
Patients with Duchenne muscular dystrophy exhibit significant, ongoing impairments in gastrointestinal (GI) function likely resulting from dysregulated nitric oxide production. Compounds increasing neuronal nitric oxide synthase expression and/or activity could improve GI dysfunction and enhance quality of life for dystrophic patients. We used video imaging and spatiotemporal mapping to identify GI dysfunction in
Four-week-old male C57BL/10 and
ST mapping identified increased contraction number in the mid and distal colon of
GI dysfunction in Duchenne muscular dystrophy has been sadly neglected as an issue affecting quality of life. ST mapping identified regional GI dysfunction in the
Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disorder resulting from mutations in the dystrophin (
The dystrophin protein, particularly the full length Dp427 isoform, forms an integral component of a multimeric protein complex termed the dystrophin-glycoprotein complex (DGC). In striated muscle, the DGC comprises the 427 kDa dystrophin protein in complex with the dystroglycans, dystrobrevins, and sarcoglycans, linking the actin cytoskeleton of the contractile apparatus to the extracellular matrix to ensure transmission of force across the muscle membrane.9,10 In addition to this integral mechanical role, components of the DGC including syntrophin, dystrobrevin, and notably neuronal nitric oxide synthase (nNOS, also known as NOS1), are implicated in signaling downstream of the DGC in striated muscle.11–18 NOS1 is localized to the sarcolemma of skeletal muscle fibers with the DGC.12 NOS1 produces nitric oxide (NO), a major endogenous mediator which in skeletal muscle, is released into the local circulation to cause vasodilation of the blood vessels, allowing for oxygenation. In DMD, sarcolemmal localization of NOS1 is lost, resulting in ischemia and damage to the muscle tissue.19 Thus, dystrophin and the DGC are key regulators of NOS1 localization and function. Forms of the DGC are expressed in the brain, and smooth muscle of the airways,20–22 but its exact role in these tissues is not well understood. The presence of a DGC in the enteric nervous system or smooth muscle of the GI system remains to be confirmed.
Studies in
Modulators of NO production have been tested for their ability to improve the dystrophic pathology in skeletal and cardiac muscle. Sildenafil, a phosphodiesterase type 5 inhibitor which modulates NO production, improves both diaphragm and heart structure and function in dystrophic mice.31–34 Additionally, L-arginine administration for 6 weeks improved dystrophic skeletal muscle pathology in the
Based on this evidence it is surprising that the effects of increasing NO production on GI function in vivo have not been examined. As compounds increasing NOS1 activity could improve GI function in muscular dystrophy and enhance quality of life for patients, we used video imaging and spatiotemporal (ST) mapping to interrogate GI dysfunction in
Animal experiments were performed in accordance with the Animal Research: Reporting of In Vivo Experiments (https://www.nc3rs.org.uk/arrive-guidelines). All experimental protocols were approved by the Animal Ethics Committee of The University of Melbourne (Approval No. AEC#1513780) and conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes as stipulated by the National Health and Medical Research Council (Australia). Experiments were performed on 4-week-old and 12-week-old male C57Bl/10ScSn (C57BL/10) and C57BL/10ScSn-Dmd
Following dissection and removal of the cecum, the oral end of the colon was attached to a cannula in a Sylgard lined organ bath, containing physiological saline at 35–37°C continuously superfused with Carbogen (95% CO2, 5% O2). Gentle pressure was applied to flush fecal pellets from cannulated colons. Fecal pellets were then collected from the organ bath into clean weigh boats for assessment of wet weight, length, and width.
Four-week-old C57BL/10 and
C57BL/10 and
A Logitech Quickcam Pro 9000 camera (Logitech, Lausanne, Switzerland) was placed above the organ bath and the video acquisition software VirtualDub 10.01 (Licensed under the GNU General Public License, developed by Avery Lee; www.virtualdub.org) used to acquire 15-minute video files of colonic contractile activity. After acquisition, .avi video files were converted into .su2 files using Scribble 2.1 (University of Melbourne in-house software), which uses an edge detection algorithm to identify colon diameter for each frame of the 15-minute video recording. The diameter of the colon at every point along its length is assigned a color, with constriction denoted warmer (red) colors and dilation assigned cooler (blue) colors. Each frame line is stacked with colon (gut) position shown on the y-axis and time across the x-axis to generate a ST map. ST maps were visualized and analyzed in a purpose-built MATLAB plugin (Analyse2; University of Melbourne in-house software) via MATLAB (2014b; The MathWorks Inc, Natick, MA, USA). ST maps were analyzed for contraction number (Supplementary Fig. 1B; depicted as yellow/red streaks) and colon diameter at the level of the proximal, mid, and distal colon. Contraction refers to the spontaneous directional activity including colonic migrating motor complexes (CMMCs) and short contractions. Constriction and resting colon diameter refer to the instantaneous measure of colon diameter indicating dilation or constriction. Contraction numbers were measured by manually counting the number of yellow/red streaks per ST map. Using the analysis software (Analyse2) within MATLAB, colon diameter measures were made by drawing a horizontal line across the spatiotemporal map at the level of the proximal, mid, and distal colon and identifying the maximal (resting colon diameter) and minimal (constricted colon diameter) colon diameter during the 15-minute recording as described previously.44 The color scale on all ST maps was kept constant to enable this comparison of the width of the colon.
