Fibroblast growth factor-2 regulates myocardial infarct repair: effects on cell proliferation, scar contraction, and ventricular function

Abstract

Fibroblast growth factor-2 (FGF2, bFGF) has been proposed to regulate wound healing and angiogenesis, but skin wound healing in FGF2-knockout (FGF2-KO) animals is only slightly delayed. To determine the role of FGF2 in myocardial infarct repair, we studied the evolution of left ventricular geometry, cell proliferation, matrix content, and cardiac function in mice lacking or overexpressing (FGF2-Tg) FGF2. Despite having no effect on initial infarct size, deletion of FGF2 resulted in reduced fibroblast proliferation and interstitial collagen deposition, decreased endothelial proliferation and vascular density, and decreased cardiomyocyte hypertrophy. Furthermore, FGF2-KO mice demonstrated a complete absence of scar contraction, resulting in increased final infarct size and marked increases in chamber size and infarct expansion. These deficits ultimately impaired left ventricular dP/dt compared with wild-type infarcted mice. Conversely, overexpression of FGF2 increased fibroblast proliferation and collagen deposition, accelerated endothelial proliferation, and enhanced cardiomyocyte hypertrophy after infarction. These changes curbed infarct expansion and preserved left ventricular function. Thus, FGF2 is an important regulator of cell proliferation, angiogenesis, collagen synthesis, myocyte hypertrophy, scar contraction, and, ultimately, left ventricular contractile function during infarct repair. FGF2 may be more important in healing of infarcts compared with skin wounds because of the mechanical stress under which infarcts heal.

Figure 1

Histology of infarct repair in FGF2-KO and FGF2-Tg mice. Noninfarcted hearts of all groups studied (A, D, G, and J) exhibited uniform thickness of the left (LV) and right (RV) ventricular walls, comparable chamber area, and normal myocyte morphology. Four days after MI (B, E, H, and K), hearts of all groups had residual necrosis (N) surrounded by granulation tissue. In all groups studied at 4 weeks after MI (C, F, I, and L), there was thinning of the ventricular wall, chamber dilation, and collagenous scar (S) formed to replace the dead myocardium. The greatest chamber dilatation and ventricular wall thinning occurred in the FGF2-KO at 4 weeks after MI. The least extensive wall thinning occurred in the FGF2-Tg hearts. Scale bar = 1 mm.

Figure 2

Postinfarct changes in LV geometry and cardiomyocyte hypertrophy. Group sizes ranged from 7 to 14 mice per time point, as listed in Tables 1 and 2. A: Four weeks after MI, the expansion index [(septal thickness/scar thickness) × (chamber area/LV area)] of FGF2-KO mice was twice that of WT mice (P = 0.03). B: Conversely, the expansion index in the FGF2-Tg group was half that of FGF2-nTg mouse hearts (P = 0.0006). C: Myocytes of WT mice exhibited a significant hypertrophic response as evidenced by an increase in myocyte cross-sectional area (XS-area, P = 0.02) whereas FGF2-KO mice showed no significant hypertrophy (P = 0.67). D: Although myocyte cross-sectional area was smaller in noninfarcted FGF2-Tg hearts than in FGF2-nTg hearts (P = 0.001), the hypertrophic response in the FGF2-Tg group (P < 0.001) was more than twice that observed in FGF2-nTg mouse hearts (P = 0.001). Each column represents ∼100 myocytes per heart (three to eight hearts per group). E: Infarcts in WT C57BL/6 mice underwent the expected wound contraction to ∼50% their initial size by 4 weeks. In contrast, infarcts from FGF2-KO mice failed to undergo wound contraction, such that they were twice as large as observed WT mice at 4 weeks. *P < 0.05 FGF2-KO versus C57 WT.

Figure 3

Myofibroblast proliferation and myocardial fibrosis. Proliferating fibroblasts were detected by double labeling with smooth muscle α-actin and BrdU antibodies (see Supplemental Figure 2 at http://ajp.amjpathol.org for histological image). Group sizes ranged from 7 to 14 mice per time point (listed in Tables 1 and 2). A: Fibroblast proliferation peaked at 4 days after MI in both FGF2-KO and WT mouse hearts; however, there was a 30% reduction in the proliferative response in FGF2-KO hearts (*P < 0.05). B: Fibroblast proliferation in FGF2-Tg mice was twice that of FGF2-nTg mice at 2 days, 4 days, and 1 week after MI (*P < 0.05). C: Collagen deposition was measured by picrosirius red staining in the noninfarcted posteroseptal region. Collagen content in FGF2-KO mice did not significantly increase after MI, whereas infarcted WT C57 hearts showed significantly increased interstitial fibrosis (*P < 0.05 versus noninfarcted). D: Collagen deposition in the FGF2-Tg group after MI was double that of the FGF2-nTg mouse hearts (P < 0.05 versus P = ns, respectively). See Supplemental Figure 3 at http://ajp.amjpathol.org for histological images.

Figure 4

Endothelial cell proliferation and vascular morphometry. Group sizes ranged from 7 to 14 mice per time point, as listed in Tables 1 and 2. A: Proliferating endothelial cells were detected by double labeling with CD31 and BrdU antibodies. See Supplemental Figure 2 (available at http://ajp. amjpathol.org) for a representative photomicrograph. Endothelial cell proliferation was significantly blunted in FGF2-KO mice, as determined by a cumulative 4-day BrdU infusion (*P < 0.05). B: Endothelial proliferation in FGF2-Tg mice (determined by pulse labeling 1 hour before sacrifice) peaked earlier than in FGF2-nTg mice (*P < 0.05). C: In WT hearts, the vascular density declined at 1 week and further declined at 4 weeks after MI (^†P < 0.001 versus noninfarcted control; ^‡P < 0.001 versus previous time point). Although the initial vascular density was comparable in noninfarcted FGF2-KO hearts, there was a more pronounced decline in density at 1 and 4 weeks after MI (*P < 0.001 versus WT C57 at same time point). D: There were no differences in vascular density between FGF2-Tg and FGF2-nTg hearts. E and F: The area occupied by vascular lumen (expressed as a percentage of the total area) did not vary among the time points or between the groups in FGF2-KO or FGF2-Tg studies. G: The average area/vessel was significantly increased at 1 (*P < 0.05) and 4 weeks (**P < 0.001) after MI in the FGF2-KO group compared with WT. H: The area/vessel increased comparably in both the FGF2-Tg and FGF2-nTg hearts after infarction.

Figure 5

LV function. The derivative of the LV pressure curve at 20 mm Hg was used as the index of contractility (dP/dt). Group sizes are shown inside the bars in parentheses. Heart rates are indicated below the group sizes in beats per minute (bpm). A: There was no significant depression of contractile function in WT heart 4 weeks after MI; however, cardiac function was impaired in the FGF2-KO group (P < 0.05). B: Cardiac function was significantly depressed at 4 weeks after MI compared with control FGF2-nTg hearts (P < 0.05); however, there was no significant diminution of contractile function in the FGF2-Tg group.