Margriet Hulsker

The Effects of Low Levels of Dystrophin on Mouse Muscle Function and Pathology

Duchenne muscular dystrophy (DMD) is a severe progressive muscular disorder caused by reading frame disrupting mutations in the DMD gene, preventing the synthesis of functional dystrophin. As dystrophin provides muscle fiber stability during contractions, dystrophin negative fibers are prone to exercise-induced damage. Upon exhaustion of the regenerative capacity, fibers will be replaced by fibrotic and fat tissue resulting in a progressive loss of function eventually leading to death in the early thirties. With several promising approaches for the treatment of DMD aiming at dystrophin restoration in clinical trials, there is an increasing need to determine more precisely which dystrophin levels are sufficient to restore muscle fiber integrity, protect against muscle damage and improve muscle function. To address this we generated a new mouse model (mdx-Xist Δhs) with varying, low dystrophin levels (3–47%, mean 22.7%, stdev 12.1, n = 24) due to skewed X-inactivation. Longitudinal sections revealed that within individual fibers, some nuclei did and some did not express dystrophin, resulting in a random, mosaic pattern of dystrophin expression within fibers. Mdx-Xist Δhs, mdx and wild type females underwent a 12 week functional test regime consisting of different tests to assess muscle function at base line, or after chronic treadmill running exercise. Overall, mdx-Xist Δhs mice with 3–14% dystrophin outperformed mdx mice in the functional tests. Improved histopathology was observed in mice with 15–29% dystrophin and these levels also resulted in normalized expression of pro-inflammatory biomarker genes, while for other parameters >30% of dystrophin was needed. Chronic exercise clearly worsened pathology, which needed dystrophin levels >20% for protection. Based on these findings, we conclude that while even dystrophin levels below 15% can improve pathology and performance, levels of >20% are needed to fully protect muscle fibers from exercise-induced damage.

Comparison of skeletal muscle pathology and motor function of dystrophin and utrophin deficient mouse strains

The genetic defect of mdx mice resembles that of Duchenne muscular dystrophy, although their functional performance and life expectancy is nearly normal. By contrast, mice lacking utrophin and dystrophin (mdx/utrn -/-) are severely affected and die prematurely. Mice with one utrophin allele (mdx/utrn +/-) are more severely affected than mdx mice, but outlive mdx/utrn -/- mice. We subjected mdx/utrn +/+, +/-, -/- and wild type males to a 12week functional test regime of four different functional tests. Mdx/utrn +/+ and +/- mice completed the regime, while mdx/utrn -/- mice died prematurely. Mdx/utrn +/- mice performed significantly worse compared to mdx/utrn +/+ mice in functional tests. Creatine kinase levels, percentage of fibrotic/necrotic tissue, morphology of neuromuscular synapses and expression of biomarker genes were comparable, whereas mdx/utrn +/- and -/- mice had increased levels of regenerating fibers. This makes mdx/utrn +/- mice valuable for testing the benefit of potential therapies on muscle function parameters.

Low dystrophin levels in heart can delay heart failure in mdx mice.

Duchenne muscular dystrophy is caused by mutations that prevent synthesis of functional dystrophin. All patients develop dilated cardiomyopathy. Promising therapeutic approaches are underway that successfully restore dystrophin expression in skeletal muscle. However, their efficiency in the heart is limited. Improved quality and function of only skeletal muscle potentially accelerate the development of cardiomyopathy. Our study aimed to elucidate which dystrophin levels in the heart are required to prevent or delay cardiomyopathy in mice. Heart function and pathology assessed with magnetic resonance imaging and histopathological analysis were compared between 2, 6 and 10-month-old female mdx-Xist(Δhs) mice, expressing low dystrophin levels (3-15%) in a mosaic manner based on skewed X-inactivation, dystrophin-negative mdx mice, and wild type mice of corresponding genetic backgrounds and gender. With age mdx mice developed dilated cardiomyopathy and hypertrophy, whereas the onset of heart pathology was delayed and function improved in mdx-Xist(Δhs) mice. The ejection fraction, the most severely affected parameter for both ventricles, correlated to dystrophin expression and the percentage of fibrosis. Fibrosis was partly reduced from 9.8% in mdx to 5.4% in 10 month old mdx-Xist(Δhs) mice. These data suggest that mosaic expression of 4-15% dystrophin in the heart is sufficient to delay the onset and ameliorate cardiomyopathy in mice.

