A Novel Functional In Vitro Model that Recapitulates Human Muscle Disorders

Abstract

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 Ca 2+ 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.

Calcium Mechanisms in Limb-Girdle Muscular Dystrophy with CAPN3 Mutations

“…Our group has recently found that mouse and human myotubes deficient for CAPN3 display decreased SERCA protein levels as well as impaired Ca 2+ reuptake into the SR [67,68]. Moreover, we also found reduced SERCA expression in muscle samples from LGMDR1 patients.…”

“…Although to date, these mechanisms are not entirely understood, there is solid evidence indicating that CAPN3 is a multifunctional protein. Different studies performed in animal models and human samples have shown that CAPN3 deficiency is associated with different features in the skeletal muscle such as oxidative damage [65,66], Ca 2+ dysregulation [67,68], sarcomere disorganization [69], mitochondrial abnormalities [66,70,71,72], abnormal muscle adaptation [73,74], and impaired muscle regeneration [71], which together would lead to inflammation, necrosis, fibrosis, atrophy, and progressive muscle degeneration, characteristic of LGMDR1 (Figure 2 and Figure 3). Indeed, patients in the early stages of the disease present an increased concentration of serum creatine kinase (CK), which is an unspecific hallmark of muscle damage [55,75].…”

“…Therefore, data from cellular models and muscle samples of patients with LGMDR1 need to be considered in order to unravel the molecular pathways involved in LGMDR1. During the last years, our group has been working on understanding these mechanisms using human biopsies as well as primary and immortalized human myotubes [67,68,104]. Below, we will review recent findings on pathogenic mechanisms associated with CAPN3 deficiency, with a particular focus on the involvement of Ca 2+ dysregulation, as summarized in Figure 5.…”

“…Interestingly, we have found that SERCA deficiency in CAPN3 knockdown myotubes resulted in increased resting intracellular [Ca 2+ ] in human myotubes, but not in mouse myotubes [67]. This is likely due to the fact that mouse muscle fibers have a higher Ca 2+ buffer capacity, since parvalbumin, a major cytosolic Ca 2+ buffer, is highly expressed in the mouse muscle [68]. In any case, SERCA mRNA levels were found unchanged in CAPN3 deficient samples, and therefore, we propose that CAPN3 is necessary to stabilize SERCA proteins and prevent their degradation, similarly to RyR1 and CaMKII.…”

Inhibition of MicroRNA 6937 Delays Photoreceptor and Vision Loss in a Mouse Model of Retinitis Pigmentosa

“…We used the non-radioactive cytotoxicity assay CytoTox 96 (Promega, Madison, WI, USA) following indications [ 38 ].…”