Immortalized human myotonic dystrophy type 1 muscle cell lines to address patient heterogeneity.

Historically, cellular models have been used as a tool to study myotonic dystrophy type 1 (DM1) and the validation of therapies in said pathology. However, there is a need for in vitro models that represent the clinical heterogeneity observed in patients with DM1 that is lacking in classical models. In this study, we immortalized three DM1 muscle lines derived from patients with different DM1 subtypes and clinical backgrounds and characterized them at the genetic, epigenetic, and molecular levels. All three cell lines display DM1 hallmarks, such as the accumulation of RNA foci, MBNL1 sequestration, splicing alterations, and reduced fusion. In addition, alterations in early myogenic markers, myotube diameter and CTCF1 DNA methylation were also found in DM1 cells. Notably, the new lines show a high level of heterogeneity in both the size of the CTG expansion and the aforementioned molecular alterations. Importantly, these immortalized cells also responded to previously tested therapeutics. Altogether, our results show that these three human DM1 cellular models are suitable to study the pathophysiological heterogeneity of DM1 and to test future therapeutic options.

A Ca2+-Dependent Mechanism Boosting Glycolysis and OXPHOS by Activating Aralar-Malate-Aspartate Shuttle, upon Neuronal Stimulation.

Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na+ and Ca2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca2+-dependent way. We find that Ca2+ upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca2+ binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca2+ uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+ flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+ release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca2+ is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca2+-dependent way, independently of effects of Ca2+ as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca2+ stimulation is blunt in the absence of Aralar, a Ca2+-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca2+-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD+/NADH ratio.

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

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.

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.

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.