Validation of DE50-MD dogs as a model for the brain phenotype of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD), a fatal musculoskeletal disorder, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin deficiency. Dog models of DMD represent key translational tools to study dystrophin biology and to develop novel therapeutics. However, characterization of dystrophin expression and function in the canine brain is lacking. We studied the DE50-MD canine model of DMD that has a missense mutation in the donor splice site of exon 50. Using a battery of cognitive tests, we detected a neurocognitive phenotype in DE50-MD dogs including reduced attention, problem-solving and exploration of novel objects. Through a combination of capillary immunoelectrophoresis, immunolabelling, qPCR and RNAScope in situ hybridization we show that regional dystrophin expression in the adult canine brain reflects that of humans, and that the DE50-MD dog lacks full length dystrophin (Dp427) protein expression but retains expression of the two shorter brain-expressed isoforms, Dp140 and Dp71. Thus, the DE50-MD dog is a translationally-relevant pre-clinical model to study the consequences of Dp427 deficiency in the brain and to develop therapeutic strategies for the neurological sequelae of DMD.

Clinical aspects, food management, and clinical evolution of a cat with hypertrophic muscular dystrophy

ABSTRACT: Hypertrophic feline muscular dystrophy (HFMD), rarely reported in the literature, is a disease caused by a hereditary recessive dystrophin deficiency linked to the X chromosome, mainly affecting young male cats. Here, we presented the clinical aspects, food management, and clinical evolution of a seven-year-old mixed-breed cat diagnosed with HFMD, having a primary history of progressive tongue protrusion.

SR Ca2+ handling in unbranched, immediately post-necrotic fast-twitch mdx fibres is similar to wt littermates

There is a lack of consensus in the literature regarding the effects of dystrophin deficiency on the Ca2+ handling properties of the sarcoplasmic reticulum (SR) in mdx mice, an animal model of Duchenne muscular dystrophy. One possible reason for this is that only a few studies control for the presence of branched fibres. Fibre branching, a consequence of degenerative-regenerative processes such as muscular dystrophy, has in itself a significant influence on the function of the SR. In our present study we attempt to detect early effects of dystrophin deficiency on SR Ca2+ handling by using unbranched fibres from the immediate post-necrotic stage in mdx mice (just regenerated following massive necrosis). Using kinetically-corrected Fura-2 fluorescence signals measured during twitch and tetanus, we analysed the amplitude, rise time and decay time of Δ[Ca2+]i in unfatigued and fatigued fibres. Decay was also resolved into SR pump and SR leak components. Fibres from mdx mice were similar in all respects to fibres from wt littermates apart from: (i) a longer rise time and slower rate of rise of [Ca2+]i during a tetanus; and (ii) a mitigation of the fall in Δ[Ca2+]i amplitude during the course of fatigue. Our findings suggest that the early effects of a loss of dystrophin on SR Ca2+ handling are only slight, and differ from the widely held view that there is significant Ca2+ pathology in mdx mice. It may be that Ca2+pathology is magnified by progressive branching and degeneration.

Orai1–STIM1 Regulates Increased Ca2+ Mobilization, Leading to Contractile Duchenne Muscular Dystrophy Phenotypes in Patient-Derived Induced Pluripotent Stem Cells

Ca2+ overload is one of the factors leading to Duchenne muscular dystrophy (DMD) pathogenesis. However, the molecular targets of dystrophin deficiency-dependent Ca2+ overload and the correlation between Ca2+ overload and contractile DMD phenotypes in in vitro human models remain largely elusive. In this study, we utilized DMD patient-derived induced pluripotent stem cells (iPSCs) to differentiate myotubes using doxycycline-inducible MyoD overexpression, and searched for a target molecule that mediates dystrophin deficiency-dependent Ca2+ overload using commercially available chemicals and siRNAs. We found that several store-operated Ca2+ channel (SOC) inhibitors effectively prevented Ca2+ overload and identified that STIM1–Orai1 is a molecular target of SOCs. These findings were further confirmed by demonstrating that STIM1–Orai1 inhibitors, CM4620, AnCoA4, and GSK797A, prevented Ca2+ overload in dystrophic myotubes. Finally, we evaluated CM4620, AnCoA4, and GSK7975A activities using a previously reported model recapitulating a muscle fatigue-like decline in contractile performance in DMD. All three chemicals ameliorated the decline in contractile performance, indicating that modulating STIM1–Orai1-mediated Ca2+ overload is effective in rescuing contractile phenotypes. In conclusion, SOCs are major contributors to dystrophin deficiency-dependent Ca2+ overload through STIM1–Orai1 as molecular mediators. Modulating STIM1–Orai1 activity was effective in ameliorating the decline in contractile performance in DMD.

