MyoScreen, a High-Throughput Phenotypic Screening Platform Enabling Muscle Drug Discovery

Abstract

Despite the need for more effective drug treatments to address muscle atrophy and disease, physiologically accurate in vitro screening models and higher information content preclinical assays that aid in the discovery and development of novel therapies are lacking. To this end, MyoScreen was developed: a robust and versatile high-throughput high-content screening (HT/HCS) platform that integrates a physiologically and pharmacologically relevant micropatterned human primary skeletal muscle model with a panel of pertinent phenotypic and functional assays. MyoScreen myotubes form aligned, striated myofibers, and they show nerve-independent accumulation of acetylcholine receptors (AChRs), excitation-contraction coupling (ECC) properties characteristic of adult skeletal muscle and contraction in response to chemical stimulation. Reproducibility and sensitivity of the fully automated MyoScreen platform are highlighted in assays that quantitatively measure myogenesis, hypertrophy and atrophy, AChR clusterization, and intracellular calcium release dynamics, as well as integrating contractility data. A primary screen of 2560 compounds to identify stimulators of myofiber regeneration and repair, followed by further biological characterization of two hits, validates MyoScreen for the discovery and testing of novel therapeutics. MyoScreen is an improvement of current in vitro muscle models, enabling a more predictive screening strategy for preclinical selection of the most efficacious new chemical entities earlier in the discovery pipeline process.

A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength

“…In vitro models of human skeletal muscle are generally incapable of studying muscle contraction, and often those that can are limited to doing so as an experimental endpoint owing to the need to remove tissues from the culture device so as to implement a force transducer for measurements 16,24 . MyoTACTIC micro-posts, as with a recently reported cell culture insert approach 35 , were designed to sustain hMMT long-term culture and to allow for non-invasive contractile force measurements of hMMTs in an easy and robust manner ( Fig.…”

“…Studies in animals 47,48,62 support a role for IGF-1 in muscle hypertrophy. Culture studies in which IGF-1 is delivered to mono-nucleated myogenic progenitors when exposed to differentiation conditions in 2D culture 16,63,64 , or to nascent myotubes in 2D 65,66 or 3D 17 culture also observed hypertrophic effects by immunohistological analysis. Interestingly, we did not find a change in the width of IGF-1 treated hMMT myotubes even at a dose reported by others to induce myotube hypertrophy 66 .…”

“…The non-invasive nature of our readouts is of critical importance since it enables longitudinal studies of drug effects on hMMTs at different time points. This challenge is faced by currently available methods where assessment of 3D tissue passive force 58 , stimulated 2D culture contractility 16 , or stimulated 3D tissue force 24,60 are implemented as endpoint assays. While there are certainly examples of 3D culture platforms that enable force measurement based on flexible-post deflection and image analysis 17,18,27,35,38 , we expect that the combined 96-well footprint, one step fabrication of a stand-alone device, and ease of use will be key elements to promote widespread adoption by other researchers.…”

“…Despite their ease of use and demonstrated predictive power in certain cases 8 , 2D models of skeletal muscle are ill-suited for in vitro studies of contractile myotubes 9 by failing to maintain structural integrity over long periods of time 10,11 , and yielding randomly oriented myotubes which limits their application for contractile force measurement [12][13][14][15] . A recent report combined 2D culture substrate micropatterning to more closely mimic the physiological environment, increase reproducibility, and provide an indirect method to assess the contractile capacity of myotubes 16 . This advancement enables scalability and high throughput drug discovery predictions.…”

Intensive Care Unit-Acquired Weakness: Not Just Another Muscle Atrophying Condition

“…that are amenable to iterative recombination for cellular and molecular analysis. The simplicity of investigations in a two-dimensional (2D) tissue culture plastic context lends to the domination of this approach for skeletal muscle drug screening efforts [ 211 ] and mechanism studies. Although differentiating human myogenic progenitors into multinucleated myotubes is straightforward in 2D culture, the cultures fail to mimic the natural tissue structure of adult human skeletal muscle tissue and lack the cell–cell and cell–extracellular environment interactions that exist in vivo [ 212 , 213 ].…”