HIPK1 Inhibition Protects against Pathological Cardiac Hypertrophy by Inhibiting the CREB‐C/EBPβ Axis

Figures

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  Figure 1. Genetic ablation of HIPK1 prevents TAC‐induced pathological cardiac hypertrophy. a) Western blot for HIPK1 in heart tissues from sham‐operated mice or mice at 2, 4, 6, and 8 weeks post TAC surgery (n = 6). b) Western blot for HIPK1 in NRCMs treated with PE or control vehicle (n = 6). c) Western blot for HIPK1 in human cardiac tissues from patients with hypertrophic cardiomyopathy and control samples (n = 3). d) Echocardiography for LV ejection fraction (EF) and fractional shortening (FS) at 8 weeks post TAC surgery (n = 13 for WT mice, n = 9 for HIPK1 KO mice). e) Heart weight (HW), heart weight/body weight (HW/BW) ratio, and heart weight/tibia length (HW/TL) ratio of mice (n = 9–13). f) Wheat germ agglutinin (WGA) staining for cardiomyocyte (CM) cross‐sectional area (n = 8,8,8,5). Scale bar = 50 µm. g) Masson staining for cardiac fibrosis area (n = 5–6). Scale bar = 100 µm. h,i) qRT‐PCR for h) ANP, BNP, and β‐MHC and i) Col1a1, Col3a1, and CTGF in mice heart tissues after sham or TAC surgery (n = 8). Data between two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among four groups were compared by one‐way ANOVA test or two‐way ANOVA test followed by…

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  Figure 2. Inhibiting HIPK1 prevents cardiomyocyte hypertrophy. a,b) Western blot for HIPK1 in NRCMs treated with a) sh‐HIPK1 or b) OE‐HIPK1 lentivirus and relative controls (n = 3). c,e) Immunofluorescent staining for α‐actinin to evaluate cardiomyocyte (CM) size in NRCM treated with c) sh‐HIPK1 or e) OE‐HIPK1 lentivirus upon PE stress (n = 4). Scale bar = 50 µm. d,f) qRT‐PCR for ANP, BNP, and β‐MHC in NRCM treated with d) sh‐HIPK1 or f) OE‐HIPK1 lentivirus upon PE stress (n = 6). Data between two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among four groups were compared by two‐way ANOVA test followed by Tukey post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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  Figure 3. HIPK1 phosphorylates CREB and activates C/EBPβ in cardiomyocytes. a) NRCMs were treated with PE, and HIPK1 expression was evaluated by Western blot in the cytoplasm and nucleus components, respectively (n = 3). b) Western blot for CREB phosphorylation and C/EBPβ isoform expression in the nucleus component of NRCMs treated with PE (n = 3). c) H9C2 cell lysates were incubated with CREB antibody, and co‐immunoprecipitation of CREB and HIPK1 was evaluated by Western blot. d,e) Western blot for HIPK1, CREB phosphorylation, and C/EBPβ isoform expressions in NRCMs transfected with d) sh‐HIPK1 or e) OE‐HIPK1 lentivirus (n = 3). f,g) qRT‐PCR for HIPK1 and C/EBPβ in NRCMs transfected with f) sh‐HIPK1 (n = 5) or g) OE‐HIPK1 (n = 6) lentivirus. h) Western blot for HIPK1, CREB phosphorylation, and C/EBPβ expressions in NRCMs transfected with OE‐HIPK1, OE‐HIPK1 (K219A, kinase‐inactive mutant form of HIPK1 by changing Lysine to Alanine at position 219), and Fugw control plasmids (n = 3). Data between two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among three groups were compared by one‐way ANOVA test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not…

