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.
Over the last few years, there has been growing interest in measuring the contractile force (CF) of engineered muscle tissues to evaluate their functionality. However, there are still no standards available for selecting the most suitable experimental platform, measuring system, culture protocol, or stimulation patterns. Consequently, the high variability of published data hinders any comparison between different studies. We have identified that cantilever deflection, post deflection, and force transducers are the most commonly used configurations for CF assessment in 2D and 3D models. Additionally, we have discussed the most relevant emerging technologies that would greatly complement CF evaluation with intracellular and localized analysis. This review provides a comprehensive analysis of the most significant advances in CF evaluation and its critical parameters. In order to compare contractile performance across experimental platforms, we have used the specific force (sF, kN/m2), CF normalized to the calculated cross-sectional area (CSA). However, this parameter presents a high variability throughout the different studies, which indicates the need to identify additional parameters and complementary analysis suitable for proper comparison. We propose that future contractility studies in skeletal muscle constructs report detailed information about construct size, contractile area, maturity level, sarcomere length, and, ideally, the tetanus-to-twitch ratio. These studies will hopefully shed light on the relative impact of these variables on muscle force performance of engineered muscle constructs. Prospective advances in muscle tissue engineering, particularly in muscle disease models, will require a joint effort to develop standardized methodologies for assessing CF of engineered muscle tissues.
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