Myofibroblasts cause heterogeneous Cx43 reduction and are unlikely to be coupled to myocytes in the healing canine infarct

Abstract

Following myocardial infarction (MI) inflammatory responses transform cardiac fibroblasts to myofibroblasts, which in vitro studies show form heterocellular gap junctions with cardiac myocytes via Connexin43 (Cx43). The ability to form heterocellular junctions in the intact heart and the impact of these junctions on propagation is unclear. We used a canine model of MI and characterized the distribution and quantity of myofibroblasts in surviving epicardial cells [epicardial border zone (EBZ)]. We found a significant increase in myofibroblasts within the EBZ and no gap junction plaques between myofibroblasts and myocytes. Because myofibroblasts produce IL-1β, which downregulates Cx43, we asked whether myofibroblast proliferation causes loss of Cx43 near myofibroblast clusters. In vitro studies showed that IL-1β caused loss of Cx43 and reduced coupling. Western blot showed a significant increase of IL-1β in the EBZ, and immunohistochemistry showed a loss of Cx43 in regions of myofibroblasts in the intact heart. Additionally, dye studies in intact heart showed no coupling between myocytes and myofibroblasts. To quantify the effect of myofibroblasts on propagation we used a two-dimensional subcellular computer model of the EBZ, which showed that heterogeneities in myofibroblast density lead to conduction abnormalities. In conclusion, an increase of myofibroblasts in the infarcted heart causes heterogeneous Cx43 levels, possibly as a result of the release of IL-1β and decreased cell-cell communication, which leads to conduction abnormalities following MI.

Fig. 1.

Electron microscopy of myofibroblasts in infarcted hearts. Normal myocardium (A) has almost no identifiable myofibroblasts, but following 5 days of coronary occlusion (B) myofibroblasts are readily identifiable by their large nuclei (N) as well as by their extensive endoplasmic reticulum (inset C) and the presence of stress fibers (inset D). Quantification of myofibroblasts (E) in different regions of the re-entrant circuits [common pathway (CCP) and outer pathway (OP)] showed that both regions had significant increases in the levels of myofibroblasts when compared with normal (0.64% in normal, 1.88% in CCP, and 2.27% in OP), but there was no difference in myofibroblasts levels in the CCP when compared with the OP, indicating that the electrical pathway does not affect myofibroblasts localization (n = 5).

Fig. 2.

Examination of cell-to-cell contacts between myofibroblasts and myocytes show that although gap junctions are identifiable between myofibroblasts (inset, *), there were no myocyte-to-myofibroblasts gap junctions (insets 1 and 2) in any section examined (1,050 μm of contact region membrane examined).

Fig. 3.

Electron micrographs of 5-day infarcted epicardial border zone (EBZ) with myocytes (*) and myofibroblasts (#) (A–F). Several examples of myocytes next of myofibroblasts showed no gap junctional plaques between these 2 cell types, even at the longitudinal ends of the myocytes (for example, see C).

Fig. 4.

A: Mardin Darby Canine Kidney (MDCK) cells contain Connexin43 (Cx43), which is found at sites of cell-cell contact (B). Treatment of 0.1 μM of IL-1β caused an internalization of Cx43, which was blocked by the IL-1β receptor antagonist IL-1ra (C). Dye spread studies showed that although control MDCK cells are highly coupled (D), IL-1β treatment caused a complete loss cell coupling (E). Inhibition of the IL-1β receptor with IL-1ra inhibited both the changes in Cx43 localization (C) and function (F). Treatment of rat neonatal myocytes (G) with 0.1 μM IL-1β (H) caused the same internalization of Cx43 as was seen in the MDCK cells that was also reversed by addition of the IL-1ra (I), suggesting that this effect of IL-1β is not cell type specific. J: quantification of the dye-spread studies (n = 3). Quantification of the level of Cx43 in cultured rat neonatal myocytes is seen in K. Values are reported as a percentage of normal.

Fig. 5.