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  Figure 4. HIPK1 knockdown inhibits pathological hypertrophy of cardiomyocytes through inactivation of the CREB‐C/EBPβ axis. a) qRT‐PCR for CREB in NRCMs transfected with OE‐CREB plasmid or control vector (n = 6). b) Immunofluorescent staining for α‐actinin in NRCMs treated with sh‐HIPK1 lentivirus and/or OE‐CREB plasmid (n = 6). Scale bar = 100 µm. c) qRT‐PCR for C/EBPβ in NRCMs transfected with OE‐C/EBPβ plasmid or control vector (n = 5). d) Immunofluorescent staining for α‐actinin in NRCMs treated with sh‐HIPK1 lentivirus and/or OE‐C/EBPβ plasmid (n = 6). Scale bar = 100 µm. e) H9C2 cell lysates were incubated with C/EBPβ antibody, and co‐immunoprecipitation of C/EBPβ with NFAT3 and GATA4 was evaluated by Western blot. f) qRT‐PCR for genes associated with pathological cardiac hypertrophy in NRCMs (n = 6). g,h) Western blot for CREB phosphorylation and C/EBPβ expressions in heart tissues from WT and HIPK1 KO mice with g) sham or h) TAC operations (n = 3). i,j) qRT‐PCR for C/EBPβ expression in heart tissues from WT and HIPK1 KO mice with i) sham or j) TAC operations (n = 5–6). Data between two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among four groups…

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  Figure 5. cTnT‐AAV9‐mediated HIPK1 knockdown prevents TAC‐induced pathological cardiac hypertrophy. a) Echocardiography for LV ejection fraction (EF) and fractional shortening (FS) at 6 weeks after TAC surgery (n = 10–11). b) Heart weight (HW), heart weight/body weight (HW/BW) ratio, and heart weight/tibia length (HW/TL) ratio of mice (n = 10–11). c) Wheat germ agglutinin (WGA) staining for cardiomyocyte (CM) cross‐sectional area (n = 6). Scale bar = 50 µm. d) Masson staining for cardiac fibrosis area (n = 6–7). Scale bar = 100 µm. e,f) qRT‐PCR for e) ANP, BNP, and β‐MHC and f) Col1a1, Col3a1, and CTGF in mice heart tissues after sham or TAC surgery (n = 6). g) Western blot for HIPK1, CREB phosphorylation, and C/EBPβ expressions in mice heart tissues with sham or TAC surgery (n = 6). Data among four groups were compared by two‐way ANOVA test followed by Tukey post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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  Figure 6. CREB overexpression attenuates the protective effect of HIPK1 knockdown against pathological cardiac hypertrophy. a) Echocardiography for LV ejection fraction (EF) and fractional shortening (FS) at 6 weeks after TAC surgery (n = 7–10). b) Heart weight (HW), heart weight/body weight (HW/BW) ratio, and heart weight/tibia length (HW/TL) ratio of mice (n = 7–10). c) Wheat germ agglutinin (WGA) staining for cardiomyocyte (CM) cross‐sectional area (n = 6). Scale bar = 50 µm. d) Masson staining for cardiac fibrosis area (n = 6–8). Scale bar = 100 µm. e,f) qRT‐PCR for e) ANP, BNP, and β‐MHC and f) Col1a1, Col3a1, and CTGF in mice heart tissues after TAC surgery (n = 7–8). g) Western blot for HIPK1, CREB phosphorylation, and C/EBPβ expressions in mice heart tissues after TAC surgery (n = 6). Data among four groups were compared by two‐way ANOVA test followed by Tukey post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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  Figure 7. Schematic illustration for the role of HIPK1 in pathological cardiac hypertrophy. Hypertrophic stress‐induced HIPK1 is present in the nucleus of cardiomyocytes, leading to activation of the CREB‐C/EBPβ axis and subsequent hypertrophic gene expression. HIPK1 inhibition prevents pathological cardiac hypertrophy through inhibiting CREB phosphorylation at Ser271 and inactivating C/EBPβ. Inhibition of HIPK1 and CREB forms a synergistic pathway in preventing pathological cardiac hypertrophy.