Contraction-Induced Loss of Plasmalemmal Electrophysiological Function Is Dependent on the Dystrophin Glycoprotein Complex

Weakness and atrophy are key features of Duchenne muscular dystrophy (DMD). Dystrophin is one of the many proteins within the dystrophin glycoprotein complex (DGC) that maintains plasmalemmal integrity and cellular homeostasis. The dystrophin-deficient mdx mouse is also predisposed to weakness, particularly when subjected to eccentric (ECC) contractions due to electrophysiological dysfunction of the plasmalemma. Here, we determined if maintenance of plasmalemmal excitability during and after a bout of ECC contractions is dependent on intact and functional DGCs rather than, solely, dystrophin expression. Wild-type (WT) and dystrophic mice (mdx, mL172H and Sgcb−/− mimicking Duchenne, Becker and Limb-girdle Type 2E muscular dystrophies, respectively) with varying levels of dystrophin and DGC functionality performed 50 maximal ECC contractions with simultaneous torque and electromyographic measurements (M-wave root-mean-square, M-wave RMS). ECC contractions caused all mouse lines to lose torque (p<0.001); however, deficits were greater in dystrophic mouse lines compared to WT mice (p<0.001). Loss of ECC torque did not correspond to a reduction in M-wave RMS in WT mice (p=0.080), while deficits in M-wave RMS exceeded 50% in all dystrophic mouse lines (p≤0.007). Moreover, reductions in ECC torque and M-wave RMS were greater in mdx mice compared to mL172H mice (p≤0.042). No differences were observed between mdx and Sgcb−/− mice (p≥0.337). Regression analysis revealed ≥98% of the variance in ECC torque loss could be explained by the variance in M-wave RMS in dystrophic mouse lines (p<0.001) but not within WT mice (R2=0.211; p=0.155). By comparing mouse lines that had varying amounts and functionality of dystrophin and other DGC proteins, we observed that (1) when all DGCs are intact, plasmalemmal action potential generation and conduction is maintained, (2) deficiency of the DGC protein β-sarcoglycan is as disruptive to plasmalemmal excitability as is dystrophin deficiency and, (3) some functionally intact DGCs are better than none. Our results highlight the significant role of the DGC plays in maintaining plasmalemmal excitability and that a collective synergism (via each DGC protein) is required for this complex to function properly during ECC contractions.

Startle responses in Duchenne muscular dystrophy: a novel biomarker of brain dystrophin deficiency

Duchenne muscular dystrophy (DMD) is characterised by loss of dystrophin in muscle. Patients affected by DMD also have variable degree of intellectual disability and neurobehavioural co-morbidities. In contrast to muscle, in which a single full-length isoform (Dp427) is produced, multiple dystrophin isoforms are produced in the brain, and their deficiency accounts for the variability of CNS manifestations, with increased risk of comorbidities in patients carrying mutations affecting the 3’ end of gene, disrupting the shorter Dp140 and Dp71 isoforms. The mdx mouse model of DMD lacks Dp427 in muscle and CNS and exhibits exaggerated startle responses to threat, linked to the deficiency of dystrophin in limbic structures such as the amygdala, which normalise with postnatal brain dystrophin-restoration therapies. A pathological startle response is not a recognised feature of DMD, and its characterisation has implications for improved clinical management and translational research.To investigate startle responses in DMD, we used a novel fear-conditioning task in an observational study of 56 males aged 7-12 years (31 DMD, mean age 9.7±1.8 years; 25 Controls, mean age 9.6±1.4 years). Trials of two neutral visual stimuli were presented to participants: one ‘safe’ cue presented alone; one ‘threat’ cue paired with an aversive noise to enable conditioning of physiological startle responses (skin conductance response, SCR; heart rate, HR). Retention of conditioned physiological responses was subsequently tested with presentation of both cues without the aversive noise in an ‘extinction’ phase. Primary outcomes were the magnitude of the initial unconditioned SCR and HR change responses to the aversive ‘threat’ and acquisition and retention of conditioned responses after conditioning. Secondary outcomes were neuropsychological measures and genotype associations.The initial (unconditioned) mean SCR to threat was greater in DMD than Controls (Mean difference 3.0 µS (95% CI 1.0, 5.1), P=.004), associated with a significant threat-induced bradycardia only in the DMD group (mean difference -5.6 bpm (95% CI 0.51, 16.9); P=.04). DMD participants found the task more aversive than Controls, consequently early termination during the extinction phase occurred in 26% of the DMD group (vs. 0% Controls; P=.007).This study provides the first evidence that boys with DMD show increased unconditioned startle responses to threat, similar to the mdx mouse phenotype that also responds to brain dystrophin restoration. Our study provides new insights into the neurobiology underlying the complex neuropsychiatric co-morbidities in DMD and defines an objective measure of this CNS phenotype, which will be valuable for future CNS-targeted dystrophin-restoration studies.

