Improvement of cardiac contractile function by peptide-based inhibition of NF-κB in the utrophin/dystrophin-deficient murine model of muscular dystrophy

Abstract

Background: Duchenne muscular dystrophy (DMD) is an inherited and progressive disease causing striated muscle deterioration. Patients in their twenties generally die from either respiratory or cardiac failure. In order to improve the lifespan and quality of life of DMD patients, it is important to prevent or reverse the progressive loss of contractile function of the heart. Recent studies by our labs have shown that the peptide NBD (Nemo Binding Domain), targeted at blunting Nuclear Factor κB (NF-κB) signaling, reduces inflammation, enhances myofiber regeneration, and improves contractile deficits in the diaphragm in dystrophin-deficient mdx mice.

Methods: To assess whether cardiac function in addition to diaphragm function can be improved, we investigated physiological and histological parameters of cardiac muscle in mice deficient for both dystrophin and its homolog utrophin (double knockout = dko) mice treated with NBD peptide. These dko mice show classic pathophysiological hallmarks of heart failure, including myocyte degeneration, an impaired force-frequency response and a severely blunted β-adrenergic response. Cardiac contractile function at baseline and frequencies and pre-loads throughout the in vivo range as well as β-adrenergic reserve was measured in isolated cardiac muscle preparations. In addition, we studied histopathological and inflammatory markers in these mice.

Results: At baseline conditions, active force development in cardiac muscles from NBD treated dko mice was more than double that of vehicle-treated dko mice. NBD treatment also significantly improved frequency-dependent behavior of the muscles. The increase in force in NBD-treated dko muscles to β-adrenergic stimulation was robustly restored compared to vehicle-treated mice. However, histological features, including collagen content and inflammatory markers were not significantly different between NBD-treated and vehicle-treated dko mice.

Conclusions: We conclude that NBD can significantly improve cardiac contractile dysfunction in the dko mouse model of DMD and may thus provide a novel therapeutic treatment for heart failure.

Figure 1

Baseline contractile function. A. Muscles from NBD treated dko mice (n = 9 muscles from n = 7 mice) exhibited a higher active developed force under baseline conditions (1.5 mM Ca²⁺, 4 Hz, 37°C) compared to muscles from vehicle treated control dko mice (n = 5 muscles from n = 4 mice). B. Maximum and minimum derivative of force (dF/dt) was higher in NBD treated mice. C. Time from stimulation to peak tension and time from peak tension to 90% relaxation were slightly, but not significantly, slower in non-treated muscles. * indicates a difference of P < 0.05 between the two groups.

Figure 2

Length-dependent activation. When the muscle was stretched from 85% of optimal length (near slack, virtually no passive tension, 37°C) to optimal length, active force development significantly increased in both NBD treated and vehicle treated groups. Repeated measures ANOVA indicated that impact of both factors, treatment and length, were significant (P < 0.05), but not the interaction, indicating unchanged length-dependent behavior after NBD treatment in dko mice. * indicates a difference of P < 0.05 between the two groups.

Figure 3

Frequency-dependent activation. A. An increase in frequency led to a decrease in force development in both muscles from NBD treated and vehicle treated dko mice. B. When normalized to their individual initial forces at 4 Hz, NBD treated muscles do not exhibit the negative force-frequency behavior displayed by the vehicle treated group at the lower frequency range at 37°C. All muscles were kept at their optimal length during this protocol. ANOVA (repeated measures) indicated that both the factors treatment and frequency, as well as the interaction between these two factors was significantly different. * indicates a difference of P < 0.05 between the two groups.

Figure 4

β-adrenergic response. The severely blunted response to the β-adrenergic agonist isoproterenol in muscles from dko mice is significantly ameliorated by NBD treatment. ANOVA (repeated measures) indicated that both the factors isoproterenol and frequency, as well as the interaction between these two factors was significantly different between NBD and vehicle treated groups. Stimulation frequency was 4 Hz, at 37°C. * indicates a difference of P < 0.05 between the two groups.

Figure 5

NBD is effective in inhibiting NF-κB in cardiac muscles from dko mice. Nuclear extracts were prepared from hearts of vehicle (n = 4) or NBD (n = 7) treated dko mice and analyzed by either EMSA (upper panel) or western blot probing for nuclear fraction p65 (bottom panels). Nonspecific band (NS) is shown on the western blot to demonstrate equivalent protein loading.

Figure 6

Histological analyses of tissue damage indicators in representative serial sections of hearts from vehicle and NBD peptide treated dko mice show similar pathology in both treatment groups. A. Hematoxylin and eosin (H&E) staining shows the presence of fibrotic scars in dko hearts from vehicle and NBD-treated groups. B. Immunostaining for collagen I shows localization of collagen in fibrotic regions. C. ER-TR7 immunostaining demonstrates fibroblasts are a major cellular infiltrate in regions of fibrosis. D. CD-45 immunostaining shows that immune cells are not detected in fibrotic scars at the time-point of analysis. Scale bar equals 50 μm.

Figure 7

Indirect comparison of functional improvement of dko myocardium by NBD treatment shows that the functional improvement in baseline cardiac contractile force (4 Hz, optimal length, 37°C) resulted in forces that are comparable with age-matched C57BL/10 wild type muscles, and relatively exceed those assessed in mdx myocardium under identical experimental conditions. Data from this study and from Ref. [10].