A Genotyping Method Combining Primer Competition PCR with HRM Analysis to Identify Point Mutations in Duchenne Animal Models

Dystrophin-null sapje zebrafish is an excellent model for better understanding the pathological mechanisms underlying Duchenne muscular dystrophy, and it has recently arisen as a powerful tool for high-throughput screening of therapeutic candidates for this disease. While dystrophic phenotype in sapje larvae can be easily detected by birefringence, zebrafish genotyping is necessary for drug screening experiments, where the potential rescue of larvae phenotype is the primary outcome. Genotyping is also desirable during colony husbandry since heterozygous progenitors need to be selected. Currently, sapje zebrafish are genotyped through techniques involving sequencing or multi-step PCR, which are often costly, tedious, or require special equipment. Here we report a simple, precise, cost-effective, and versatile PCR genotyping method based on primer competition. Genotypes can be resolved by standard agarose gel electrophoresis and high-resolution melt assay, the latter being especially useful for genotyping a large number of samples. Our approach has shown high sensitivity, specificity, and reproducibility in detecting the A/T point mutation in sapje zebrafish and the C/T mutation in the mdx mouse model of Duchenne. Hence, this method can be applied to other single nucleotide substitutions and may be further optimized to detect small insertions and deletions. Given its robust performance with crude DNA extracts, our strategy may be particularly well-suited for detecting single nucleotide variants in poor-quality samples such as ancient DNA or DNA from formalin-fixed, paraffin-embedded material.We thank Anna Wojtalewicz and Leire Escudero for technical help, and Dr. Javier Ruiz-Ederra for helpful discussions on HRM assay. This work was supported by funding from Health Institute Carlos III (ISCIII) and the European Regional Development Fund (ERDF/ESF, "Investing in your future"), Grant PI17/00676; from Duchenne Parent Project Spain (DPPE); from the Basque Government (2016111091); and from the University of the Basque Country UPV/EHU (PPGA19/58). H.L.-F. and L.M.-M. hold predoctoral fellowships from the University of the Basque Country, J.L.-E. holds a predoctoral fellowship from the Basque Government, and A.V.-I. holds a Ramon y Cajal contract funded by the Spanish Ministry of Economy and Competitivenes

A Novel Functional In Vitro Model that Recapitulates Human Muscle Disorders

Here, we aim to address the increasing need for a suitable human muscle in vitro model in order to advance in the knowledge of muscle pathophysiology and test novel therapies for muscle disorders. Our model is based on a simple 2D culture method that yields highly mature human myotubes under optimized environmental conditions. Culture conditions that produced functional and contractile human myotubes with an extended lifetime consisted in extracellular matrix overlay and addition of several trophic factors to the differentiation medium. In this work, we describe the generation of suitable models of muscular dystrophies (limb-girdle muscular dystrophy type 2A—LGMD2A and Duchenne) by silencing expression of key proteins in these myotubes. Western blot and immunocytochemical analyses demonstrated similar features between our knockdown human myotubes and dystrophic muscles in vivo, which support the general validity of our cellular models. We also found that both dystrophic models present higher resting cytosolic Ca2+ levels than controls, which support a common underlying deficit in calcium homeostasis. This novel human in vitro system would allow for high-throughput screening of new treatments for these muscular dystrophies as well as for other neuromuscular disorders. In addition, our model could be used to advance in our understanding of human skeletal muscle pathophysiology

Targeting the Ubiquitin-Proteasome System in Limb-Girdle Muscular Dystrophy With CAPN3 Mutations

