[2S06m] 15. Advanced Cell and Cell Culture Engineering for Biopharmaceuticals and Functional Food - Part 4

11:10 AM - 11:30 AM JST(2:10 AM - 2:30 AM UTC)

[2ACCE-05] AI-based quantification of myotube contractility and sarcomere maturation in 2D cultures

○HyeonJun Choe¹, Minami Yamamoto¹, Masamichi Kamihira¹ (1. Kyushu university (Japan))

Keywords:

Skeletal muscle,Myotube contraction,AI-based image analysis,Sarcomere organization,Imidazole dipeptides

[Background]
Accurate and scalable assessment of skeletal muscle function is essential for drug discovery and for understanding the mechanisms underlying muscle degeneration and aging. While three-dimensional (3D) muscle constructs enable direct measurement of contractile force, their low throughput and experimental complexity limit broader application. Two-dimensional (2D) myotube cultures provide a scalable alternative; however, quantitative evaluation of contractile behavior and structural maturation remains challenging because conventional analyses often rely on manual and subjective assessments. This study aimed to develop an automated analytical framework to quantify myotube contractility and structural characteristics in 2D cultures.
[Method]
An AI-based analysis system, the Myotube Contraction Analysis System (MyoCAS), was developed to automatically quantify displacement generated by myotube contraction in 2D time-lapse microscopy. The system tracks myotube motion on a frame-by-frame basis to calculate maximum contractile displacement. In parallel, the number of spatially distinct regions exhibiting displacement was quantified to evaluate the spatial extent of contractile activity.To investigate the structural basis of contractile activity, sarcomere organization was analyzed using deep learning–based segmentation and classified into premyofibril, nascent myofibril, and mature myofibril stages. In addition, myotube regions were segmented to quantify myotube area, enabling evaluation of muscle hypertrophy and differentiation independent of contractile measurements. Using this analytical framework, the effects of the imidazole dipeptides carnosine, anserine, and homocarnosine were evaluated across a range of concentrations.
[Results]
Treatment with testosterone increased contractile displacement, whereas dexamethasone decreased displacement, demonstrating that MyoCAS sensitively detects pharmacological modulation of myotube contractility. Treatment with imidazole dipeptides also increased contractile displacement. Structural analysis further indicated that increased displacement was associated with more advanced sarcomere organization. In addition, myotube area and sarcomere organization enabled quantitative evaluation of morphological changes associated with muscle growth and structural maturation.
[Discussion]
These findings suggest that contractile displacement quantified by MyoCAS reflects the underlying sarcomere organization and structural maturation of myotubes. The integration of AI-based motion analysis with deep learning–based structural and morphological analyses provides a comprehensive approach to evaluating both functional and structural characteristics of skeletal muscle in 2D culture systems.
[Conclusion]
MyoCAS provides a robust and scalable platform for integrated functional and structural phenotyping of skeletal muscle. This system enables high-content analysis of myotube contractility, sarcomere organization, and myotube morphology, offering a useful framework for muscle biology studies and compound screening in 2D myotube cultures.