Casein Kinase-2 Interacting Protein-1 Regulates Physiological Cardiac Hypertrophy via Inhibition of Histone Deacetylase 4 Phosphorylation

Figures

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  FIGURE 1. CKIP-1 expression changes in the hearts of mice after 21 days swimming exercise. (A) Schematic representation of swimming exercise induced physiological cardiac hypertrophy model in mice. (B) CKIP-1 mRNA levels assessed by quantitative polymerase chain reaction in mouse heart tissues from WT mice with 21 days swimming exercise, normalized to GAPDH. n = 7 replicates from rest mice and n = 8 replicates from swimming mice. (C) Western blot analysis of CKIP-1 expression in total protein extracts from rest and swimming exercise hearts with 21 days training (n = 3). (D) Quantification of CKIP-1 protein levels. Values are means ± SEM, ∗P < 0.05, ∗∗P < 0.01. Statistical differences between two groups were determined by the unpaired two-tailed Student’s t-test. CKIP-1, casein kinase-2 interacting protein-1; WT, wild type; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  FIGURE 2. Physiological stress-swimming exercise causes pathological cardiac remodeling in CKIP-1-deficient mice. (A) Top, representative images of hematoxylin and eosin staining in hearts from WT and CKIP-1 KO aged (2-month-old) mice after rest or swimming exercise. Scale bars, 50 μm. Bottom, representative images of heart sections stained with Masson’s trichrome. Scale bars, 50 μm. (B) Heart weight (HW) to body weight (BW) ratios in WT and CKIP-1 KO mice with rest or swimming exercise. (C) Quantification of LV weight/body weight. (D–F) Cardiac function was monitored via echocardiography at 3 weeks after swimming exercise: (D) representative M-mode echocardiographic images of each study group at 3 weeks; (E) ejection fraction (EF) in WT-Rest, KO-Rest, WT-Swimming, and KO-Swimming mice; (F) fractional shortening (FS) in WT and CKIP-1 KO mice after rest or swimming exercise. (G–L) Quantitative analysis of the diastolic and systolic left ventricular internal diameter (LVIDd and LVIDs), LV posterior wall thickness (LVPWd and LVPWs), and LV anterior wall thickness (LVAWd and LVAWs) from WT and KO mice by echocardiography after swimming exercise. Values are means ± SEM, n = 8∼12, ∗P < 0.05, ∗∗P…

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  FIGURE 3. Myocardial CKIP-1 overexpression does not affect physiological cardiac remodeling in response to swimming exercise. (A) CKIP-1 TG mice and WT littermates at 2 months of age were subjected to rest or swimming exercise. Histological sections from hearts were stained with H&E (scale bars, 50 μm) and MTT to detect fibrosis (scale bars, 50 μm). The ratios of heart weight to body weight (B) and LV mass to body weight (C) in WT and CKIP-1 TG mice after swimming exercise. Representative echocardiographic M-mode images (D), ejection fraction (E), and fractional shortening (F) evaluated by echocardiography in anesthetized WT and CKIP-1-TG mice with rest or swimming exercise. (G–L) Transthoracic echocardiography evaluating the left ventricular structure of WT and TG mice following swimming. Values are means ± SEM, n = 4∼9, ∗P < 0.05, ∗∗P < 0.01. Statistical differences among groups were analyzed by two-way analysis of variance (ANOVA) followed by the Bonferroni procedure. CKIP-1, casein kinase-2 interacting protein-1; WT, wild type; TG, transgenic; H&E, hematoxylin and eosin; MTT, Masson’s trichrome.

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  FIGURE 4. CKIP-1 deficiency exacerbates HDAC4 phosphorylation during exercise induced physiological cardiac hypertrophy. CKIP-1, phosphorylated-HDAC4 (S632), phosphorylated-HDAC4 (S246), and HDAC4 protein expression were measured by Western blot (A) and quantification of p-HDAC4 (S632), p-HDAC4 (S246), and HDAC4 levels in WT and CKIP-1 KO mice in response to swimming exercise (B). (C–E) Quantitative polymerase chain reaction analyses of the mRNA levels of hypertrophic marker genes (ANP, BNP, and β-MHC) in the indicated groups. Values are means ± SEM, n = 4∼6, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Statistical differences among groups were analyzed by two-way analysis of variance (ANOVA) followed by the Bonferroni procedure. CKIP-1, casein kinase-2 interacting protein-1; WT, wild type; KO, knockout; HDAC4, histone deacetylase 4; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, myosin heavy chain β.

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  FIGURE 5. Overexpression of CKIP-1 inhibits the phosphorylation of HDAC4. (A) Western blotting showing the expression levels of CKIP-1, phosphorylated HDAC4 (S632), phosphorylated HDAC4 (S246), and the total protein expression levels of HDAC4. (B) Quantification of p-HDAC4 (S632), p-HDAC4 (S246), and HDAC4 levels. (C–E) Transcript levels of hypertrophic marker genes (ANP, BNP, and β-MHC) in the hearts of WT and CKIP-1 TG mice after swimming exercise. Values are means ± SEM, n = 4∼6, ∗P < 0.05, ∗∗P < 0.01. Statistical differences among groups were analyzed by two-way analysis of variance (ANOVA) followed by the Bonferroni procedure. CKIP-1, casein kinase-2 interacting protein-1; WT, wild type; TG, transgenic; HDAC4, histone deacetylase 4; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, myosin heavy chain β.

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  FIGURE 6. Model for the mechanism of CKIP-1 regulation of cardiac remodeling by inhibition of HDAC4 phosphorylation during swimming.