Fig 1: Cardiomyocyte‐specific Pkm2 (pyruvate kinase M2) deficiency activated RAC1 (rho family, small GTP binding protein)‐MAPK signaling pathway in the progression of heart failure. A and B, Western blot and quantification of phosphorylation of p38, p38, p‐ERK, ERK, phosphorylation of JNK, and JNK in neonatal rat cardiomyocytes treated with si‐Pkm2 for 72 hours. β‐actin served as loading control. C through F, Western blot and quantification of p‐p38, p38, p‐ERK, ERK, p‐JNK, and JNK expression in heart tissues from Pkm2 conditional knockout and Pkm2f/f mice 6 weeks after sham or transverse aortic constriction surgery. β‐actin served as loading control. Minimum of n=6 per group. cKO indicates conditional knockout; Pkm2, pyruvate kinase M2; RAC1, rho family, small GTP binding protein; p‐p38, phosphorylation of p38; and p‐JNK, phosphorylation of JNK. Values represent as the mean±SEM; **P<0.01, ***P<0.001, ****P<0.0001.
Fig 2: PKM2 (pyruvate kinase M2) acted as a protein kinase to inhibit RAC1 (rho family, small GTP binding protein) activation.(A through C) Western blot and quantitation of PKM2 and PKM1 (pyruvate kinase M1) expression in neonatal rat cardiomyocytes after Pkm2 knockdown. β‐actin served as loading control, n=4. D, Relative pyruvate production determined by an absorbance assay in NRCMs after Pkm2 knockdown, n=4. E, Immunoprecipitation (IP) assay using anti‐PKM2 antibody in neonatal mouse cardiomyocytes. F, Immunoprecipitation assay using anti‐RAC1 antibody in neonatal mouse cardiomyocytes. G, Representative immunofluorescence images showing the colocalization of PKM2 (green) and RAC1 (red) in adult mouse cardiomyocytes and NRCMs by confocal immunofluorescence analysis (scale bars, 25 μm in adult mouse cardiomyocytes; 10 μm in NRCMs). Line profile analyses showing the distribution and intensity. The Pearson coefficient was measured from the images using ImageJ software (n=21). H, Representative Western blot and Coomassie brilliant blue staining showing the phosphorylation of RAC1 by PKM2 in vitro. Phosphoenolpyruvate was used as the phosphate donor. I, Western blot and quantitation of p‐RAC1 (S71) expression in NRCMs after Pkm2 knockdown. β‐actin served as loading control. J, Western blot and quantitation of RAC1 protein expression in the cycloheximide chase experiment. β‐actin served as loading control. AMCMs indicates adult mouse cardiomyocytes; IB, immunoblot; IgG, immunoglobulin G; IP, Immunoprecipitation; NC, negative control; NRCMs, neonatal rat cardiomyocytes; PKM1, pyruvate kinase M1; PKM2, pyruvate kinase M2; RAC1, rho family, small GTP binding protein; TAC, transverse aortic constriction; and WGA, wheat germ agglutinin. Values represent as the mean±SEM; *P<0.05, **P<0.01,****P<0.0001.
