Table 1.
Sequences of primers for regular PCR.
Table 2.
Sequences of primers for real-time PCR.
Figure 1.
Ankrd1/CARP upregulation in response to Angiotensin II stimulation.
(A) Representative images of cultured neonatal rat ventricular cardiomyocytes (NRVCs) stained with rhodamine phalloidin and 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI), and the cell size increased when treated with Angiotensin II (Ang II 1 µM) for 24h. (B) Hypertrophic marker atrial natriuretic peptide (ANP) detected by Real-time PCR using GAPDH as an internal control (*P<0.05 vs. the control group). Expression changes of Ankrd1 in response to Ang II in cultured NRVCs were detected by PCR (C) or real-time PCR (D). (E) Western blot analysis of time-dependent changes of CARP expression in NRVCs exposed to Ang II. *P<0.05 vs. Ang II-untreated group, #P<0.01 vs. Ang II 24 h group. Repeat times n = 3. Data are mean ± SEM.
Figure 2.
Subcellular translocation of CARP in response to treatment with Ang II or Ad-Ankrd1.
(A) Expression changes of calpain 1 in response to Ang II stimulation in cultured neonatal rat ventricular cardiomyocytes (NRVCs) detected by real-time PCR, the insert represents amplification curve of calpain 1 and β-actin, *P<0.05. (B) Confocal microscopic subcellular distribution of CARP (green) in response to Ang II stimulation in NRVCs. Immunostaining with F-actin (red) was used to confirm the cardiomyocytes, and DAPI (blue) was used to stain the nuclei. Bar = 10 µm. (C) Dose-dependent infective efficiency of Ad-Ankrd1 in cultured NRVCs detected by the green fluorescence of co-expressed EGFP. MOI: multiplicity of infection. (D) Western blot analysis of CARP protein levels in response to different dose of Ad-Ankrd1 infection (*P<0.01 vs. Ad-EGFP group, n = 3). (E) Representative EGFP fluorescence microscopic photos of LV myocardium at 1 week after intramyocardial injection of vehicle (non-transfection), Ad-EGFP or Ad-Ankrd1 delivery. (F) Western blot analysis of CARP protein levels in response to intramyocardial injection of Ad-EGFP or Ad-Ankrd1 at 1 week after sham operation. (G) Subcellular location of forced Ankrd1 expression in Ad-Ankrd1 transfected cardiomyocytes. The green fluorescence was emitted from the report gene EGFP which was constructed in adenovirus carrying Ankrd1. Bar = 10 µm.
Figure 3.
Effect of Ad-Ankrd1 transfection on myocyte hypertrophy in NRVCs.
Hypertrophic markers of ANP (A) and β-MHC (B) gene expressions in response to Ang II stimulation in the presence of Ad-Ankrd1 or Ad-EGFP (10 MOI) were detected by Real-time PCR (*P<0.01 vs. control, #P<0.01 vs. Ad-EGFP + Ang II, n = 5). (C) Representative pictures of cultured cardiomyocytes used for calculation of cross section area and quantitative results (*P<0.01 vs. control (1st bar), #P<0.01 vs. Ad-Ankrd1+ Ang II, n = 100 in each group). (D) PCR results of early growth response 1 (egr-1) in response to Ad-Ankrd1 treatment. (E) Western blot analysis of calcineurin in cardiomyocytes in response to different treatment (*P<0.01 vs. control (1st bar), #P<0.01 vs. Ad-EGFP + Ang II group). (F) Nuclear translocation of nuclear factor of activated T cells (NFAT) induced by Ad-Ankrd1 transfection in the absence of Ang II. Every experiment was repeated at least 3 times. Dose of Ang II was 1 µM. Scale bar = 10 µm for panel C and E. Data are mean ± SEM.
Figure 4.
Myocardial injection of Ad-Ankrd1 in mice promotes cardiac hypertrophy.
Cardiac overexpression of Ankrd1 was achieved by myocardial injection of Ad-Ankrd1 at 2 weeks after TAC, and the mice in each group were sacrificed at 4 weeks after the initial surgery. (A) Representative pictures of Western blot for CARP (upper panel), immunohistochemistry of myocardial CARP (2nd line, scale bar = 50 µm), whole hearts (3rd line, scale bar = 3 mm) and myocyte cross-sectional area stained with rhodamine-conjugated wheat germ agglutinin (low panel, scale bar = 30 µm) from each group. (B) Expression levels of CARP in each group. (C) HW/BW ratio at 4 weeks after surgery in different groups. (D) Myocyte surface area was calculated from 100 cells in each group. (E) ANP gene expression detected by Real-time PCR using β-actin as an internal control. (F) Western blot analysis of calcineurin expression in heart from each group. (G) Semi-quantitative analysis of calcineurin expression. For panel B-E, G, *P<0.01 vs. sham, #P<0.01 vs. TAC + Ad-EGFP, n = 4–9 in each group. Data are mean ± SEM.
Figure 5.
Myocardial injection of AAV-sh-Ankrd1 in mice inhibits cardiac hypertrophy.
(A) Infective efficiency of AAV-sh-Ankrd1 in cultured NRVCs for 96 h detected by the green fluorescence of co-expressed EGFP (zsGreen). (B) Western blot analysis of CARP protein levels in response to AAV-sh-Ankrd1 or AAV-scramble infection (*P<0.01 vs. scramble group, n = 5). (C) Representative fluorescence microscopic pictures of left ventricular myocardium 5 weeks after intramyocardial AAV-sh-Ankrd1 delivery. (D) Heart weigh/body weight ratio (HW/BW) in TAC or Sham mice treated with AAV-scramble or AAV-sh-Ankrd1. *P<0.01 vs. the corresponding Sham (TAC -) group, #P<0.01 vs. TAC + scramble group, n = 5 in each group. TAC or Sham was persisted for 1 week.
Figure 6.
Olmesartan downregulates CARP and ameliorates cardiomyocyte hypertrophy.
(A) Treatment with RNH6270 (RNH 1 µM, active form of olmesartan) reduced Ang II (1 µM) -induced increased of CARP protein in NRVCs (*P<0.05 vs. control, #P<0.05 vs. Ang II. n = 3). (B) ANP mRNA expression in NRVCs exposed to Ang II (1 µM) stimulation in the presence/absence of RNH (1 µM). (C) Representative picture of whole heart from different group (scale bar = 2 mm). (D) HW/BW ratio was significantly lower in TAC mice treated with olmesartan medoxomil (OM) in comparison with untreated TAC mice, while addition of Ad-Ankrd1 partially blocked the antihypertrophic effect of OM. (*P<0.01 vs. Sham, #P<0.01 vs. TAC, &P<0.05 vs. TAC + OM, n = 4–6 in each group). (E) Olmesartan treatment for 4 weeks reduced the myocardial expression of CARP in TAC mice (*P<0.05 vs. Sham, #P<0.05 vs. TAC). Data are mean ± SEM.