[EN] LGMDR1 is caused by mutations in the CAPN3 gene that encodes calpain 3 (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Our previous findings show that CAPN3 deficiency leads to reduced SERCA levels through increased protein degradation. This work investigates the potential contribution of the ubiquitin-proteasome pathway to increased SERCA degradation in LGMDR1. Consistent with our previous results, we observed that CAPN3-deficient human myotubes exhibit reduced SERCA protein levels and high cytosolic calcium concentration. Treatment with the proteasome inhibitor bortezomib (Velcade) increased SERCA2 protein levels and normalized intracellular calcium levels in CAPN3-deficient myotubes. Moreover, bortezomib was able to recover mutated CAPN3 protein in a patient carrying R289W and R546L missense mutations. We found that CAPN3 knockout mice (C3KO) presented SERCA deficits in skeletal muscle in the early stages of the disease, prior to the manifestation of muscle deficits. However, treatment with bortezomib (0.8 mg/kg every 72 h) for 3 weeks did not rescue SERCA levels. No change in muscle proteasome activity was observed in bortezomib-treated animals, suggesting that higher bortezomib doses are needed to rescue SERCA levels in this model. Overall, our results lay the foundation for exploring inhibition of the ubiquitin-proteasome as a new therapeutic target to treat LGMDR1 patients. Moreover, patients carrying missense mutations in CAPN3 and presumably other genes may benefit from proteasome inhibition by rescuing mutant protein levels. Further studies in suitable models will be necessary to demonstrate the therapeutic efficacy of proteasome inhibition for different missense mutations.This research was funded by Diputación Foral de Gipuzkoa (AV-I, 2018-000117-01-B, 2019-00362-01-B); Fundación Gangoiti Barrera, Ministerio de Ciencia e Innovación (AV-I,PID 2020-119780RB-I00), the Basque Government (AV-I, ETORTEK-KK-2019/00093); the University of the Basque Country (AV-I, GIU20/057), and Instituto de Salud Carlos III, co-funded by European Regional Development Fund/ European Social Fund, “Investing in your future” (AV-I, PI17/00676; AL, PI17/01841). JL-E held a PhD fellowship from the Basque Government, LM-M holds a PhD fellowship from the UPV/EHU and GG holds a Juan de la Cierva- Incorporación 2019 contract funded by the Spanish Ministry of Science and Innovation

Characterization and pharmacological modulation of calcium handling proteins in Limb-Girdle Muscle Dystrophy type R1

Páginas 114-122 confidenciales. Tesis completa174 p.-- Tesis censurada 167 p.Limb-girdle muscle dystrophy type R1 (LGMDR1) is the most common form of limb-girdle muscle dystrophy, currently with no effective treatment. LGMDR1 is caused by mutations in the CAPN3 gene and is characterized by reduced mobility, making daily live activities challenging. CAPN3 gene encodes calpain 3 protein (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Although the main function of CAPN3 in skeletal muscle and the pathophysiological mechanisms underlying the disease remain to be clarified, previous studies suggest that dysregulation of calcium (Ca2+) homeostasis is involved in the pathogenic mechanisms of this muscular dystrophy. In fact, our previous data suggest that CAPN3 deficiency may trigger calcium handling proteins dysfunction such as Sarco-Endoplasmic Reticulum ATPase pumps (SERCA) due to its over-ubiquitination and its consequent exacerbated degradation by Ubiquitin-Proteasome System (UPS). In the present work, we have characterized SERCA expression in different human in vitro LGMDR1 models observing that CAPN3 dysfunction generates SERCA protein downregulation, leading to increase basal intracellular calcium levels. Moreover, CAPN3-deficient LHCN-M2 human myotubes show upregulated sarcoplasmic reticulum (SR) stress markers. We have targeted SERCA degradation inhibiting UPS by Bortezomib (BTZ) treatment, which recovers not only SERCA2 protein expression but also mutated CAPN3 expression, restoring at least its structural function, and consequently, normalizing resting cytosolic Ca2+ levels. Despite the mild phenotype observed in C3KO mouse model, SERCA protein expression is reduced particularly in the diaphragm, as in C3 null rats, which show slightly more severe phenotype. However, due to the general modest phenotype of LGMDR1 animal models, the narrow therapeutic window between effectiveness and toxicity of BTZ treatment, and the high susceptibility of the rat model to Bortezomib, SERCA protein expression is not restored in the treated animal models. In summary, further studies should focus on using other approaches to stabilize SERCA2 protein in the skeletal muscle, which ideally may also target the maintenance of mutant CAPN3. Furthermore, generation of new animal models with human-like CAPN3 mutations may be needed to analyse preservation of mutant CAPN3. These models would also help understand the different pathophysiological mechanisms underlying this disease and move forward in the search of new therapies

