Fibrosis, which is the pathologic accumulation of extracellular matrix (ECM), is one of the main traits of Duchenne muscular dystrophy (DMD) and many other muscle pathologies. Fibrotic ECM differs from healthy ECM in its structural properties, specifically density, orientation, and cross-linking, often which resulting in a stiffer ECM compared to healthy muscle. Excessive stiffness of muscle the cause of muscle contractures that are common in DMD and other neuromuscular disorders. Similarly, we are also interested in the collagen architecture and structural differences between fibrotic muscle and healthy muscle. They hypothesis is that these aspects of collagen architecture are disrupted in fibrosis and responsible for the increase in stiffness, rather than simply the excess collagen present. The parent R01 investigates how ECM architecture is organized by FAPs and also directs FAP fate. Aim 3 of the parents R01 investigates how ECM architecture influences muscle passive mechanical stiffness in coordination with microdystrophin gene therapy. However, many aspects of ECM architecture may be dynamically altered with muscle stretch. As in tendon, we would predict that as tissue undergoes stretch the collagen fibers would align along the direction of stretch and once straightened contribute largely to passive force. However, integrated muscle mechanics with detailed muscle architecture that is necessary to understand how dynamic changes in muscle architecture dictate mechanical properties has not been demonstrated previously. This supplement would address that need by leveraging Gabriel’s technical expertise and equipment available to design a system that is capable of simultaneously measuring muscle mechanics while imaging muscle ECM architecture. This is an extension of the parent R01 through the additional of this technical capability, which would allow investigation of how deformable the ECM architecture is in the fibrotic condition of muscular dystrophy along with the extent to which microdystrophin gene therapy recovers matrix dynamic changes. The primary component of ECM architecture that is anticipated to change with stretch would be collagen alignment and straightness, however, the level of interactions and fiber diameter may also dynamically shift. The overall hypothesis is that collagen architecture is dynamically shifted by muscle stretch, but that these shifts are decreased in dystrophic muscle, and recovered with early treatment of microdystrophin gene therapy.