The extracellular matrix of dystrophic mouse diaphragm accounts for the majority of its passive stiffness and is resistant to collagenase digestion.

The healthy skeletal muscle extracellular matrix (ECM) has several functions including providing structural integrity to myofibers, enabling lateral force transmission, and contributing to overall passive mechanical properties. In diseases such as Duchenne Muscular dystrophy, there is accumulation of ECM materials, primarily collagen, which results in fibrosis. Previous studies have shown that fibrotic muscle is often stiffer than healthy muscle, in part due to the increased number and altered architecture of collagen fibers within the ECM. This would imply that the fibrotic matrix is stiffer than the healthy matrix. However, while previous studies have attempted to quantify the extracellular contribution to passive stiffness in muscle, the outcomes are dependent on the type of method used. Thus, the goals of this study were to compare the stiffness of healthy and fibrotic muscle ECM and to demonstrate the efficacy of two methods for quantifying extracellular-based stiffness in muscle, namely decellularization and collagenase digestion. These methods have been demonstrated to remove the muscle fibers or ablate collagen fiber integrity, respectively, while maintaining the contents of the extracellular matrix. Using these methods in conjunction with mechanical testing on wildtype and D2. mice, we found that a majority of passive stiffness in the diaphragm is dependent on the ECM, and the D2. diaphragm ECM is resistant to digestion by bacterial collagenase. We propose that this resistance is due to the increased collagen cross-links and collagen packing density in the ECM of the D2. diaphragm. Taken altogether, while we did not find increased stiffness of the fibrotic ECM, we did observe that the D2. diaphragm conveyed resistance against collagenase digestion. These findings demonstrate how different methods for measuring ECM-based stiffness each have their own limitations and can produce different results.