Dystrophin deficiency impairs vascular structure and function in the canine model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by dystrophin deficiency. Vascular dysfunction has been suggested as an underlying pathogenic mechanism in DMD. However, this has not been thoroughly studied in a large animal model. Here we investigated structural and functional changes in the vascular smooth muscle and endothelium of the canine DMD model. The expression of dystrophin and endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), and the structure and function of the femoral artery from 15 normal and 16 affected adult dogs were evaluated. Full-length dystrophin was detected in the endothelium and smooth muscle in normal but not affected dog arteries. Normal arteries lacked nNOS but expressed eNOS in the endothelium. NOS activity and eNOS expression were reduced in the endothelium of dystrophic dogs. Dystrophin deficiency resulted in structural remodeling of the artery. In affected dogs, the maximum tension induced by vasoconstrictor phenylephrine and endothelin-1 was significantly reduced. In addition, acetylcholine-mediated vasorelaxation was significantly impaired, whereas exogenous nitric oxide-induced vasorelaxation was significantly enhanced. Our results suggest that dystrophin plays a crucial role in maintaining the structure and function of vascular endothelium and smooth muscle in large mammals. Vascular defects may contribute to DMD pathogenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: Duchenne muscular dystrophy; canine model; dystrophin; eNOS; nNOS; vasculature; vasoconstriction; vasorelaxation.

Figure 1.. Histological evaluation of dystrophin, eNOS and nNOS expression and NOS activity in normal and DMD dog femoral arteries.

(A) Representative photomicrographs of HE staining (top panels) and dystrophin immunostaining (bottom panels). Double arrowhead marks the location of the tunica media. Dys (R17), Immunostaining was performed using an antibody that recognizes dystrophin spectrin-like repeat 17. (B) Representative photomicrographs of dystrophin and CD31 immunostaining. Arrow, non-specific fluorescence signal at the internal elastic lamina; Dys (CT), Immunostaining was performed using an antibody that recognizes the C-terminal domain of dystrophin; CD31, Immunostaining was performed using an antibody that recognizes CD31, an endothelial marker. (C) Representative photomicrographs of in situ NOS activity staining. NOS activity is revealed by intense dark blue staining. (D) Representative photomicrographs of eNOS immunostaining. Arrowhead, eNOS expression in the tunica intima; Arrow, non-specific fluorescence signal at the internal elastic lamina; 2^nd Ab, Staining was performed in the absence of the primary antibody. (E) Representative photomicrographs of nNOS immunostaining. Arrow, non-specific fluorescence signal at the internal elastic lamina; 2^nd Ab, Staining was performed in the absence of the primary antibody. (F) Representative nNOS immunostaining photomicrographs of normal and DMD skeletal muscle. Scale bar applies to all the images in the same panel.

Figure 2.. Biochemical evaluation of dystrophin, eNOS and nNOS expression in normal and DMD dog femoral arteries.

(A) Representative photomicrographs of the cropped dystrophin, eNOS, and α-tubulin western blots from endothelial and smooth muscle fractions. α-tubulin was used as the loading control. Raw data are shown in supplementary material, Figure S5. (B) Densitometry quantification of eNOS expression in endothelial cells of normal (n=5) and DMD (n=5) dogs. Asterisk, significantly different between normal and DMD. (C) Representative photomicrographs of the cropped utrophin, CD31, α-tubulin, and α-smooth muscle actin (SMA) from endothelial and smooth muscle fractions. (D) Representative photomicrographs of the cropped dystrophin, nNOS, and α-tubulin western blots from endothelial and smooth muscle fractions. α-tubulin was used as the loading control. Uncropped blots are shown in supplementary material, Figure S5.

Figure 3.. Quantitative characterization of normal and DMD dog femoral arteries by anatomic and histological measurements.

