PKD knockdown inhibits pressure overload-induced cardiac hypertrophy by promoting autophagy via AKT/mTOR pathway

Abstract

Growing evidence shows that protein kinase D (PKD) plays an important role in the development of pressure overload-induced cardiac hypertrophy. However, the mechanisms involved are not clear. This study tested our hypothesis that PKD might mediate cardiac hypertrophy by negatively regulating autophagy using the technique of PKD knockdown by siRNA. Cardiac hypertrophy was induced in 8-week old male C57BL/6 mice by transverse aortic constriction (TAC). TAC mice were then divided into five groups receiving the treatments of vehicle (DMSO), an autophagy inducer rapamycin (1 mg/kg/day, i.p.), control siRNA, lentiviral PKD siRNA (2×10⁸ transducing units/0.1 ml, i.v. injection in one day after surgery, and repeated in 2 weeks after surgery), and PKD siRNA plus 3-methyladenine (3-MA, an autophagy inhibitor, 20 mg/kg/day, i.p.), respectively. Four weeks after TAC surgery, echocardiographic study, hematoxylin and eosin (HE) staining, and Masson's staining showed mice with TAC had significantly hypertrophy and remodeling compared with sham animals. Treatments with PKD siRNA or rapamycin significantly ameliorated the cardiac hypertrophy and dysfunction. Moreover, PKD siRNA increased cardiac autophagic activity determined by electron micrographic study and the biomarkers by Western blot, accompanied with the downregulated AKT/mTOR/S6K signaling pathway. All the cardiac effects of PDK knockdown were inhibited by co-treatment with 3-MA. These results suggest that PKD is involved in the development of cardiac hypertrophy by inhibiting cardiac autophagy via AKT/mTOR pathway.

Keywords: AKT/mTOR pathway; PKD knockdown; autophagy; cardiac hypertrophy; pressure overload.

Fig 1

TAC-induced cardiac hypertrophy with various treatments. (A) Timeline of study protocol. (B) Representative gross heart pictures. (C) Heart weight/body weight (HW/BW) and (D) left ventricle weight/body weight (LVW/BW) ratios. (E) Representative M-mode echocardiographic images after 4 weeks of TAC. TAC, transverse aorta constriction; V, vehicle; NC, negative control; Rap, rapamycin. Data are mean±SEM, n=8-9/group. * P<0.05 vs. sham; # P<0.05 vs. vehicle or NC siRNA treated TAC mice; … P<0.05 vs. TAC-PKD siRNA.

Fig 2

H&E staining of left ventricles and real-time PCR. (A) Representative images of H&E staining in left ventricle transverse sections. (B) Quantification results of myocyte cross-sectional areas (CSA). (C) ANF mRNA level in the heart. (D) BNP mRNA level in the heart. TAC, transverse aorta constriction; V, vehicle; NC, negative control; Rap, rapamycin. Data are mean±SEM, n=8-9/group. * P<0.05 vs. sham; # P<0.05 vs. vehicle or NC siRNA treated TAC mice; … P<0.05 vs. TAC-PKD siRNA.

Fig 3

Fibrosis in left ventricles. (A) Representative images of Masson's Trichrome staining in left ventricles, the blue stands for fibrosis. (B) Quantification results of the cardiac fibrosis area. TAC, transverse aorta constriction; V, vehicle; NC, negative control; Rap, rapamycin. Data are mean±SEM, n=8-9/group. * P<0.05 vs. sham; # P<0.05 vs. vehicle or NC siRNA treated TAC mice; … P<0.05 vs. TAC-PKD siRNA.

Fig 4

Biomarkers of cardiac autophagy. (A) Representative images of Western blot for LC3, Beclin 1, and p62. (B) Quantification of the signal densities from Western blot. TAC, transverse aorta constriction; V, vehicle; NC, negative control; Rap, rapamycin. Data are mean±SEM, n=8-9/group. * P<0.05 vs. sham; # P<0.05 vs. vehicle or NC siRNA treated TAC mice; … P<0.05 vs. TAC-PKD siRNA.

Fig 5

Autophagic vacuoles in cardiomyocytes. (A) Representative electron micrographs showing autophagic vacuoles in cardiomyocytes of TAC mice with various treatments (magnification: x10,000). Arrows indicate autophagic vacuoles. (B) Quantification of the number of autophagosomes/cell in different groups (a random number of 30 cells were selected for each group). TAC, transverse aorta constriction; V, vehicle; NC, negative control; Rap, rapamycin. Data are mean±SEM, n=8-9/group. * P<0.05 vs. sham; # P<0.05 vs. vehicle or NC siRNA treated TAC mice; … P<0.05 vs. TAC-PKD siRNA.

Fig 6

Signaling pathways regulating cardiac autophagy. (A) Representative images of Western blot for phosphorylated and total Akt, mTOR, S6K, AMPK, and PKD in the theart. (B) Quantification of the signal densities from Western blot. TAC, transverse aorta constriction; V, vehicle; NC, negative control; Rap, rapamycin. Data are mean±SEM, n=8-9/group. * P<0.05 vs. sham; # P<0.05 vs. vehicle or NC siRNA treated TAC mice; … P<0.05 vs. TAC-PKD siRNA.