Synd4 induced cardiomyocytes hypertrophy via Akt phosphorylation. (A–C) Western blot showed no changes of (A) calcineurin and (B and C) its downstream targets, NFAT3 in myocardium between WT and synd4–/– mice, as well as between sedentary and exercise mice. Data are shown as a ratio to β-actin or histone H3 and are expressed as a fold-increase when compared with sedentary synd4–/– mice. N = 6, ANOVA; (D) Western blot showed decreased S473 and T308 Akt phosphorylation in synd4–/– mice after exercise. Data are shown as a ratio to total Akt and are expressed as a fold-increase when compared with WT mice. *P < 0.05, synd4–/– mice versus WT mice, N = 6, ANOVA; Ad-synd4 was used to activate synd4 in cultured cardiomyocyte. (E) Immunofluorescence assay showed synd4 overexpression could induce cardiomyocyte enlargement. A6730 (Akt inhibitor, 5 μmol/L) and calphostin C (PKC inhibitor, 100 nmol/L) could attenuate synd4 depended cardiomyocyte enlargement. *P < 0.05, Ad-Lacz versus Ad-synd4, Ad-synd4 + A6730 versus Ad-synd4, Ad-synd4 + calphostin C versus Ad-synd4; N = 3 experiments, ANOVA; (F) Western blot showed synd4 overexpression could increase Akt phosphorylation in S473 and T308. Calphostin C could blunt synd4-induced Akt phosphorylation. Data are shown as a ratio to total Akt and are expressed as a fold-increase when compared with Ad-Lacz groups. *P < 0.05, Ad-Lacz versus Ad-synd4, Ad-synd4 + calphostin C versus Ad-synd4; N = 3 experiments, ANOVA.