Exploration of Detrimental Effect of AGEs on 3D Skeletal Muscle Model in Microphysiological System

Abstract

Skeletal muscle is vital for mobility and body support, and its deterioration significantly impacts the quality of life. Diabetes and aging contribute to skeletal muscle degeneration, with advanced glycation end-products (AGEs) accumulating in the body and adversely affecting muscle health [1]. AGEs-mediated signaling pathways lead to issues like muscular atrophy, degeneration, impaired regeneration, inflammation, and apoptosis. While the biochemical effects of AGEs on skeletal muscle are actively explored, there is a lack of mechanical assessment despite the muscles being essential mechanical components. Moreover, a three-dimensional (3D) in vitro skeletal muscle model to directly study the impact of AGEs has yet to be included. Previous studies made progress in understanding the impact of AGEs in vitro. However, their two-dimensional experiments were limited to mimicking in vivo conditions [2-4]. Establishing a robust 3D skeletal muscle model is essential for biochemical and mechanical analysis, shedding light on the mechanism of AGEs and facilitating anti-AGEs substance development. To address this, we introduced a skeletal muscle-on-a-chip system, suitable for modeling 3D skeletal muscle tissue and measuring contractile function triggered by electric pulse stimulation. To verify the toxicity of AGEs on skeletal muscle tissue, skeletal muscle cells were exposed to AGEs, which resulted in significant morphological and functional changes in skeletal muscle cells. Contractility assays using a microfluidic system showed a notable decrease in the contractile force of AGEs-treated muscle tissue, pointing to weakened strength and contractility. The response rate to electric pulse stimulation was also significantly reduced in the AGEs-treated group, confirming further functional impairment. These findings highlight the detrimental effects of AGEs on skeletal muscle morphology and function (Figure 3). To assess the biochemical effects of AGEs on skeletal muscle tissue, we conducted several assays. Initially, we stained F-actin and myosin heavy chain (MHC) for the skeletal muscle functionality and senescent marker SA β-gal, which increased with a longer duration of AGEs treatment (Figure 1). Western blot analysis revealed that AGEs had varying effects on protein expression depending on the timing and duration of treatment. Treating AGEs at the beginning of differentiation resulted in increased pRB, p53, and p21, along with unchanged MHC levels, suggesting a negative impact on muscle-specific protein synthesis indicating impaired differentiation and muscle senescence (Figure 2). Further, our RNA sequencing demonstrated the detrimental effects of AGEs on cell adhesion, Ca2+ channels, proliferation, and differentiation (Figure 5). To investigate the correlation between the effect of AGEs and fluorescence lifetime imaging microscopy (FLIM), we analyzed skeletal muscle tissue with and without AGEs treatment. FLIM revealed increased collagen and NADH intensity and changes in fluorescence lifetime for NADH and collagen in the AGEs-treated group, indicating alterations in metabolic state and redox status associated with detrimental effects of AGEs (Figure 4). Our groundbreaking 3D in vitro disease model, created with an organ-on-a-chip system, offers a unique opportunity to comprehensively investigate the impact of AGEs on skeletal muscle. This system is hoped to contribute to the understanding of involved mechanisms and paves the way for targeted anti-AGEs strategies.