CDK9 binds and activates SGK3 to promote cardiac repair after injury via the GSK-3β/β-catenin pathway

Figures

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  FIGURE 1. CDK9 interacts with SGK3 and enhances SGK3 activity. (A–D) Silver staining of a gel showing SGK3 and the control IgG pulldown proteins (CDK9, 42 kDa, red arrow) (A). Co-immunoprecipitation reveals that CDK942 interacts with SGK3 in the H9C2 cell line (B), neonatal CMs (C), and P1 heart (D). (E) Co-localization of SGK3 and CDK9 in neonatal myocardium was detected by immunofluorescence. (F) SGK3, phosphorylated (p-) CDK9 and CDK9 proteins were detected by Western blot in CMs transfected with Ad5: cTnT-SGK3 (MOI = 50). (G) CDK9, SGK3 Thr320 and SGK3 proteins were detected by Western blot in CMs transfected with Ad5: cTnT-CDK9 (MOI = 50). CMs indicate cardiomyocytes, MW indicates molecular weight, MOI indicates a multiplicity of infection; statistical significance was calculated with the unpaired Student t-test; data are presented as mean ± SEM, **P ≤ 0.01, ***P ≤ 0.001.

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  FIGURE 2. Expression pattern and subcellular localization of CDK9. (A) CDK9 proteins of ventricular myocardium detected by western blot in P1–P56 mice. (B) mRNA expression levels of CDK9 in embryo and P1–P56 mice. (C) CDK9 proteins of ventricular myocardium detected by western blot in P7 and P7-AR mice. (D) mRNA expression levels of CDK9 in P7 and P7-AR mice. (E) SGK3 and CDK9 proteins of ventricular myocardium in P1 and P56 mice submitted to nucleocytoplasmic fractionation. MW indicates molecular weight, AR indicates apical resection; statistical significance was calculated with the unpaired Student t-test; data are presented as mean ± SEM, *P ≤ 0.05, ***P ≤ 0.001; each dot indicates a biological replicate.

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  FIGURE 3. CDK9 promotes cardiomyocyte (CM) proliferation in vitro. (A) Knockdown and overexpression of CDK9 proteins confirmed by western blot in neonatal CMs treated with Ad5: cTnT-CON, Ad5: cTnT-CDK9 or Ad5: cTnT-CDK9 RNAi. (B) Knockdown and overexpression of CDK9 mRNA confirmed by qRT-PCR. (C–E) CM proliferation quantified in neonatal CMs transfected with Ad5: cTnT-CON or Ad5: cTnT-CDK9 by immunofluorescence for DNA synthesis (C), cell-cycle activity (D), and mitosis (E) (n = 6). (F–H) CM proliferation was quantified in neonatal CMs treated with Ad5: cTnT-CDK9 RNAi by immunofluorescence for DNA synthesis (F), cell-cycle activity (G), and mitosis (H) (n = 6). (I) CM cell cycle activity was detected by flow cytometry analysis after transfected with Ad5: cTnT-CON RNAi, Ad5: cTnT-CDK9, or Ad5: cTnT-CDK9 RNAi (CDK9i) (n = 3). White arrows in representative pictures point to the proliferating CMs; scale bars, 20 μm; CMs indicate cardiomyocytes, MW indicates molecular weight; statistical significance was calculated with the unpaired Student t-test; data are presented as mean ± SEM, *P ≤ 0.05, **P ≤ 0.01, ##P ≤ 0.01, ***P ≤ 0.001; each dot indicates a biological replicate.

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  FIGURE 4. CDK9 knockdown impairs myocardial regeneration after AR in neonatal mice. (A) CDK9 protein of ventricular myocardium in neonatal mice injected Ad5: cTnT-CON RNAi or Ad5: cTnT-CDK9 RNAi (CDK9i) after AR surgery detected by western blot at 6 days after surgery. (B–D) CM proliferation in neonatal myocardial tissue after intra-myocardial injection with CDK9i or CONi quantified by immunofluorescence for DNA synthesis (B), cell-cycle activity (C), mitosis (D) (n = 3). (E) Comparison of scar size by Masson staining between the CONi and CDK9i groups at 22 days after AR surgery (n = 6). (F) LVEF and LVFS in P1 AR mice at 1 and 22 days after CDK9i or CONi injection was detected by echocardiography (n = 8). (G) Overall survival rate in neonatal mice from AR + CONi and AR + CDK9i groups (n = 15). Scale bars, 500 μm; CM indicates cardiomyocyte, MW indicates molecular weight; statistical significance was calculated with the unpaired Student t-test; data are presented as mean ± SEM, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; each dot indicates a biological replicate.

