Glycations on Decellularized Muscle Matrix Reduce Muscle Regeneration and Increase Inflammation.

Abstract

Volumetric muscle loss (VML) due to traumatic injury results in the abrupt loss of contractile units, stem cells, and connective tissue, leading to long-term muscle dysfunction and reduced regenerative potential. Muscle connective tissue contains a proregenerative extracellular matrix (ECM), and our lab harnesses the regenerative capacity of decellularized muscle matrix (DMM) to treat VML, a condition with limited treatment options. However, a major limitation is that muscle often comes from aged donors. Previous work from our lab showed that aged donor muscle contains higher levels of advanced glycation end-product (AGE) cross-links compared to muscle from younger donors. This study aimed to determine whether increased AGE cross-links reduce the regenerative capacity of DMM. To test this, we first generated AGEs in DMM with direct D-ribose incubation. We then removed ∼35% of the gastrocnemius muscle in a model and treated it with either AGE-DMM or standard DMM (no AGEs), comparing results to controls. Although muscle force results remained unchanged between AGE-DMM and DMM, AGEs led to reduced muscle mass in histological sections, fewer fibers, and smaller fiber diameters. AGEs also increased collagen levels in histology, but protein assays showed reduced collagen production. We investigated the canonical receptor for AGEs, the receptor for AGEs (RAGE), and found elevated levels in AGE-treated VML compared to DMM alone, along with increased levels of the noncanonical receptor galectin-3. Both RAGE and galectin-3 are associated with inflammation, and proteomics revealed higher inflammatory markers in AGE-treated muscle than in DMM alone. In conclusion, our data suggest that AGEs impair the regenerative potential of DMM, highlighting the importance of considering donor age when sourcing muscle for DMM therapies. Impact Statement This study investigates advanced glycation end-product cross-links in skeletal muscle extracellular matrix (ECM) as a way to model its deleterious effects on muscle regeneration in vivo. We demonstrate here that ECM glycations reduce muscle regeneration, enhance inflammatory markers, reduce ECM protein production, and proteomic analysis identified unique targets that could be explored in future research endeavors.