.

Not applicable. No datasets were generated or analysed during the current study.