Fig 1.
Lentiviral overproduction of APN inhibited Ang II-induced cardiac hypertrophy.
For lentiviral vector-mediated gene transfer, wild-type Wistar rats were treated with a lentiviral vector expressing APN (LV-APN) or with a control virus (LV-Ctr) delivered through the jugular vein 3 d before infusion with Ang II. Ang II was infused (200 ng/kg/min) for 28 days into those rats. (A) Cross-sectional area of the cardiomyocytes in rats was assessed by hematoxylin and eosin (H&E) staining. Bar represents 20 μm. (B) Quantification of cell size and (C) heart weight / body weight ratio. Total RNA was isolated from the left ventricles of rats, and subjected to real-time RT-PCR for ANF (D) and BNP (E). Amplification curves were normalized to β-actin. All samples were analyzed in triplicate and expressed as the mean ± SD. (**, p< 0.01, n = 6 for each group).
Fig 2.
Echcardiography showed that lentiviral vector-mediated APN overexpression improve Ang II stimulated hypertrophy.
(A) M-mode tracings. Representative photographs were shown. The left ventricular end-diastolic posterior wall thickness (LVPWd) and end-diastolic interventricular septal thickness (IVSd) were shown in (B) and (C). (D), ejection fraction of left ventricle (LVEF). Data represent mean ± SD. (**, p < 0.01. n = 6).
Fig 3.
APN attenuated cardiac hypertrophy in vitro and reversed miR-133a downregulation by Ang II in vivo and in vitro.
(A) Lentiviral—mediated APN overexpression and supplement of recombinant APN reversed miR-133a downregulation by Ang II in vivo. (B) NRVMs underwent 100 nM Ang II stimulation in the presence or absence of APN (2 or 5 μg/ml) with 1.0 mCi/ml [3H]-leucine for 24 h. APN was applied to NRVMs 60 min before Ang II stimulation. To remove the possibility that the increased protein synthesis is hyperplasia, the data were normalized to cell numbers. (C) Total RNA was isolated from the NRVMs and subjected to real-time RT-PCR for ANF and (D) BNP, and (E) miR-133a. Amplification curves were normalized to β-actin or U6 snRNA (**, p < 0.01. *, p < 0.05, n = 6).
Fig 4.
APN upregulated miR-133a through AMPK pathway in the Ang II mediated hypertrophic responses.
(A) Phosphorylated (p-) AMPK and AMPK level were measured by Western blot in NRVMs. (B) miR-133a level was detected by real-time PCR. NRVMs were transduced with lentiviral AMPK shRNA or Scramble controls and then treated with the indicated regents. Amplification curves were normalized to U6 snRNA (**, p< 0.01. ##, p < 0.01 vs control group. n = 3).
Fig 5.
APN upregulated miR-133a levels by inhibiting ERK1/2 phosphorylation.
(A) p-ERK and ERK expression level were determined by Western blot. (B) miR-133a level was detected by real-time RT-PCR. The miR-133a expression level was normalized to U6 expression following the ΔΔCT method. (**, p< 0.01, n = 3).
Fig 6.
AdipoR1 was responsible in mediating APN signals.
(A) AdipoR1 was inhibited by Ang II stimulation as determined by qPCR. Western blot was performed to determine phosphorylation of AMPK (B) and (C) ERK. (D) miR-133a level was determined by qRT-PCR (**, p < 0.01).
Fig 7.
APN suppressed increased CTGF expression caused by Ang II.
(A) CTGF levels were measured in the rat left ventricle by immunohistochemistry using CTGF antibodies. (B) CTGF expression in NRVMs were determined by Western blot (**, p< 0.01. n = 3).