Gait characterization in golden retriever muscular dystrophy dogs using linear discriminant analysis

Background: Accelerometric analysis of gait abnormalities in golden retriever muscular dystrophy (GRMD) dogs isof limited sensitivity, and produces highly complex data. The use of discriminant analysis may enable simpler andmore sensitive evaluation of treatment benefits in this important preclinical model.Methods: Accelerometry was performed twice monthly between the ages of 2 and 12 months on 8 healthy and20 GRMD dogs. Seven accelerometric parameters were analysed using linear discriminant analysis (LDA). Manipulationof the dependent and independent variables produced three distinct models. The ability of each model to detect gaitalterations and their pattern change with age was tested using a leave-one-out cross-validation approach.Results: Selecting genotype (healthy or GRMD) as the dependent variable resulted in a model (Model 1) allowing agood discrimination between the gait phenotype of GRMD and healthy dogs. However, this model was not sufficientlyrepresentative of the disease progression. In Model 2, age in months was added as a supplementary dependentvariable (GRMD_2 to GRMD_12 and Healthy_2 to Healthy_9.5), resulting in a high overall misclassification rate (83.2%).To improve accuracy, a third model (Model 3) was created in which age was also included as an explanatory variable.This resulted in an overall misclassification rate lower than 12%. Model 3 was evaluated using blinded data pertainingto 81 healthy and GRMD dogs. In all but one case, the model correctly matched gait phenotype to the actualgenotype. Finally, we used Model 3 to reanalyse data from a previous study regarding the effects ofimmunosuppressive treatments on muscular dystrophy in GRMD dogs. Our model identified significant effect ofimmunosuppressive treatments on gait quality, corroborating the original findings, with the added advantages ofdirect statistical analysis with greater sensitivity and more comprehensible data representation.Conclusions: Gait analysis using LDA allows for improved analysis of accelerometry data by applying adecision-making analysis approach to the evaluation of preclinical treatment benefits in GRMD dogs

Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy

Autosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy characterized by centrally located nuclei in muscle fibers. CNM results from mutations in the gene encoding dynamin 2 (DNM2), a large GTPase involved in endocytosis, intracellular membrane trafficking, and cytoskeleton regulation. We developed a knock-in mouse model expressing the most frequent DNM2-CNM mutation; i.e. the KI-Dnm2R465W model. Heterozygous (HTZ) KI-Dnm2 mice progressively develop muscle atrophy, impairment of contractile properties, histopathological abnormalities, and elevated cytosolic calcium concentration. Here, we aim at better characterizing the calcium homeostasis impairment in extensor digitorum longus (EDL) and soleus muscles from adult HTZ KI-Dnm2 mice. We demonstrate abnormal contractile properties and cytosolic Ca2+ concentration in EDL but not soleus muscles showing that calcium impairment is correlated with muscle weakness and might be a determinant factor of the spatial muscle involvement. In addition, the elevated cytosolic Ca2+ concentration in EDL muscles is associated with an increased sarcolemmal permeability to Ca2+ and releasable Ca2+ content from the sarcoplasmic reticulum. However, amplitude and kinetics characteristics of the calcium transient appear unchanged. This suggests that calcium defect is probably not a primary cause of decreased force generation by compromised sarcomere shortening but may be involved in long-term deleterious consequences on muscle physiology. Our results highlight the first pathomechanism which may explain the spatial muscle involvement occurring in DNM2-related CNM and open the way toward development of a therapeutic approach to normalize calcium content

TRPC3, but not TRPC1, as a good therapeutic target for standalone or complementary treatment of DMD

International audienceBackground Duchenne muscular dystrophy (DMD) is an X-linked inherited disease caused by mutations in the gene encoding dystrophin that leads to a severe and ultimately life limiting muscle-wasting condition. Recombinant adeno-associated vector (rAAV)-based gene therapy is promising, but the size of the full-length dystrophin cDNA exceeds the packaging capacity of a rAAV. Alternative or complementary strategies that could treat DMD patients are thus needed. Intracellular calcium overload due to a sarcolemma permeability to calcium (SPCa) increase is an early and critical step of the DMD pathogenesis. We assessed herein whether TRPC1 and TRPC3 calcium channels may be involved in skeletal muscle SPCa alterations and could represent therapeutic targets to treat DMD.Methods All experiments were conducted in the DMD mdx rat, an animal model that closely reproduces the human DMD disease. We measured the cytosolic calcium concentration ([Ca 2+ ] c ) and SPCa in EDL ( Extensor Digitorum Longus ) muscle fibers from age-matched WT and DMD mdx rats of 1.5 to 7 months old. TRPC1 and TRPC3 expressions were measured in the EDL muscles at both the mRNA and protein levels, by RT-qPCR, western blot and immunocytofluorescence analysis.Results As expected from the malignant hyperthermia like episodes observed in several DMD mdx rats, calcium homeostasis alterations were confirmed by measurements of early increases in [Ca 2+ ] c and SPCa in muscle fibers. TRPC3 and TRPC1 protein levels were increased in DMD mdx rats. This was observed as soon as 1.5 months of age for TRPC3 but only at 7 months of age for TRPC1. A slight but reliable shift of the TRPC3 apparent molecular weight was observed in DMD mdx rat muscles. Intracellular localization of both channels was not altered. We thus focused our attention on TRPC3. Application of Pyr10, a specific inhibitor of TRPC3, abolished the differences between SPCa values measured in WT and DMD mdx . Finally, we showed that a rAAV-microdystrophin based treatment induced a high microdystrophin expression but only partial prevention of calcium homeostasis alterations, skeletal muscle force and TRPC3 protein increase.Conclusions All together our results show that correcting TRPC3 channel expression and/or activity appear to be a promising approach as a single or as a rAAV-based complementary therapy to treat DMD

Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca2+ sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca2+ sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca2+ transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E′/A′, and higher E/E′ ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca2+ sensitivity, faster Ca2+ transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca2+ sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca2+ transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities