Figure 1.
Overexpression of CARP inhibits phenylephrine-induced cardiomyocyte hypertrophy.
(A) Primary neonatal rat cardiomyocytes were infected with adenoviruses expressing GFP or Myc-tagged CARP-GFP at the indicated MOIs. After 24 hours, CARP and Myc-tagged CARP levels were evaluated using Western blotting. (B) α-actin staining showed that adenoviral-mediated overexpression of CARP inhibited phenylephrine-induced cardiomyocyte hypertrophy. Cells were infected with Ad-GFP as a negative control. Nuclei were stained with DAPI. (C) Quantification of the cell surface areas shown in (B). 100–120 random cells were measured in each group. *P<0.001 relative to phenylephrine+Ad-GFP (PE+Ad-GFP), #P<0.01, compared with con+vehicle; (D) The effect of CARP overexpression on the levels of mRNA of α-actin, β-MHC, and ANF in cardiomyocytes exposed to phenylephrine or not. mRNA levels were measured via semi-quantitative RT-PCR; GAPDH was employed as an internal control; (E) Quantification of mRNA levels in (D). ***P<0.001, compared with Ad-GFP, ###P<0.001, compared with PE+Ad-GFP.
Figure 2.
Pressure overload-induced cardiac hypertrophy is attenuated in CARP Tg mice.
WT and CARP Tg mice were subjected to TAC or a sham operation. Cardiac hypertrophy and function were assessed by echocardiography. After 4 weeks, the mice were sacrificed for assessment of cardiac hypertrophy. (A) Representative examples of M-mode echocardiograms of hearts from WT and CARP Tg mice subjected to TAC or a sham operation. (B) The ratio of heart weight to body weight (HW/BW). (C) The ratio of heart weight to tibia length (HW/TL). (D) Staining of heart sections from WT and CARP Tg mice subjected to TAC or a sham operation. Upper panel: Gross heart morphology; middle panel: H&E-stained longitudinal sections; lower panel: PSR-stained sections. (E) H&E staining of sections from the left ventricular myocardium of WT and CARP Tg mice subjected to TAC or a sham operation. Scale bar = 20 µm. (F) Quantification of cross-sectional areas of the cardiomyocytes shown in (E). (G) Quantification of collagen areas in the myocytes shown in (D).
Figure 3.
Isoproterenol-induced cardiac hypertrophy is attenuated in CARP Tg mice.
WT and CARP Tg mice were continuously infused with vehicle (100 µmol/L ascorbic acid) or isoproterenol at a rate of 30 mg/kg/day using a subcutaneously implanted mini-osmotic pump. After 14 days, mice were sacrificed for assessment of cardiac hypertrophy. (A) Representative examples of M-mode echocardiograms of hearts from WT and CARP Tg mice infused with isoproterenol (ISO) or vehicle (sham). (B) The ratio of heart weight to body weight (HW/BW). (C) The ratio of heart weight to tibia length (HW/TL). (D) Staining of hearts from WT and CARP Tg mice infused with ISO or vehicle. Upper panel: Gross heart morphology; middle panel: H&E-stained longitudinal sections; lower panel: PSR-stained sections. (E) H&E staining of sections from the left ventricular myocardia of WT and CARP Tg mice infused with ISO or vehicle. Scale bar = 20 µm. (F) Quantification of cross-sectional areas in the cardiomyocytes shown in (E). (G) Quantification of collagen areas in the myocytes shown in (D). (H) Changes in the expression levels of mRNAs transcribed from the ANF, β-MHC, and α-actin genes after infusion of isoproterenol in WT and CARP Tg mice. mRNA expression levels were quantitated using real-time PCR.
Figure 4.
Overexpression of CARP inhibits the activation of MEK/ERK signaling by hypertrophic stimuli.
(A) Western blotting of heart protein extracts to examine the levels of various phosphorylated and total kinases in WT and CARP Tg mice following TAC or a sham operation. A representative Western blot (from one of three independent experiments) is shown. (B) Western blotting of protein extracts to detect various phosphorylated and total kinase levels in parental and CARP-overexpressing cardiomyocytes subjected to phenylephrine treatment or not. A representative Western blot (from one of three independent experiments) is shown. (C) Quantification of the expression of the p-MEK1/2 and p-ERK1/2 proteins shown in (B) at 30 min after commencement of PE treatment.
Figure 5.
Upregulated TGF-β/Smad signaling is inhibited in CARP Tg mice following TAC.
(A) Quantitative detection of TGF-β1, -β2, and -β3 mRNA expression by real-time PCR in hearts from WT and CARP Tg mice subjected to TAC or a sham operation. (B) ELISA measurements of TGF-β1 levels in heart homogenates from WT and CARP Tg mice subjected to TAC or a sham operation. (C) Western blotting to detect phosphorylated and total Smad3 protein in heart protein extracts from WT and CARP Tg mice subjected to TAC or a sham operation. (D) Quantification of the level of phosphorylated Smad 3 shown in (C).
Figure 6.
Addition of exogenous human TGF-β1 rescues the attenuation in cardiomyocyte hypertrophy evident upon CARP overexpression.
(A) Overexpression of CARP inhibited phenylephrine-induced TGF-β1 secretion by cardiomyocytes. Cultured neonatal rat cardiomyocytes were infected with adenoviruses containing CARP/GFP or GFP alone and next exposed to 10 µM phenylephrine for 24 hours. The levels of TGF-β1 in culture supernatants were next determined using a double-antibody sandwich ELISA method and normalized to the concentrations in the corresponding cardiomyocytes. TGF-β1 secretion from cardiomyocytes infected with adenovirus/GFP alone was taken to be unity in each experiment. n = 4. *P<0.05, compared with the GFP control at an MOI of 50; #P<0.05, compared with the GFP control at an MOI of 50/PE. (B) Supplementation with exogenous TGF-β1 reversed the inhibitory effect of CARP on hypertrophic markers up-regulation in cardiomyocytes in response to phenylephrine. Cultured cardiomyocytes were infected with adenoviruses containing GFP alone or CARP/GFP/c-myc and next treated with phenylephrine, human TGF-β1, or phenylephrine plus human TGF-β1, for 24 hours. TRIzol was added and total RNA was extracted for determination of the levels of mRNA encoding ANF and β-MHC using real-time PCR. n = 3. **P<0.01, ***P<0.001, compared with GFP alone; #P<0.05, ###P<0.001. (C) Western blotting to explore expression of CARP and c-myc in the cardiomyocytes of (B) above. Data from one of four independent experiments are shown. (D) The inhibitory effect of CARP overexpression on increase in cardiomyocyte size induced by phenylephrine was reversed by addition of hTFG-β1. Cultured cardiomyocytes infected with GFP or GFP/CARP adenoviruses were treated the same way as described in (B) and fixed in 4% (v/v) formaldehyde. Then F-actin was stained with Rhodamine phalloidin and myocyte size was assessed using a microscope equipped with a 200× objective and appropriate epifluorescence filters. 100 random cells were measured in each group and four independent experiments were performed. hTGF-β1, human TGF-β1; MOI, multiplicity of infection; PE, phenylephrine.