Western blot analysis shows an increase in IL-1β in the EBZ of 5-day (5d) infarcted heart (A). Immunohistochemical studies indicate that normal heart has very few myofibroblasts (B) but has abundant Cx43, which is homogeneously expressed in the epicardium at the myocyte intercalated disks (C). Overlay images can be seen in D. In contrast, epicardial tissue from the 5-day infarct shows large myofibroblast clusters (E). Cx43 is found in a heterogeneous distribution in the same tissue section (F) with positive staining only seen in regions devoid of α-smooth muscle actin (SMA) staining. The overlay image shows that regions of myocytes intermixed with myofibroblasts have very low levels of Cx43, whereas regions in which myofibroblasts are missing still contain Cx43 (G). Higher magnification images of the border region of α-SMA staining (H) clearly shows that the myocytes have lateralized Cx43 (I). Overlay image shows the intercalation of a myocyte with lateralized Cx43 at the border of the α-SMA positive region (J). AU, arbitrary units.

Fig. 6.

Dye spread assay in intact canine infarcted heart. A: Lucifer Yellow (green) in myocytes of the EBZ of a 48-h infarct did not pass into myofibroblasts (red), indicating that these 2 cells types are not coupled in the intact heart after 48 h of infarction. B: myofibroblasts (red) were also not coupled directly to myocytes (green) in EBZ of a 5-day infarct.

Fig. 7.

Simulation of the paracrine effect of myofibroblasts (Myofib) on myocytes in the computer model. A: region of the simulated tissue in the absence of myofibroblasts. Red lines identify the boundaries of myocytes. Gap junctions are shown in blue. B: effect of myofibroblasts with an average density of ∼25% on the same tissue as in A. Note that if myofibroblast clusters (red squares) are located on top of nodes, which contain gap junctions (see white arrows for example), gap junctions are eliminated to simulate the experimental finding that in the presence of myofibroblasts Cx43 is reduced. C: reduction in gap junctions for different myofibroblast densities.

Fig. 8.

Effect of myofibroblast proliferation on conduction velocity when myofibroblasts are located on top of myocytes and exert a purely paracrine effect on myocytes by reducing gap junctions and cell-to-cell conductance. Activation maps during longitudinal propagation (from left to right) in the absence of myofibroblasts (A), for a density of 25% (B) and for a density of 50% (C) for a basic cycle length of 250 ms are shown. D: longitudinal conduction velocity for different densities of myofibroblasts. E: space constant in the longitudinal direction for different densities of myofibroblasts.

Fig. 9.

Effect of heterogeneities in myofibroblast (Myofib) density on propagation when myofibroblasts are located on top of myocytes and exert a purely paracrine effect on myocytes by reducing gap junctions and cell-to-cell conductance. A: propagation of a premature impulse (S1S2 = 196 ms) in a preparation with a myofibroblast density of ∼50%. The propagating impulse does not block. B: block of a premature impulse (S1S2 = 196 ms) at the boundary between an area with myofibroblasts (density 50%) and an area without myofibroblasts.

Fig. 10.

Effect of myofibroblast proliferation on conduction velocity when myofibroblasts displace myocytes. Activation maps during longitudinal propagation (from left to right) in the absence of myofibroblasts (A), for a density of 15% (B) and for a density of 25% (C) for a basic cycle length of 250 ms. D: longitudinal conduction velocity for different densities of myofibroblasts when myofibroblasts displace myocytes (solid line) and when myocytes are located on top of myocytes (dashed line). E: space constant in the longitudinal direction for different densities of myofibroblasts when myofibroblasts displace myocytes (solid line) and when myocytes are located on top of myocytes (dashed line).

Fig. 11.

Effect of heterogeneities in myofibroblast (Myofib) density on propagation when myofibroblasts displace myocytes. A: propagation of a premature impulse (S1S2 = 198 ms) in a preparation with a myofibroblast density of ∼15%. The propagating impulse does not block. B: block of a premature impulse (S1S2 = 198 ms) at the boundary between an area with myofibroblasts (density 15%) and an area without myofibroblasts.