Fig 3: RAC1 (rho family, small GTP binding protein) inhibition mitigated pressure overload‐induced heart failure. A, Representative M‐mode echocardiography, gross appearance of whole hearts (scale bar, 1 mm), heart cross‐sections stained with hematoxylin and eosin (scale bar, 1 mm), histological analysis of heart sections by picrosirius red staining (scale bar, 50 μm) and cell boundaries demarcated with wheat germ agglutinin staining (scale bar, 25 μm) from Pkm2 conditional knockout and Pkm2 f/f mice after transverse aortic constriction surgery cotreated with saline or NSC23766. B, Quantitative analyses of echocardiography showing ejection fraction and fractional shortening, n=9 to 10. C, The ratio of heart weight to body weight, n=9 to 10. D, Statistical results for myocardial interstitial fibrosis analyzed by ImageJ software. n=6 to 7. E, Statistical results for the cell cross‐sectional area, n=9 to 10. F, Western blot and quantification of β‐MHC expression in hearts of Pkm2 conditional knockout and Pkm2f/f mice after transverse aortic constriction surgery cotreated with saline or NSC23766. β‐actin served as loading control. β‐MHC indicates β‐myosin heavy chain; cKO, conditional knockout; EF, ejection fraction; FS, fractional shortening; HE, hematoxylin and eosin; HW/BW, heart weight to body weight; Pkm2, pyruvate kinase M2; PSR, picrosirius red; RAC1, rho family, small GTP binding protein; TAC, transverse aortic constriction; and WGA, wheat germ agglutinin. Values represent as the mean±SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 4: Cardiomyocyte‐specific overexpression of PKM2 (pyruvate kinase M2) ameliorated pressure overload‐induced heart failure. A, Schematic illustration of adeno‐associated virus type 9‐cardiac troponin T‐mPkm2 injection in mice after surgery. B, Representative M‐mode echocardiography, gross appearance of whole hearts (scale bar, 1 mm), heart cross‐sections stained with hematoxylin and eosin (scale bar, 1 mm), histological analysis of heart sections by picrosirius red staining (scale bar, 50 μm) and cell boundaries demarcated with wheat germ agglutinin staining (scale bar, 25 μm) from littermate injected adeno‐associated virus type 9‐cardiac troponin T‐null or adeno‐associated virus type 9‐cardiac troponin T‐mPkm2 virus 1 week after transverse aortic constriction surgery. C and D, Quantitative analyses of echocardiography showing ejection fraction, fractional shortening, and heart rate, n=6 to 9. E, Statistical results for myocardial interstitial fibrosis analyzed by ImageJ software, n=6 to 9. F, Statistical results for the cell cross‐sectional area, n=6 to 9. G, The ratio of heart weight to body weight, n=6 to 9. H, Western blot and quantification of β‐MHC, RAC1 (rho family, small GTP binding protein), phosphorylation of p38, p38, phosphorylation of JNK, and JNK expression of cardiomyocyte‐specific overexpression of Pkm2 after transverse aortic constriction surgery. Β‐actin served as loading control, n=6. AAV9 indicates adeno‐associated virus type 9; β‐MHC, β‐myosin heavy chain; cTnT, cardiac troponin T; HE, hematoxylin and eosin; HW/BW, heart weight to body weight; mPkm2 indicates mouse Pkm2 gene; p‐p38, phosphorylation of p38; p‐JNK, phosphorylation of JNK; PSR, picrosirius red; TAC, transverse aortic constriction; and WGA, wheat germ agglutinin. Values represent as the mean±SEM, ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 5: Cardiomyocyte‐specific Pkm2 (pyruvate kinase M2) knockout exacerbated pressure overload‐induced heart failure in vivo. A, Representative M‐mode echocardiography, gross appearance of whole hearts (scale bar, 1 mm), heart cross‐sections stained with hematoxylin and eosin (scale bar, 1 mm), histological analysis of heart sections by picrosirius red staining (scale bar, 50 μm), and cell boundaries demarcated with wheat germ agglutinin staining (scale bar, 25 μm) of Pkm2 conditional knockout and Pkm2f/f mice 6 weeks after sham or transverse aortic constriction surgery. B, Quantitative analyses of echocardiography showing ejection fraction, fractional shortening, left ventricular internal end‐systolic diameter, and left ventricular internal end‐diastolic diameter , n=9 to 18. C, The ratio of heart weight to body weight, n=9 to 17. D, The sections were stained with wheat germ agglutinin to measure the cross‐sectional area of cardiomyocytes, n=6 to 7. E, Statistical results of myocardial interstitial fibrosis analyzed by ImageJ software, n=9 to 15. F, Western blot and quantification of β‐MHC expression of Pkm2 conditional knockout and Pkm2f/f mice 6 weeks after sham or transverse aortic constriction surgery. β‐actin served as loading control; n=5 to 9. β‐MHC indicates β‐myosin heavy chain; cKO, conditional knockout; EF, ejection fraction; FS, fractional shortening; HE, hematoxylin and eosin; HW/BW, heart weight to body weight; LVID, d, LV internal end‐diastolic diameter; LVID, s, LV internal end‐systolic diameter; Pkm2, pyruvate kinase M2; Pkm2 f/f, Pkm2 gene flanked by 2 loxP sites; PSR, picrosirius red; RAC1, rho family, small GTP binding protein; TAC, transverse aortic constriction; and WGA, wheat germ agglutinin. Values represent as the mean±SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Supplier Page from Abcam for Recombinant Human Rac1 protein (His tag N-Terminus)