(A) Anatomic quantification of the axial length, outer diameter, inner diameter, wall thickness, and lumen size to wall thickness ration of normal (n=15) and DMD (n=16) dog femoral artery rings. (B) Histological quantification of the thickness of the tunica media and tunica adventitia, and the ratio of the tunica media thickness to tunica adventitia thickness of normal (n=7) and DMD (n=11) dog femoral arteries. Asterisk, significantly different between normal and DMD.

Figure 4.. Dystrophin deficiency altered vasoconstriction and vasodilation responses in the intact dog femoral artery ring.

Vasoconstriction was evaluated by the developed and specific tension following stimulation with different doses of phenylephrine or endothelin-1. Vasodilation was evaluated by the percentage of vasorelaxation following stimulation with different doses of acetylcholine or sodium nitroprusside. (A) Quantification of the developed tension induced by different doses of phenylephrine. Each data point shows the mean ± SEM of n=15 normal and n=16 DMD dog artery rings. (B) Quantification of the specific tension induced by different doses of phenylephrine. Each data point shows the mean ± SEM of n=15 normal and n=16 DMD dog artery rings. (C) Quantification of the developed tension induced by different doses of endothelin-1. Each data point shows the mean ± SEM of n=6 normal and n=6 DMD dog artery rings. (D) Quantification of the specific tension induced by different doses of endothelin-1. Each data point shows the mean ± SEM of n=6 normal and n=6 DMD dog artery rings. (E) Quantification of the percentage of vasorelaxation induced by different doses of acetylcholine. Each data point shows the mean ± SEM of n=15 normal and n=13 to 16 DMD dog artery rings. (F) Quantification of the percentage of vasorelaxation induced by different doses of sodium nitroprusside. Each data point shows the mean ± SEM of n=15 normal and n=14 to 16 DMD dog artery rings. Asterisk, significantly different between normal and DMD at the same drug concentration. Note, results shown in panels A, B, E and F are not identical to the results shown in the control groups in supplementary material, Figures S7, S9, and S10. This is due to sample size difference. Only a subset of dogs (6 normal and 6 DMD) samples used in Figure 4 underwent denudation, L-NAME treatment, and L-NAME/Indomethacin co-treatment. The control group data shown in supplementary material, Figure S7 are from the same dogs that were also evaluated under denuded, L-NAME treated, and L-NAME/Indomethacin co-treated conditions.

Figure 5.. Impact of endothelial removal, L-NAME treatment, and combined L-NAME/indomethacin treatment on phenylephrine-induced or endothelin-1 induced vasoconstriction in normal and DMD dog femoral artery rings.

(A) Phenylephrine-induced developed tension (top panels) and specific tension (bottom panels) of the femoral artery rings after mechanical removal of the endothelium (denuded, left panels), in the presence of L-NAME (middle panels), and in the presence of both L-NAME and indomethacin (right panels). (B) Endothelin-1 induced developed tension (top panels) and specific tension (bottom panels) of the femoral artery rings after mechanical removal of the endothelium (denuded, left panels), in the presence of L-NAME (middle panels), and in the presence of both L-NAME and indomethacin (right panels). n = 6 for both normal and DMD. Asterisk, significantly different between normal and DMD at the same concentration of the vasoconstrictor (phenylephrine or endothelin-1).

Figure 6.. Impact of endothelial removal, L-NAME treatment, and combined L-NAME/indomethacin treatment on acetylcholine-induced or sodium nitroprusside-induced vasorelaxation in normal and DMD dog femoral artery rings.

(A) Acetylcholine-induced (left panels) relaxation of the femoral artery rings after mechanical removal of the endothelium (denuded, top panel), in the presence of L-NAME (middle panel), and in the presence of both L-NAME and indomethacin (bottom panel). (B) Sodium nitroprusside-induced (right panels) relaxation of the femoral artery rings after mechanical removal of the endothelium (denuded, top panel), in the presence of L-NAME (middle panel), and in the presence of both L-NAME and indomethacin (bottom panel). n = 6 for both normal and DMD. Asterisk, significantly different between normal and DMD at the same concentration of the vasodilator (acetylcholine or sodium nitroprusside).