Comprehensive Gene-Expression Survey Identifies Wif1 as a Modulator of Cardiomyocyte Differentiation

During chicken cardiac development the proepicardium (PE) forms the epicardium (Epi), which contributes to several non-myocardial lineages within the heart. In contrast to Epi-explant cultures, PE explants can differentiate into a cardiomyocyte phenotype. By temporal microarray expression profiles of PE-explant cultures and maturing Epi cells, we identified genes specifically associated with differentiation towards either of these lineages and genes that are associated with the Epi-lineage restriction. We found a central role for Wnt signaling in the determination of the different cell lineages. Immunofluorescent staining after recombinant-protein incubation in PE-explant cultures indicated that the early upregulated Wnt inhibitory factor-1 (Wif1), stimulates cardiomyocyte differentiation in a similar manner as Wnt stimulation. Concordingly, in the mouse pluripotent embryogenic carcinoma cell line p19cl6, early and late Wif1 exposure enhances and attenuates differentiation, respectively. In ovo exposure of the HH12 chicken embryonic heart to Wif1 increases the Tbx18-positive cardiac progenitor pool. These data indicate that Wif1 enhances cardiomyogenesis.

Preclinical Studies on Intestinal Administration of Antisense Oligonucleotides as a Model for Oral Delivery for Treatment of Duchenne Muscular Dystrophy

Antisense oligonucleotides (AONs) used to reframe dystrophin mRNA transcripts for Duchenne muscular dystrophy (DMD) patients are tested in clinical trials. Here, AONs are administered subcutaneously and intravenously, while the less invasive oral route would be preferred. Oral delivery of encapsulated AONs supplemented with a permeation enhancer, sodium caprate, has been successfully used to target tumor necrosis factor (TNF)-α expression in liver. To test the feasibility of orally delivered AONs for DMD, we applied 2′-O-methyl phosphorothioate AONs (with or without sodium caprate supplementation) directly to the intestine of mdx mice and compared pharmacokinetics and -dynamics with intravenous, intraperitoneal, and subcutaneous delivery. Intestinally infused AONs were taken up, but resulted in lower plasma levels compared to other delivery routes, although bioavailability could be largely improved by supplementation of sodium caprate. After intestinal infusion, AON levels in all tissues were lower than for other administration routes, as were the ratios of target versus nontarget organ levels, except for diaphragm and heart where comparable levels and ratios were observed. For each administration route, low levels of exon skipping in triceps was observed 3 hours post-AON administration. These data suggest that oral administration of naked 2′-O-methyl phosphorothioate AONs may be feasible, but only when high AON concentrations are used in combination with sodium caprate.

Accurate Dystrophin Quantification in Mouse Tissue; Identification of New and Evaluation of Existing Methods.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder primarily affecting males. This disorder is caused by mutations in the DMD gene that abolish dystrophin protein function. Many therapeutic approaches for DMD aim at recovery of the dystrophin protein in muscle fibers of affected patients, rendering accurate dystrophin quantification important. Several methods have been reported to detect and quantify dystrophin restoration in preclinical and clinical trials. We here evaluated the applicability of dystrophin specific enzyme-linked immunosorbent assays (ELISA) and a TaqMan protein assay, benchmarking them against Western blotting analysis. Despite numerous optimization attempts, in our hands the background signals in the ELISA and TaqMan protein assays were too high to allow dystrophin quantification. By contrast, the Western blot approach was able to detect dystrophin levels as low as 0.2% in a reproducible manner. We provide a Western blot protocol that allows sensitive and accurate dystrophin quantification in preclinical studies.

Voluntary exercise improves muscle function and does not exacerbate muscle and heart pathology in aged Duchenne muscular dystrophy mice

Duchenne muscular dystrophy is a severe muscle wasting disease, characterized by a severely reduced lifespan in which cardiomyopathy is one of the leading causes of death. Multiple therapies aiming at dystrophin restoration have been approved. It is anticipated that these therapies will maintain muscle function for longer and extend the ambulatory period, which in turn will increase the cardiac workload which could be detrimental for cardiac function. We investigated the effects of voluntary running exercise in combination with low dystrophin levels on function and pathology of skeletal muscle and heart. We divided 15.5-month old female mdx (no dystrophin), mdx-XistΔhs (varying low dystrophin levels) and wild type mice (BL10-WT and XistΔhs-WT) to either a sedentary or voluntary wheel running regime and assessed muscle function at 17.5 months of age. Thereafter, a cardiac MRI was obtained, and muscle and heart histopathology were assessed. We show that voluntary exercise is beneficial to skeletal muscle and heart function in dystrophic mice while not affecting muscle pathology. Low amounts of dystrophin further improve skeletal muscle and cardiac function. These findings suggest that voluntary exercise may be beneficial for skeletal muscle and heart in DMD patients, especially in conjunction with low amounts of dystrophin.

