Absence of dystrophin in mice reduces NO-dependent vascular function and vascular density: total recovery after a treatment with the aminoglycoside gentamicin - PubMed (original) (raw)

Comparative Study

Absence of dystrophin in mice reduces NO-dependent vascular function and vascular density: total recovery after a treatment with the aminoglycoside gentamicin

Laurent Loufrani et al. Arterioscler Thromb Vasc Biol. 2004 Apr.

Abstract

Objective: Mutations in the dystrophin gene causing Duchenne's muscular dystrophy (DMD) lead to premature stop codons. In mice lacking dystrophin (mdx mice), a model for DMD, these mutations can be suppressed by aminoglycosides such as gentamicin. Dystrophin plays a role in flow (shear stress)-mediated endothelium-dependent dilation (FMD) in arteries. We investigated the effect of gentamicin on vascular contractile and dilatory functions, vascular structure, and density in mdx mice.

Methods and results: Isolated mice carotid and mesenteric resistance arteries were mounted in arteriographs allowing continuous diameter measurements. Mdx mice showed lower nitric oxide (NO)-dependent FMD and endothelial NO synthase (eNOS) expression as well as decreased vascular density in gracilis and cardiac muscles compared with control mice. Treatment with gentamycin restored these parameters. In contrast, smooth muscle-dependent contractions as well as endothelium-dependent or -independent dilation were not affected by dystrophin deficiency or by gentamicin treatment.

Conclusions: Dystrophin deficiency induces a selective defect in flow-dependent mechanotransduction, thus attenuating FMD and eNOS expression, and may contribute to low arteriolar density. These findings open important perspectives regarding the mechanism involved in the pathophysiology of genetic diseases related to premature stop codons such as DMD.

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Figures

Figure 1

Figure 1

Immunolocalization of dystrophin in carotid arteries (revealed by peroxidase, a) and in mesenteric resistance arteries (Texas-red immunofluorescence, b) was performed using Dys2 anti-dystrophin antibodies. Arteries were isolated from control, mdx mice or mdx mice treated for two weeks with gentamicin. In mesenteric resistance arteries dystrophin (Dys2) was localized to the plasma membrane of endothelial cells, using immunofluorescence and confocal microscopy. This was performed in arteries perfused under a pressure of 75 mmHg and a flow of 50 μl/min (c). (n=6 mice per group). Due to the movement generated by the perfusion of the arteries in physiological conditions, the scanning of the vascular wall was performed at a speed of 10 images/sec. The expression of dystrophin was determined in carotid arteries, using western-blot analysis. Dys1, Dys2 and Dys3 antibodies were directed against C-terminus, N-terminus and mid-rod domains of dystrophin, respectively (d and e). (n=8 mice per group). *P <0.001; two-factor ANOVA, versus control. #P <0.001; two-factor ANOVA, non-treated versus gentamicin-treated mdx mice.

Figure 1

Figure 1

Immunolocalization of dystrophin in carotid arteries (revealed by peroxidase, a) and in mesenteric resistance arteries (Texas-red immunofluorescence, b) was performed using Dys2 anti-dystrophin antibodies. Arteries were isolated from control, mdx mice or mdx mice treated for two weeks with gentamicin. In mesenteric resistance arteries dystrophin (Dys2) was localized to the plasma membrane of endothelial cells, using immunofluorescence and confocal microscopy. This was performed in arteries perfused under a pressure of 75 mmHg and a flow of 50 μl/min (c). (n=6 mice per group). Due to the movement generated by the perfusion of the arteries in physiological conditions, the scanning of the vascular wall was performed at a speed of 10 images/sec. The expression of dystrophin was determined in carotid arteries, using western-blot analysis. Dys1, Dys2 and Dys3 antibodies were directed against C-terminus, N-terminus and mid-rod domains of dystrophin, respectively (d and e). (n=8 mice per group). *P <0.001; two-factor ANOVA, versus control. #P <0.001; two-factor ANOVA, non-treated versus gentamicin-treated mdx mice.

Figure 2

Figure 2

Passive vascular responses to pressure in carotid (left panel) and mesenteric resistance arteries (right panel) isolated from control or mdx mice treated for two weeks with gentamicin (genta, 34 mg/kg/day/14days). Arteries were submitted to stepwise increases in pressure when bathed in a Ca2+-free physiological salt solution containing EGTA (2 mmol/L) and sodium nitroprusside (10 μM), thus defining the passive arterial diameter, expressed as active tone (passive diameter - active diameter 6,14 (a, b). Arterial wall thickness was determined the same arteries (c, d). Cross-sectional compliance (e and f) was calculated from the diameter values shown in a and b. n=10 per group. *P <0.05; two-factor ANOVA.

Figure 3

Figure 3

Myogenic tone determined in carotid (a) and mesenteric resistance arteries (b) isolated from control or mdx mice treated for two weeks with gentamicin (genta, n=10 per group). Responses to flow were then determined in mesenteric (c) and carotid arteries (d) under a pressure of 75 mmHg. In another group of experiments, the same experiments (flow-mediated dilation) were performed in vimentin-null mice mesenteric arteries (e). *P <0.05; two-factor ANOVA, mdx versus control (c and d) or vim+/+ versus vim−/− (e)

Figure 4

Figure 4

Effect of NO-synthesis blockade with L-NAME (LN) on flow-mediated dilation in mesenteric resistance arteries isolated from control (a) or mdx mice (b) treated for two weeks with gentamicin (genta). Data are represented as μm dilation (a and b) and as percentage inhibition of flow-mediated dilation by L-NAME (in control, c, and mdx, d, mice) (n=10 per group). NO-synthase expression, determined in carotid arteries is shown in e. #P <0.001; two-factor ANOVA, non-treated versus gentamicin-treated mice. *P <0.01; two-factor ANOVA, versus control.

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