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  FIGURE 5. CDK9 overexpression promotes myocardial regeneration after MI in adult mice. (A) CDK9 protein of ventricular myocardium in adult mice injected with AAV9: cTnT-CDK9 or AAV9: cTnT-CON after MI detected by western blot (MI + CDK9 or MI + CON). (B) Left: cardiac morphology in adult mice subjected to MI + CDK9 or MI + CON at 14 dpi. Right: heart weight/Body weight (HW/BW) ratio in adult mice subjected to MI + CDK9 or MI + CON at 14 dpi (n = 6). (C) CM size in adult mice subjected to MI + CDK9 or MI + CON at 14 dpi was detected by wheat germ agglutinin (WGA) immunofluorescence (n = 6). (D,E) CM proliferation in adult myocardial tissue subjected to MI + CDK9 or MI + CON quantified by immunofluorescence for mitosis (D) and cell-cycle activity (E) (n = 6); white arrows point to the proliferating CMs. (F) Comparison of scar size by Masson staining between the CON and CDK9 groups at 28 days after MI (n = 6). (G) LVEF, LVFS, LV Mass and LV Vol d/s in adult MI mice at 4 and 28 days after CDK9 injection was detected by echocardiography (n = 7). (H) Overall survival rate in adult mice from MI + CON and MI + CDK9 groups (n = 14). Scale bars, 20/50 μm; CM indicates cardiomyocyte, MW indicates…

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  FIGURE 6. CDK9 regulates myocardial regeneration and repair through SGK3/GSK-3β/β-catenin axis in vitro. (A) p-SGK3, p-GSK3β, β-catenin and cyclin D1 proteins of ventricular myocardium in adult mice injected with Ad5: cTnT-CDK9 (CDK9) after MI detected by western blot. (B) p-SGK3, p-GSK3β, β-catenin and cyclin D1 proteins of neonatal CMs were detected by western blot after being transfected with CDK9 and/or Ad5:SGK3 RNAi (SGK3i). (C–E) CM proliferation is quantified by immunofluorescence for DNA synthesis (C), cell-cycle activity (D), and mitosis (E) in CMs transfected with CDK9 and/or SGK3i (n = 6). Scale bars, 20 μm; CMs indicate cardiomyocytes, MW indicates molecular weight; statistical significance was calculated with the unpaired Student t-test; data are presented as mean ± SEM, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; each dot indicates a biological replicate.

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  FIGURE 7. CDK9 regulates myocardial regeneration and repair through SGK3/GSK-3β/β-catenin axis in vivo. (A) p-SGK3, p-GSK3β and β-catenin proteins in neonatal myocardial tissue after intra-myocardial injection with Ad5: cTnT-CDK9 RNAi (CDK9i) and/or Ad5: cTnT-SGK3 (SGK3) after AR detected by western blot. (B–D) CM proliferation in neonatal AR myocardial tissue after intraperitoneal injection with CDK9i and/or SGK3 quantified by immunofluorescence for DNA synthesis (B), cell-cycle activity (C), mitosis (D) (n = 3). Scale bars, 50 μm; CMs indicates cardiomyocytes, AR indicates apical resection, MW indicates molecular weight; statistical significance was calculated with one-way ANOVA; data are presented as mean ± SEM, **P ≤ 0.01, ***P ≤ 0.001; each dot indicates a biological replicate.

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  FIGURE 8. Research pattern diagram. Model depicting CDK9 binds to SGK3 and activates SGK3 and its downstream GSK-3β/β-catenin/cyclin D1 pathways to regulate the CM cycle activity, promoting CM proliferation and myocardial regeneration (CMs indicate cardiomyocytes).