Indices of Defective Autophagy in Whole Muscle and Lysosome Enriched Fractions From Aged D2-mdx Mice

Duchenne muscular dystrophy (DMD) is a fatal, progressive muscle disease caused by the absence of functional dystrophin protein. Previous studies in mdx mice, a common DMD model, identified impaired autophagy with lysosomal insufficiency and impaired autophagosomal degradation as consequences of dystrophin deficiency. Thus, we hypothesized that lysosomal abundance would be decreased and degradation of autophagosomes would be impaired in muscles of D2-mdx mice. To test this hypothesis, diaphragm and gastrocnemius muscles from 11 month-old D2-mdx and DBA/2J (healthy) mice were collected. Whole muscle protein from diaphragm and gastrocnemius muscles, and protein from a cytosolic fraction (CF) and a lysosome-enriched fraction (LEF) from gastrocnemius muscles, were isolated and used for western blotting. Initiation of autophagy was not robustly activated in whole muscle protein from diaphragm and gastrocnemius, however, autophagosome formation markers were elevated in dystrophic muscles. Autophagosome degradation was impaired in D2-mdx diaphragms but appeared to be maintained in gastrocnemius muscles. To better understand this muscle-specific distinction, we investigated autophagic signaling in CFs and LEFs from gastrocnemius muscles. Within the LEF we discovered that the degradation of autophagosomes was similar between groups. Further, our data suggest an expanded, though impaired, lysosomal pool in dystrophic muscle. Notably, these data indicate a degree of muscle specificity as well as model specificity with regard to autophagic dysfunction in dystrophic muscles. Stimulation of autophagy in dystrophic muscles may hold promise for DMD patients as a potential therapeutic, however, it will be critical to choose the appropriate model and muscles that most closely recapitulate findings from human patients to further develop these therapeutics.

Dystrophin deficiency disrupts muscle clock expression and mitochondrial quality control in mdx mice

Impaired oxidative capacity and mitochondrial function contribute to the dystrophic pathology in muscles of Duchenne muscular dystrophy (DMD) patients and in relevant mouse models of the disease. Emerging evidence suggests an association between disrupted core clock expression and mitochondrial quality control, but this has not been established in muscles lacking dystrophin. We examined the diurnal regulation of muscle core clock and mitochondrial quality control expression in dystrophin-deficient C57BL/10ScSn-Dmdmdx (mdx) mice, an established model of DMD. Male C57BL/10 (BL/10; n=18) and mdx mice (n=18) were examined every 4 hours beginning at the dark cycle. Throughout the entire light-dark cycle, extensor digitorum longus (EDL) muscles from mdx mice had decreased core clock mRNA expression (Arntl, Cry1, Cry2, Nr1d2; p<0.05) and disrupted mitochondrial quality control mRNA expression related to biogenesis (decreased; Ppargc1a, Esrra; p<0.05), fission (increased; Dnm1l; p<0.01), fusion (decreased; Opa1, Mfn1; p<0.05) and autophagy/mitophagy (decreased: Bnip3; p<0.05; increased: Becn1; p<0.05). Cosinor analysis revealed a decrease in the rhythmicity parameters mesor and amplitude for Arntl, Cry1, Cry2, Per2, and Nr1d1 (p<0.001) in mdx mice. Diurnal oscillations in Esrra, Sirt1, Map1lc3b and Sqstm1 were absent in mdx mice, along with decreased mesor and amplitude of Ppargc1a mRNA expression (p<0.01). The expression of proteins involved in mitochondrial biogenesis (decreased: PPARGC1A, p<0.05) and autophagy/mitophagy (increased: MAP1LC3BII, SQSTM1, BNIP3; p<0.05) were also dysregulated in tibialis anterior muscles of mdx mice. These findings suggest that dystrophin deficiency in mdx mice impairs the regulation of the core clock and mitochondrial quality control, with relevance to DMD and related disorders.