Collagen Content and Cross‐links Scale with Passive Stiffness in Dystrophic Mouse Diaphragm, but are not Altered with Administration of Collagen Cross‐linking Inhibitor Beta‐aminopropionitrile

Abstract

In fibrotic skeletal muscle, such as that seen in Duchenne Muscular Dystrophy (DMD), there is upregulation of extracellular matrix (ECM) material, primarily collagen, and a severe change in muscle mechanics that often leads to contracture. Collagen cross-links, which can make the ECM stiffer, are increased in fibrotic models, such as the mdx mouse model of DMD. The objective of this study was to test the effects of cross-link inhibitor beta-aminopropionitrile (BAPN) on skeletal muscle ECM of wildtype (wt) and mdx mice. We hypothesized that BAPN treatment would reduce the amount of collagen cross-links and passive stiffness in skeletal muscle. Wt (N=9) and mdx (N=13) mice were given daily BAPN or phosphate-buffered saline (PBS) injections starting at 16-19 weeks of age for 4 weeks up to sacrifice. Soleus (SOL), extensor digitorum longus (EDL), and diaphragm (DP) muscles were collected for muscle active and passive mechanical measurement and imaged for collagen alignment data. Muscle collagen content and cross-linking were analyzed via hydroxyproline and collagen solubility assay. Across genotypes and muscles, BAPN treatment had no significant effects on collagen content or cross-links. Mdx mice showed reduced specific tension across muscles as expected (Fig 1A). The mdx DP had markedly higher dynamic stiffness than wt (+149%), without genotype differences in EDL and SOL, but BAPN increased stiffness in the mdx SOL (+100%) (Fig 1B). Total collagen content was increased across muscles in mdx mice, with the most dramatic effect again in the DP (+210%) compared to EDL (+62%) and SOL (+30%) (Fig 1C). Mdx mice also had significantly greater collagen cross-linking in DP (+9.3%) and EDL (+38%) muscles as measured by collagen solubility (Fig 2A). The dynamic stiffness of DP muscles was significantly related to total collagen content (R2 = 0.25) (Fig 1D), and cross-linking index of solubility also significantly scaled with dynamic stiffness across the DP (R2=0.19) (Fig 2B). BAPN treatment increased orientation index, a measure for collagen alignment, in wt SOL (+162%), but did not have any other effects on alignment. Orientation index was significantly increased in mdx EDL and SOL compared to wt (EDL: +76%; SOL: +45%), but not in the DP (Fig 2C). Dynamic stiffness across all muscles was significantly related to orientation index (R2 = 0.06), but this relationship was not significant in the DP (Fig 2D). These data show that the relationship between passive muscle stiffness and collagen is muscle-dependent. The DP had the strongest relationships with more collagen and cross-linking related to increased stiffness across conditions while also being the most fibrotic muscle. However, collagen alignment in the DP was not related to stiffness, implicating other factors contributing to stiffness in fibrosis. Overall, administration of BAPN had little or no impact on muscle stiffness, collagen content, cross-linking, and collagen alignment. Thus, the delivery or action of BAPN was not effective in reducing collagen cross-links within muscle.