Characterization and pharmacological modulation of calcium handling proteins in Limb-Girdle Muscle Dystrophy type R1

Páginas 114-122 confidenciales. Tesis completa174 p.-- Tesis censurada 167 p.Limb-girdle muscle dystrophy type R1 (LGMDR1) is the most common form of limb-girdle muscle dystrophy, currently with no effective treatment. LGMDR1 is caused by mutations in the CAPN3 gene and is characterized by reduced mobility, making daily live activities challenging. CAPN3 gene encodes calpain 3 protein (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Although the main function of CAPN3 in skeletal muscle and the pathophysiological mechanisms underlying the disease remain to be clarified, previous studies suggest that dysregulation of calcium (Ca2+) homeostasis is involved in the pathogenic mechanisms of this muscular dystrophy. In fact, our previous data suggest that CAPN3 deficiency may trigger calcium handling proteins dysfunction such as Sarco-Endoplasmic Reticulum ATPase pumps (SERCA) due to its over-ubiquitination and its consequent exacerbated degradation by Ubiquitin-Proteasome System (UPS). In the present work, we have characterized SERCA expression in different human in vitro LGMDR1 models observing that CAPN3 dysfunction generates SERCA protein downregulation, leading to increase basal intracellular calcium levels. Moreover, CAPN3-deficient LHCN-M2 human myotubes show upregulated sarcoplasmic reticulum (SR) stress markers. We have targeted SERCA degradation inhibiting UPS by Bortezomib (BTZ) treatment, which recovers not only SERCA2 protein expression but also mutated CAPN3 expression, restoring at least its structural function, and consequently, normalizing resting cytosolic Ca2+ levels. Despite the mild phenotype observed in C3KO mouse model, SERCA protein expression is reduced particularly in the diaphragm, as in C3 null rats, which show slightly more severe phenotype. However, due to the general modest phenotype of LGMDR1 animal models, the narrow therapeutic window between effectiveness and toxicity of BTZ treatment, and the high susceptibility of the rat model to Bortezomib, SERCA protein expression is not restored in the treated animal models. In summary, further studies should focus on using other approaches to stabilize SERCA2 protein in the skeletal muscle, which ideally may also target the maintenance of mutant CAPN3. Furthermore, generation of new animal models with human-like CAPN3 mutations may be needed to analyse preservation of mutant CAPN3. These models would also help understand the different pathophysiological mechanisms underlying this disease and move forward in the search of new therapies

Calcium Mechanisms in Limb-Girdle Muscular Dystrophy with CAPN3 Mutations

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a rare disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness of shoulder, pelvic, and proximal limb muscles that usually appears in children and young adults and results in loss of ambulation within 20 years after disease onset in most patients. The pathophysiological mechanisms involved in LGMDR1 remain mostly unknown, and to date, there is no effective treatment for this disease. Here, we review clinical and experimental evidence suggesting that dysregulation of Ca2+ homeostasis in the skeletal muscle is a significant underlying event in this muscular dystrophy. We also review and discuss specific clinical features of LGMDR1, CAPN3 functions, novel putative targets for therapeutic strategies, and current approaches aiming to treat LGMDR1. These novel approaches may be clinically relevant not only for LGMDR1 but also for other muscular dystrophies with secondary calpainopathy or with abnormal Ca2+ homeostasis, such as LGMD2B/LGMDR2 or sporadic inclusion body myositis.This research was funded by Instituto de Salud Carlos III, co-funded by European Regional Development Fund/European Social Fund, "Investing in your future" (A.V.-I., PI17/00676; A.L.d.M., PI17/01841); the Basque Government (A.V.-I., 2016111091) and Diputacion Foral de Gipuzkoa (A.V.-I., 2018-000117-01-B and 2019-00362-01-B). A.V.-I. holds a Ramon y Cajal contract funded by the Spanish Ministry of Economy and Competitiveness, and J.L.-E. holds a PhD fellowship from the Basque Government