Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor necrosis factor alpha therapy

Abstract

Myocardial fibrosis caused by maladaptive extracellular matrix (ECM) remodeling is implicated in the dysfunction of the failing heart. Matrix metalloproteinases (MMPs) regulate ECM remodeling, and are regulated by cytokines. Transgenic mice with cardiac-specific overexpression of tumor necrosis factor alpha (TNF-alpha) (TNF1.6) develop heart failure. We hypothesized that modulation of TNF-alpha and/or MMP activity might alter the myocardial ECM remodeling process and the development of heart failure. To test this hypothesis, we took advantage of the TNF1.6 mice and studied soluble and total collagens and collagen type profiling by using hydroxyproline quantification, Sircol collagen assay, Northern blot analysis, and immunohistochemistry and studied myocardial function by using echocardiography. Progressive ventricular hypertrophy and dilation in the TNF1.6 mice were accompanied by a significant increase in MMP-2 and MMP-9 activity, an increase in collagen synthesis, deposition, and denaturation, and a decrease in undenatured collagens. In young TNF1.6 mice, these changes in the ECM were associated with marked diastolic dysfunction as demonstrated by significantly reduced transmitral Doppler echocardiographic E/A wave ratio. Anti-TNF-alpha treatment with adenoviral vector expressing soluble TNF-alpha receptor type I attenuated both MMP-2 and MMP-9 activity, prevented further collagen synthesis, deposition and denaturation, and preserved myocardial diastolic function in young, but not old, TNF1.6 mice. The results suggest a critical role of TNF-alpha and MMPs in myocardial matrix remodeling and functional regulation and support the hypothesis that both TNF-alpha and MMPs may serve as potential therapeutic targets in the treatment of heart failure.

Figure 1

Gelatin zymography of ventricular extracts of neonates (A), 12-week-old (B), 48-week-old (C) wild-type (WT) and TNF1.6 mice. (D) Summary of quantitative data of MMP-2 and MMP-9 activity in 12- and 48-week-old mouse heart (n = 4 each). *, P < 0.01 compared with respective wild type.

Figure 2

Modulation of the activity of MMPs in the TNF1.6 mouse heart. Gelatin zymography demonstrates that AdTNFRI treatment abolished MMP-9 gelatinolytic activity after 2 weeks (lane 5) and reduced MMP-2 activity to wild-type level after 6 weeks (lane 6). Controls are negative without known MMPs (lane 1), FVB wild type (lane 2), TNF1.6 (lane 3), and TNF1.6 treated with AdLacZ control vector (lane 4).

Figure 3

Representative Northern blot analysis results of collagen proα1(I) and proα1(III). The same blot was sequentially hybridized with proα1(I), proα1(III), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probes. The bar graphs summarize the quantitative results of proα1(I) and proα1(III) mRNA levels normalized to that of GAPDH in both 12- and 48-week-old wild-type (WT), TNF1.6, and TNF1.6 mice treated with AdTNFRI (n = 4 each). *, P < 0.05 compared with wild type; †, P < 0.05 compared with TNF1.6.

Figure 4

(On the opposite page.) Indirect immunofluorescence of myocardial type I and type III collagens. (A–F) Type I collagen. (G–L) Type III collagen. (A–C and G–I) 12-week-old mice; (D–F and J–L) 48-week-old mice. Both type I and type III collagens appear increased in TNF-α transgenic (B, E, H, and K) relative to wild-type mice (A, D, G, and J), which were prevented by AdTNFRI treatment in 12-week-old mice (C and I) but not in 48-week-old mice (F and L). Original magnification: ×200. Quantified collagen volume fraction shows that the increased collagens I and III were prevented by AdTNFRI treatment in 12-week-old mice (n = 3) (M and N). *, P < 0.05 compared with wild type; †, P < 0.05 compared with TNF1.6.

Figure 5

Changes in collagens in the mouse heart. (A) Reduction in undenatured collagen in TNF1.6 mice was partially prevented by AdTNFRI treatment. (B) Total soluble collagen content was increased in 12-, 24-, and 48-week-old TNF1.6 mouse heart, and the increase was prevented by AdTNFRI treatment in 12-week-old TNF1.6 mice. (C) Gradual increase in total collagen content was seen in both wild-type and TNF1.6 mice, but was more apparent in TNF1.6 mice. The collagen accumulation in 12-week-old TNF1.6 mouse heart was prevented by AdTNFRI treatment. n = 8 in each group. *, P < 0.05 compared with wild type; †, P < 0.05 compared with TNF1.6.

Figure 6

Echocardiographic measurement of the geometry and function of the mouse heart. LV dimensions were calculated from measurements of end-diastolic diameter (arrow 1), end-systolic diameter (arrow 2), anterior wall (arrow 3), and posterior wall (arrow 4), and E/A wave ratio was calculated as shown in schematic M-mode (a) and Doppler (b) echocardiographic tracings. Ventricular hypertrophy in 12-week-old TNF1.6 mice (d), ventricular dilation in 48-week-old TNF1.6 mice (j) as compared with respective wild-type mice (c and i). AdTNFRI treatment had no effect on ventricular hypertrophy or dilation (e and k), but the reduced E/A ratio in 12-week-old TNF1.6 mice (g) was prevented by the treatment (h).