Reduced expression of Cx43 attenuates ventricular remodeling after myocardial infarction via impaired TGF-beta signaling

Abstract

In addition to mediating cell-to-cell electrical coupling, gap junctions are important in tissue repair, wound healing, and scar formation. The expression and distribution of connexin43 (Cx43), the major gap junction protein expressed in the heart, are altered substantially after myocardial infarction (MI); however, the effects of Cx43 remodeling on wound healing and the attendant ventricular dysfunction are incompletely understood. Cx43-deficient and wild-type mice were subjected to proximal ligation of the anterior descending coronary artery and followed for 6 days or 4 wk to test the hypothesis that reduced expression of Cx43 influences wound healing, fibrosis, and ventricular remodeling after MI. We quantified the progression of infarct healing by measuring neutrophil expression, collagen content, and myofibroblast expression. We found significantly reduced transformation of fibroblasts to myofibroblasts at 6 days and significantly reduced collagen deposition both in the infarct at 6 days and at 4 wk in the noninfarcted region of Cx43-deficient mice. As expected, transforming growth factor (TGF)-beta, a profibrotic cytokine, was dramatically upregulated in MI hearts, but its phosphorylated comediator (pSmad) was significantly downregulated in the nuclei of Cx43-deficient hearts post-MI, suggesting that downstream signaling of TGF-beta is diminished substantially in Cx43-deficient hearts. This diminution in profibrotic TGF-beta signaling resulted in the attenuation of adverse structural remodeling as assessed by echocardiography. These findings suggest that efforts to enhance the expression of Cx43 to maintain intercellular coupling or reduce susceptibility to arrhythmias should be met with caution until the role of Cx43 in infarct healing is fully understood.

Fig. 1.

A, top: representative trichrome-stained tissue sections from wild-type (WT; left) and connexin43 (Cx43)-deficient (Cx43^+/−; right) hearts demonstrate more unresorbed necrotic myocardium (dark purple) in the infarct region of the left ventricle in Cx43^+/− hearts 6 days postmyocardial infarction (post-MI). Bottom, histograms showing that the area of the unresorbed myocardium was significantly greater in the infarct regions from both the apex (*P = 0.005) and midway between the base and apex (mid; *P = 0.008) in Cx43-deficient hearts. Bars = 100 μm. B, top: representative images of neutrophil infiltration as assessed by counting myeloperoxidase-positive (MPO⁺) cells (pink) and total cellular nuclei (blue) within the infarct. Bottom, histograms showing that MPO⁺ cells were significantly more abundant in the infarct regions of Cx43-deficient hearts (*P = 0.005). Bars = 100 μm.

Fig. 2.

A, top: representative images of picrosirius red-stained collagen (pink) and fast green-stained noncollagenous protein (primarily myocytes, green) in the infarct regions of WT (left) and Cx43^+/− (right) hearts 6 days post-MI. Collagen deposition was significantly reduced in Cx43^+/− infarcts as indicated by the reduced amount of Sirius red-stained collagen (top right) and by the histograms (bottom; *P = 0.016). Bars = 100 μm. B, top: representative images of picrosirius red-stained collagen in the remote, noninfarcted regions of the left ventricular free wall 4 wk post-MI (left: WT; right: Cx43^+/−). Bottom, summarized data showing significantly reduced collagen content in the noninfarcted free wall (*P = 0.003) and noninfarcted septum (*P < 0.001) of Cx43-deficient hearts. Bars = 50 μm.

Fig. 3.

A, top: representative images of bromodeoxyuridine-positive cells (pink nuclei) and total Hoechst-stained nuclei (blue) in the infarct regions of WT (left) and Cx43^+/− (right) hearts 6 days post-MI. Bottom: histograms showing that cell proliferation was significantly greater in the infarcts of Cx43^+/− hearts measured at the apex (*P = 0.02). Cell proliferation tended to increase in more basal sections (mid) but did not reach statistical significance. Bars = 50 μm. B, top: representative fluorescence images of α-smooth muscle actin-immunoreactive signals indicative of myofibroblasts (green) in the infarct regions of WT (left) and Cx43^+/− (right) hearts 6 days post-MI. Total cellular nuclei are shown in blue. Myofibroblast expression was significantly reduced in Cx43^+/− hearts as shown in the fluorescence image (top right) and in the histograms (bottom; *P = 0.002). Bars = 100 μm.

Fig. 4.

Top: representative immunoblot of transforming growth factor (TGF)-β ligand (arrows at 12 and 24 kDa) showing increased TGF-β in the infarct regions (Inf; lanes 1 and 3) of both WT and Cx43^+/− ventricular homogenates compared with noninfarcted regions (NI; lanes 2 and 4) and compared with sham-operated mice (lanes 5 and 6) 6 days post-MI. The actin blot (40 kDa) was used as a loading control to normalize TGF-β protein expression. Bottom: summarized data indicating that the increase in TGF-β was nearly identical in WT and Cx43^+/− infarcts (see text). *P = 0.005–0.009.

Fig. 5.

A: representative fluorescence images showing reduced phosphorylated (p)Smad-immunoreactive signals in Cx43^+/− (right) compared with WT (left) hearts in both the infarct scar (top) and remote, noninfarcted area (bottom) 6 days post-MI. Bars = 50 μm. B: summarized data indicating that the pSmad signal was significantly smaller in both Cx43^+/− infarcted (left; *P = 0.018) and noninfarcted (right; *P = 0.022) areas 6 days post-MI compared with those observed in WT hearts.