Screening of anti‐atrophic peptides by using photo‐cleavable peptide array and 96‐well scale contractile human skeletal muscle atrophy models

Abstract

Skeletal muscle atrophy is characterized by decreases in protein content, myofiber diameter, and contractile force generation. As muscle atrophy worsens the quality of life, the development of anti‐atrophic substances is desirable. In this study, we aimed to demonstrate a screening process for anti‐atrophic peptides using photo‐cleavable peptide array technology and human contractile atrophic muscle models. We developed a 96‐well system and established a screening process with less variability. Dexamethasone‐induced human atrophic tissue was constructed in the system. Eight peptides were selected from the literature and used for the screening of peptides for preventing the decrease of the contractile forces of tissues. The peptide QIGFIW, which showed preventive activity, was selected as the seed sequence. As a result of amino acid substitution, we obtained QIGFIQ as a peptide with higher anti‐atrophic activity. These results indicate that the combinatorial use of the photo‐cleavable peptide array technology and 96‐well screening system could comprise a powerful approach to obtaining anti‐atrophic peptides, and suggest that the 96‐well screening system and atrophic model represent a practical and powerful tool for the development of drugs/functional food ingredients.

l-Anserine Increases Muscle Differentiation and Muscle Contractility in Human Skeletal Muscle Cells

“…Microdevices for measuring the contractile force of the engineered skeletal muscle tissues were fabricated as reported previously. ,, Briefly, molds made of polytetrafluoroethylene were immersed in precured polydimethylsiloxane (SILPOT 184, Dow Corning Toray, Tokyo, Japan) and then placed in a vacuum chamber to remove air bubbles. After baking at 70 °C for 1 h, the molds were removed.…”

“…Skeletal muscle tissue was constructed on a microdevice as reported previously . Briefly, a microdevice was placed in a well of a 96-well plate.…”

“…Skeletal muscle tissue was constructed on a microdevice as reported previously. 20 Briefly, a microdevice was placed in a well of a 96-well plate. Next, 12 μL of a mixture containing a cell suspension and a hydrogel solution with a cell concentration of 2 × 10 6 cells/mL was poured into the microdevice and incubated at 37 °C for 30 min.…”

“…17−19 More recently, we developed a 96-well contractile force-measuring system with engineered human skeletal muscle tissues and were able to screen for antiatrophic peptides using this system. 20 In the present study, we evaluated the effects of L-anserine on human skeletal muscle cells. First, we examined the effects of L-anserine on the differentiation of skeletal muscle cells in two-dimensional (2D) culture by analyzing the myotube widths and gene expression levels.…”

“…In vitro studies using skeletal muscle cells are useful for directly evaluating the effects of functional materials on skeletal muscle, because unlike in in vivo studies, the effects of complex organ–organ interactions do not need to be considered. Although rodent-derived skeletal muscle cells have been widely used in in vitro studies, it is preferable to use human-derived skeletal muscle cells for evaluating functional materials for food ingredients and drugs, because cells from different species may respond differently to the materials. , Skeletal muscle tissues constructed using human skeletal muscle cells can be employed as a tool for evaluating functional materials in vitro. , In particular, the contractile force, which can be measured noninvasively, is considered to be a useful index for tissue characterization, because it reflects the complex interactions of multiple second messenger pathways and structural proteins. , We previously developed microdevices to measure the contractile force of engineered skeletal muscle tissues. − More recently, we developed a 96-well contractile force-measuring system with engineered human skeletal muscle tissues and were able to screen for antiatrophic peptides using this system …”