Evidence for impaired neurovascular transmission in a murine model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by mutations in the dystrophin gene. Little is known about how blood flow control is affected in arteriolar networks supplying dystrophic muscle. We tested the hypothesis that mdx mice, a murine model for DMD, exhibit defects in arteriolar vasomotor control. The cremaster muscle was prepared for intravital microscopy in pentobarbital sodium-anesthetized mdx and C57BL/10 control mice (n ≥ 5 per group). Spontaneous vasomotor tone increased similarly with arteriolar branch order in both mdx and C57BL/10 mice [pooled values: first order (1A), 6%; second order (2A), 56%; and third order (3A), 61%] with no difference in maximal diameters between groups measured during equilibration with topical 10 μM sodium nitroprusside (pooled values: 1A, 70 ± 3 μm; 2A, 31 ± 3 μm; and 3A, 19 ± 3 μm). Concentration-response curves to acetylcholine (ACh) and norepinephrine added to the superfusion solution did not differ between mdx and C57BL/10 mice, nor did constriction to elevated (21%) oxygen. In response to local stimulation from a micropipette, conducted vasodilation to ACh and conducted vasoconstriction to KCl were also not different between groups; however, constriction decayed with distance (P < 0.05) whereas dilation did not. Remarkably, arteriolar constriction to perivascular nerve stimulation (PNS) at 2, 4, and 8 Hz was reduced by ∼25-30% in mdx mice compared with C57BL/10 mice (P < 0.05). With intact arteriolar reactivity to agonists, attenuated constriction to perivascular nerve stimulation indicates impaired neurovascular transmission in arterioles controlling blood flow in mdx mice.

Fig. 1.

Dystrophin is absent and utrophin is upregulated in skeletal muscle of mdx mice. Representative immunofluorescence staining for dystrophin (left column) and utrophin (right column) of rectus abdominus muscle fibers C57BL/10 (top row) and mdx mice (bottom row). Alternate serial sections (thickness, 8 μm) were immunolabeled for dystrophin and utrophin. Dystrophin was expressed at the perimeter of skeletal muscle fibers from C57BL/10 mice but was absent in mdx mice. Utrophin was not expressed in muscle fibers of C57BL/10 mice but was expressed at the perimeter of muscle fibers in mdx mice. Scale bar (=100 μm) applies to all images.

Fig. 2.

Dilation to ACh and constriction to norepinephrine (NE) are intact in arterioles of mdx mice. Cumulative concentration-response curves in 1A, 2A, and 3A arterioles of mdx mice (■) and C57BL/10 (○) mice to ACh (A) and NE (B). Resting baseline (B) and maximum [sodium nitroprusside (SNP)] diameters shown for reference. Note difference in ordinate scales between arteriolar branch orders. Responses to ACh and NE were not significantly different between mdx (n = 7) and C57BL/10 mice (n = 6).

Fig. 3.

Conducted vasodilation is intact in mdx mice. Using microiontophoresis, ACh was delivered from a micropipette positioned on a 3A as illustrated at top; direction of blood flow indicated by arrow. Diameter changes were evaluated locally and at observation sites (indicated by *) located 500 μm upstream in the 3A and at 1,000 and 2,000 μm upstream in the parent 2A. A: baseline diameter at rest. B: diameter change at local and remote sites. Vasomotor tone (e.g., in 3A) was not significantly different between mdx and C57BL/10 mice (C57BL/10, 64 ± 2%; mdx, 67 ± 2%). Neither resting diameters nor diameter changes were significantly different between mdx mice and C57BL/10 mice (n = 5 per group).

Fig. 4.

Conducted vasoconstriction is intact in mdx mice. Using pressure ejection, KCl was delivered onto an arteriole from a micropipette positioned on an unbranched 2A segment as illustrated at top; direction of blood flow indicated by arrow. Diameter was recorded locally and at observation sites (indicated by *) located 500, 1,000, and 2,000 μm upstream. A: baseline diameters at rest. B: diameter change at local and remote sites. Neither resting diameters nor diameter changes were significantly different between mdx mice and C57BL/10 mice (n = 8 for C57BL/10; n = 12 for mdx). There was a main effect of distance on the amplitude of vasoconstriction, which progressively decreased from the site of stimulation (†P < 0.01).

Fig. 5.

Arteriolar constriction to perivascular nerve stimulation (PNS) is attenuated consistently in mdx mice. A: representative records of arterioles from C57BL/10 and mdx mice stimulated at 8 Hz. B: vasoconstriction during PNS increased with stimulus frequency and was attenuated consistently in mdx compared with C57BL/10 mice. *mdx significantly different from C57BL/10, P < 0.05. There was a main effect of frequency on the amplitude of vasoconstriction (†P < 0.01).