Proximal, mid, and distal colon segments were snap frozen in Eppendorf tubes in liquid nitrogen and stored at −80°C. Lysis buffer (50 mM TrisHCl, 150 mM NaCl, 1mM EDTA, 1% (volume/volume) Triton X-100, 2 mM Na3VO4, 10 mM NaF, 1 μM protease inhibitor cocktail) was added at a ratio of 10:1 buffer to tissue weight. Samples were minced with small scissors and homogenized using a PT 2100 Polytron (Kinematica, Switzerland) (15 second homogenization, 45 second intervals, repeated 3 times) on ice. Lysates were centrifuged (10 000g, 4°C, 10 minutes) and protein concentration was determined using a DC Protein Assay (Bio-Rad, Hercules, CA, USA), with lysates diluted to 1 mg/mL and 4 × Laemmli sample buffer added to each sample. Samples were denatured (95°C for 3 minutes) and 15 μg of total protein loaded onto 26-well Bio-Rad Criterion gels (4–15%; Bio-Rad) alongside Precision Plus Protein Dual Color Standard (Bio-Rad). Gels were run at 100V at room temperature and proteins were transferred to polyvinylidene difluoride (PVDF; Immobilon-P, Merck Millipore, Darmstadt, Germany) membranes via wet transfer at 100 V for 90 minutes in ice cold transfer buffer. Membranes were blocked in 1× Tris buffered saline with 0.1% Tween-20 (TBST)/5% bovine serum albumin (BSA; Sigma Aldrich), followed by overnight incubation at 4°C in primary antibodies. Membranes were washed in TBST (3 × 5 minutes), followed by 1 hour incubation at room temperature in secondary antibody and washed in TBST (4 × 10 minutes). Membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) on a ChemiDoc MP Imaging System (Bio-Rad). Total protein stains were completed using the BLOT-FastStain kit according to manufacturer’s instructions (G-Biosciences, St. Louis, MO, USA) and quantification performed using Image Lab 4.1 software (Bio-Rad).
The following primary antibodies were used throughout the experiments in TBST/5% BSA: mouse-anti-dystrophin MANEX1011B (clone 1C7) (deposited by Glenn E Morris; Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA, USA; 1:1000) and mouse-α-β-dystroglycan (Leica Biosystems, Wetzlar, Germany; 1:5000). Horseradish peroxidase-conjugated sheep-anti-mouse IgG (GE Healthcare Life Sciences, Marlborough, MA, USA) was used at 1:5000 in TBST/5% BSA to detect dystrophin and 1:10000 in TBST/5% BSA to detect β-dystroglycan.
Proximal, mid, and distal colon segments were snap frozen in Eppendorf tubes in liquid nitrogen and stored at −80°C. NOS activity was measured using a colorimetric Nitric Oxide Synthase activity assay kit (#ab211083; Abcam, Cambridge, UK) as per the manufacturers’ instructions.
Data were analyzed between the 2 groups for the effect of genotype using Student’s
Prior to initiating a dietary intervention, we first sought to confirm and characterize the GI phenotype in
After 8 weeks of oral amino acid supplementation no significant differences in colon length (Supplementary Fig. 2A), fecal pellet number (Supplementary Fig. 2B), length (Supplementary Fig. 2C), or fecal pellet width (Supplementary Fig. 2D) were observed between groups, except for a trend for
ST mapping of video recordings of colon contractions ex vivo revealed no difference in the number of orally-initiated CMMC contractions between C57BL/10 and
Altered GI contractile activity in the
Although expression of the full length Dp427 kDa dystrophin protein has been reported in the interstitial cells of Cajal, enteric neurons, smooth muscle, and myoid cells of the gut by immunofluorescent studies,45 there remains a lack of knowledge regarding the function of dystrophin in these cells, particularly in relation to the presence of a DGC. By western immunoblotting, Dp427 expression was confirmed in proximal, mid, and distal colon segments from C57BL/10 mice, and was absent in colon segments from
As the DGC localizes NOS1 to the sarcolemma and NOS1 inhibition increases the contraction number in the colon, we also examined NOS activity in the proximal, mid, and distal colon from C57BL/10 and
GI dysfunction in DMD has been sadly neglected by researchers despite it being one of the most important clinical issues impacting quality of life for patients. Using ST mapping we identified regional GI dysfunction in the form of increased contractions in the distal colon in a murine model of DMD at both 4 and 12 weeks of age. Exogenous administration of L-arginine to isolated
Although Dp427 expression has been identified in the mouse colon,45,46 no studies had shown a functional DGC in the colon. Western immunoblotting showed decreased protein expression of β-dystroglycan in colons of
While exogenous administration of arginine has previously been shown to improve GI function in isolated distal colon segments in vitro,30 this is the first study to demonstrate that dietary supplementation with arginine, and to a lesser extent citrulline, can improve colon motility which has important therapeutic relevance to DMD patients. In the present study, dietary intervention was performed for a period of 8 weeks, since arginine35,38 and citrulline50 supplementation are under investigation for the treatment of skeletal muscle pathology in DMD, and are therefore likely to be administered as chronic supplements. However, as acute in vitro arginine supplementation also improves the function of colons from
While both citrulline and arginine supplementation reduced the contraction number in the distal colon of
In the colon, NO both inhibits CMMC pacemaker activity54–56 (where NO inhibition results in increased contractile activity) and modulates mechanical tone25 (regulating constriction and dilation of the colon). NOS inhibition, through the exogenous administration of NOLA, increased the contraction number in colons from C57BL/10 but not
Previous studies on colon dysfunction in
In summary, ST mapping identified regional GI dysfunction and increased contraction number in the colon of
The authors thank Associate Professor Jess Nithianantharajah from The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Australia, for supplying C57BL/6 mice.
Note: To access the supplementary figures mentioned in this article, visit the online version of
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