G.P.84

Duchenne muscular dystrophy is a severe muscle wasting disorder, caused by the loss of dystrophin. Since dystrophin is needed to maintain fiber stability during contractions, its absence results in fragile fibers, which are prone to exercise induced damage. In contrast to DMD patients, the mdx mouse model is less severely affected due, at least in part, to overexpression of the dystrophin homologue utrophin. To determine the role of utrophin in maintaining muscle strength and integrity, we performed in vivo force measurements in the tibialis anterior (TA) of 8week old dystrophic mice expressing utrophin on two ( mdx ), one ( mdx-utrn+/ −) or zero alleles ( mdx-utrn − / −). The specific force was significantly lower in all dystrophic compared to wild type mice, and mdx-utrn − / − mice performed significantly worse than mdx and mdx-utrn+/ − mice. Although mdx mice produced lower forces than wild type mice, the force frequency relationship curves followed a similar pattern. A substantial increase in force was observed upon stimulation with higher frequencies (>80Hz) in mdx mice. Contrastingly, lower frequencies ( mdx-utrn+/ − mice, while their maximum specific force was comparable to mdx mice. This suggested that the proportions of slow and fast fibers differed between the dystrophic models. Histologically, we confirmed a shift towards slow fibers in the TAs of mdx-utrn+/ − and −/− mice, whereas in mdx and wild type mice predominantly fast fibers were found. Eccentric (lengthening) contractions provoked a large force drop in mdx mice, while wild type mice were resistant. Interestingly, slow muscle fibers of mdx-utrn+/ − mice enabled better maintenance of force during eccentric contractions than seen in mdx mice. Mdx-utrn − / − mice did not show a drop in force as they were already very weak at protocol initiation. These results suggest that loss of utrophin provokes a shift towards slow fibers which partly preserves muscle function in heterozygotes.

T.P.26 Low dystrophin levels increase survival and improve pathology and motor function in dystrophin/utrophin double knockout mice

Abstract In Duchenne Muscular Dystrophy (DMD) patients muscle fibers are susceptible to exercise-induced injury due to absence of functional dystrophin. No cure is available, but in the last decade major progress has been made in the challenge to restore dystrophin expression in DMD patients. It is unknown how much dystrophin is needed to slow or prevent disease progression. To elucidate this, we generated mdx-Xist Δhs utrn − / − mice in which skewed X-inactivation results in expression of variable, low dystrophin levels in a utrophin negative background. These mice ( n =20) underwent a 12week functional test regime after which histopathology was assessed. Dystrophin levels of 3–10% already significantly improved performance of two and four limb hanging wire tests and histopathology, while 10–17% further normalized this towards wild type. For improvement in grip strength higher dystrophin levels are needed. Most striking was the effect of already very modest dystrophin levels in maintenance of basic muscle function and protection against death from overall weakness. Whereas mdx/utrn − / − mice did not live beyond 12weeks, 62% of the mice expressing 3–10% dystrophin and all mice expressing 10–17% dystrophin survived 16weeks. A survival study in 42 mdx-Xist Δhs utrn − / − mice assessing skeletal muscle function and histopathology showed a median survival extension to 26weeks in mice with 3–10% dystrophin, while mice with 10–30% lived even longer. Biomarkers, skeletal muscle and heart function, and histopathology were significantly improved in mice with 3–10% dystrophin and further improvement was achieved with 10–30% dystrophin. These results suggest that even very low dystrophin levels already may have beneficial effects, and that survival and improvement of endurance efforts may be amongst the early effects of treatment. This underscores the urgency to develop better clinical readouts for the non-ambulatory phase.

P1.44 Development of heart failure in mice with low dystrophin levels

MR data acquisition is performed on a Philips 3T Achieva, Siemens Verio, or Siemens TIM Trio system. Reproducibility of the MR measures have been evaluated for the soleus using MRI-T2 (Day 1: 43 ± 9 ms, Day 2: 44 ± 9 ms; CV 2.2 ± 1.9%), T2 of H2O from spectroscopic relaxometry (Day 1: 30.3 ± 2.6 ms, Day 2: 30.4 ± 2.9 ms; CV 2.1 ± 1.5%), and the ratio of lipid/(lipid + water) using H-spectroscopy (Day 1: 0.32 ± 0.23 ms, Day 2: 0.32 ± 0.22 ms; CV 4.8 ± 2.8%). In summary, the MR measures implemented in this multisite study are highly reproducible in children with DMD and controls. These noninvasive measures show promise for evaluating disease progression and treatment in DMD subjects, and are continuing to be evaluated in this multi-center study.