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. 2005 Jan 11;102(2):437-42.
doi: 10.1073/pnas.0408704102. Epub 2004 Dec 29.

Inhibition of cardiac myofibroblast formation and collagen synthesis by activation and overexpression of adenylyl cyclase

James S Swaney  1 , David M Roth, Erik R Olson, Jennifer E Naugle, J Gary Meszaros, Paul A Insel
Affiliations

Inhibition of cardiac myofibroblast formation and collagen synthesis by activation and overexpression of adenylyl cyclase

James S Swaney et al. Proc Natl Acad Sci U S A. .

Abstract

Transformation of fibroblasts to myofibroblasts, characterized by expression of alpha-smooth muscle actin (alpha-SMA) and production of extracellular matrix (ECM) components, is a key event in connective tissue remodeling. Approaches to inhibit this transformation are needed in tissues, such as the heart, where excessive ECM production by cardiac fibroblasts (CFs) causes fibrosis, myocardial stiffening, and cardiac dysfunction. We tested whether adenylyl cyclase (AC) activation (increased cAMP levels) modulates the transformation of adult rat CF to myofibroblasts, as assessed by immunofluorescent microscopy, immunoblotting, and collagen synthesis. A 24-h incubation of CF with TGF-beta or angiotensin II increased alpha-SMA expression, which was inhibited by the AC agonist forskolin and a cAMP analog that activates protein kinase A. Treatment with forskolin blunted serum-, TGF-beta-, and angiotensin II-stimulated collagen synthesis. CFs engineered to overexpress type 6 AC had enhanced forskolin-promoted cAMP formation, greater inhibition by forskolin of TGF-beta-stimulated alpha-SMA expression, and a decrease in the EC(50) of forskolin to reduce serum-stimulated collagen synthesis. The AC stimulatory agonist adrenomedullin inhibited collagen synthesis in CF that overexpressed AC6 but not in controls. Thus, AC stimulation blunts collagen synthesis and, in parallel, the transformation of adult rat CF to myofibroblasts. AC overexpression enhances these effects, "uncovering" an inhibition by adrenomedullin. These findings implicate cAMP as an inhibitor of ECM formation by means of blockade of the transformation of CF to myofibroblasts and suggest that increasing AC expression, thereby enhancing cAMP generation through stimulation of receptors expressed on CF, could provide a means to attenuate and prevent cardiac fibrosis and its sequelae.

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Figures

Fig. 1.
Fig. 1.
Forskolin and CPT-cAMP blunt collagen synthesis of adult rat cardiac fibroblasts. Shown are collagenase-sensitive [3H]proline incorporation by fibroblasts grown for 48 h in serum-free media and then stimulated for 48 h with serum-free media (control) or 2.5% in the absence or presence of forskolin (Fsk, 10 μM), CPT-cAMP (100 μM), CPT-cAMP (100 μM), or adrenomedullin (AM, 1 μM) (A) or serum-free media alone or with Ang II (100 nM) or TGF-β (10 ng/ml) in the presence or absence of Fsk (10 μM) (B). (C) Cardiac fibroblasts were cultured and serum-starved as noted above and subsequently treated with the indicated agents for 48 h before protein isolation (see Materials and Methods for lysis buffer composition). Densitometry and quantitation for C was performed on the pro-α1 (I) band (top band). Values represent mean ± SEM of at least four experiments performed in triplicate and compared by using a paired t test. #, P < 0.05 in response to forskolin or CPT-cAMP.
Fig. 2.
Fig. 2.
α-SMA expression in CF of different passage number and in response to TGF-β and Ang II. (A) α-SMA expression was measured by immunoblot analysis using adult rat CF at passages 2–5. CFs at passage 1 were split from confluent monolayers at a ratio of 1:3 and maintained in the presence of 10% FBS for 48 h before collection of whole cell lysates. After immunoblotting for α-SMA (Upper), the blot was stripped and reprobed with GAPDH as a loading control (Lower). (B) α-SMA expression was measured by using CF maintained in the presence of 10% FBS, with and without isobutylmethylxanthine (IBMX; 200 μM). After immunoblotting for α-SMA, the blot was stripped and reprobed with vimentin as a loading control, and quantitation of α-SMA immunoreactive bands was performed. (C) Immunohistochemistry was performed by using CF (passage 2) grown for 48 h in serum-free media and then for 24 h in serum free media alone (control), Ang II (100 nM), or TGF-β (10 ng/ml) in the presence or absence of Fsk (10 μM). Cells were stained for α-SMA (green) and nuclear staining of DNA with DAPI (blue). (D) α-SMA protein expression with these same treatments was verified by immunoblot. Data are expressed as fold change relative to control. Values represent mean ± SEM of at least three experiments and compared by using a paired t test and ANOVA with post hoc multiple comparison tests. #, P < 0.05 as compared to control; *, P < 0.05 in response to forskolin.
Fig. 3.
Fig. 3.
Overexpression of AC6 enhances forskolin- or adrenomedullin-stimulated cAMP production. cAMP production was measured by RIA using CF grown for 48 h in serum-free media and then stimulated for 10 min with 2.5% FBS alone (A Inset) or in the presence of the indicated concentrations of forskolin (A) or adrenomedullin (B). CF were incubated with an adenovirus expressing either LacZ (control) or AC6 for 24 h before stimulation. Values represent mean ± SEM of at least three experiments and were compared by using a paired t test and ANOVA. *, P < 0.05 compared to 2.5% FBS; #, P < 0.05 compared to LacZ cells.
Fig. 4.
Fig. 4.
Overexpression of AC6 enhances forskolin- and adrenomedullin-promoted inhibition of collagen synthesis. Collagenase-sensitive [3H]proline incorporation was measured by using adult rat CF grown for 48 h in serum-free media and then stimulated for 48 h with 2.5% FBS in the absence (A Inset)or presence of the indicated concentrations of forskolin (A) or adrenomedullin (B). CF were incubated with an adenovirus expressing either LacZ (control) or AC6 for 24 h before stimulation. Data are normalized for [3H]proline incorporation into cells grown under serum-free conditions. Values represent mean ± SEM of at least three experiments and were compared by using a paired t test and one-way ANOVA. *, P < 0.05 compared to 2.5% FBS; #, P < 0.05 compared to LacZ cells.
Fig. 5.
Fig. 5.
AC6 overexpression enhances forskolin- and adrenomedullin-mediated inhibition of TGF-β-stimulated α-SMA expression. Immunohistochemistry was performed by using adult rat CF that overexpressed either LacZ (A) or AC6 (B). CF (passage 2) were grown for 48 h in serum-free media and then for 24 h in serum-free media alone (control) or with TGF-β (10 ng/ml) in the presence or absence of forskolin (Fsk, 1 μM) or adrenomedullin (AM, 0.1 μM). CF were incubated with an adenovirus expressing either LacZ (control) or AC6 for 24 h before stimulation. For immunohistochemical studies, CF were stained for α-SMA (green), F-actin (phalloidin, red), vimentin (red), and DNA (DAPI, blue). Overlap between α-SMA and F-actin staining is also represented (yellow). (C) α-SMA and vimentin protein expression in these same treatments were verified by immunoblot analysis. Quantitation of immunoreactive bands was performed; α-SMA expression was normalized to that of vimentin. Values represent mean ± SEM of at least four experiments and compared by using a paired t test. *, P < 0.05 compared to TGF-β;#, P < 0.05 compared to LacZ cells.
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