A robust split-luciferase-based cell fusion screening for discovering myogenesis-promoting molecules

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Skeletal muscle atrophy significantly impacts quality of life.
• Myogenesis-promoting chemicals are crucial for developing pharmaceuticals to treat muscle atrophy.
• Existing screening methods may lack robustness and quantitative accuracy.

Purpose of the Study:

• To develop and validate a robust, quantitative bioluminescence-based assay for screening myogenesis-promoting compounds.
• To identify novel myogenesis-promoting agents from a library of bioactive small molecules.
• To establish a high-throughput screening platform for myogenesis modulators.

Main Methods:

• Utilized two stable C2C12 myoblast cell lines engineered with split luciferase fragments and a DnaE intein indicator.
• Cell fusion during myogenesis triggers luciferase reconstitution, inducing measurable bioluminescence.
• Applied the assay to screen 1191 pharmacologically active small molecules using high-throughput screening.

Main Results:

• The bioluminescence intensity directly and quantitatively correlated with the extent of cell fusion and myogenesis.
• Identified two compounds, Imatinib and Doxazosin mesylate, as potent myogenesis-promoting agents.
• Demonstrated the assay's robustness and quantitative accuracy through simple luminescence measurements.

Conclusions:

• The developed bioluminescence assay provides a reliable method for evaluating myogenesis.
• Imatinib and Doxazosin mesylate are identified as promising candidates for skeletal muscle atrophy treatment.
• The assay system is highly adaptable for high-throughput screening of both myogenesis-promoting and inhibiting molecules.