De novo revertant fiber formation and therapy testing in a 3D culture model of Duchenne muscular dystrophy skeletal muscle.

The biological basis of Duchenne muscular dystrophy (DMD) pathology is only partially characterized and there are still few disease-modifying therapies available, therein underlying the value of strategies to model and study DMD. Dystrophin, the causative gene of DMD, is responsible for linking the cytoskeleton of muscle fibers to the extracellular matrix beyond the sarcolemma. We posited that disease-associated phenotypes not yet captured by two-dimensional culture methods would arise by generating multinucleated muscle cells within a three-dimensional (3D) extracellular matrix environment. Herein we report methods to produce 3D human skeletal muscle microtissues (hMMTs) using clonal, immortalized myoblast lines established from healthy and DMD donors. We also established protocols to evaluate immortalized hMMT self-organization and myotube maturation, as well as calcium handling, force generation, membrane stability (i.e., creatine kinase activity and Evans blue dye permeability) and contractile apparatus organization following electrical-stimulation. In examining hMMTs generated with a cell line wherein the dystrophin gene possessed a duplication of exon 2, we observed rare dystrophin-positive myotubes, which were not seen in 2D cultures. Further, we show that treating DMD hMMTs with a ß1-integrin activating antibody, improves contractile apparatus maturation and stability. Hence, immortalized myoblast-derived DMD hMMTs offer a pre-clinical system with which to investigate the potential of duplicated exon skipping strategies and those that protect muscle cells from contraction-induced injury. STATEMENT OF SIGNIFICANCE: Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder that is caused by mutation of the dystrophin gene. The biological basis of DMD pathology is only partially characterized and there is no cure for this fatal disease. Here we report a method to produce 3D human skeletal muscle microtissues (hMMTs) using immortalized human DMD and healthy myoblasts. Morphological and functional assessment revealed DMD-associated pathophysiology including impaired calcium handling and de novo formation of dystrophin-positive revertant muscle cells in immortalized DMD hMMTs harbouring an exon 2 duplication, a feature of many DMD patients that has not been recapitulated in culture prior to this report. We further demonstrate that this "DMD in a dish" system can be used as a pre-clinical assay to test a putative DMD therapeutic and study the mechanism of action.

TGFß signaling curbs cell fusion and muscle regeneration.

Muscle cell fusion is a multistep process involving cell migration, adhesion, membrane remodeling and actin-nucleation pathways to generate multinucleated myotubes. However, molecular brakes restraining cell-cell fusion events have remained elusive. Here we show that transforming growth factor beta (TGFß) pathway is active in adult muscle cells throughout fusion. We find TGFß signaling reduces cell fusion, regardless of the cells' ability to move and establish cell-cell contacts. In contrast, inhibition of TGFß signaling enhances cell fusion and promotes branching between myotubes in mouse and human. Exogenous addition of TGFß protein in vivo during muscle regeneration results in a loss of muscle function while inhibition of TGFßR2 induces the formation of giant myofibers. Transcriptome analyses and functional assays reveal that TGFß controls the expression of actin-related genes to reduce cell spreading. TGFß signaling is therefore requisite to limit mammalian myoblast fusion, determining myonuclei numbers and myofiber size.