Uremia Impedes Skeletal Myocyte Myomixer Expression and Fusogenic Activity: Implication for Uremic Sarcopenia.

In patients with chronic kidney disease (CKD), skeletal muscle mass and function are known to occasionally decline. However, the muscle regeneration and differentiation process in uremia has not been extensively studied. In mice with CKD induced by adenine-containing diet, the tibialis anterior muscle injured using a barium chloride injection method recovered poorly as compared to control mice. In the cultured murine skeletal myocytes, stimulation with indoxyl sulfate (IS), a representative uremic toxin, morphologically jeopardized the differentiation, which was counteracted by L-ascorbic acid (L-AsA) treatment. Transcriptome analysis of cultured myocytes identified a set of genes whose expression was down-regulated by IS stimulation but up-regulated by L-AsA treatment. Gene silencing of myomixer, one of the genes in the set, impaired myocyte fusion during differentiation. By contrast, lentiviral overexpression of myomixer compensated for a hypomorphic phenotype caused by IS treatment. The split-luciferase technique demonstrated that IS stimulation negatively affected early myofusion activity that was rescued by L-AsA treatment. Lastly, in mice with CKD compared with control mice, myomixer expression in the muscle tissue in addition to the muscle weight after the injury was reduced, both of which were restored with L-AsA treatment. Collectively, data showed that the uremic milieu impairs the expression of myomixer and impedes the myofusion process. Considering frequent musculoskeletal injuries in uremic patients, defective myocyte fusion followed by delayed muscle damage recovery could underlie their muscle loss and weakness.

Novel cell-based system to assay cell-cell fusion during myotube formation.

A live assay tool has been established to uncover the precise molecular mechanisms underlying complex cell fusion events in myoblasts. The novel cell-based assay, HiMy (HiBiT-based myoblast fusion), utilizes a recently developed split-luciferase technology. The assay successfully detected cell fusion in differentiating C2C12 myoblast cultures. This allowed us to measure mixing of the cytoplasm, which occurred several hours after the initiation of C2C12 differentiation. Unlike what was reported earlier, the fusion was detected a few hours after the initiation of differentiation. Thus, this assay is sensitive enough to monitor fusion events before they become detectable using conventional methods. Furthermore, a panel of laboratory compounds, including a variety of inhibitors of cellular enzymes or activities, were assayed using the HiMy assay. Lovastatin, a cholesterol biogenesis inhibitor, decreased HiMy activity by approximately 50%. In contrast, mevalonolactone, a precursor for cholesterol synthesis, increased fusion activity. These results confirmed the previous finding that the amount of cellular cholesterol positively correlates with the rate of myoblast fusion during myogenesis. These results indicate that the novel cell fusion assay is a quick, accurate, and robust method to monitor intercellular fusion events.