Key result
A virus-free transfection method using JetPrime in C2C12 cells successfully produced functional human cardiac myosin with gliding velocities of 1.5-1.8 µm/s, similar to virus-based methods.
Why the study?
Expressing striated muscle myosin II heavy chain standardly relies on viral infection in C2C12 cells, which is work-intensive, hazardous, and limits fast generation of mutations.
A novel virus-free transfection method enables efficient expression and purification of functional human cardiac myosin in mammalian cells, simplifying the study of myosin mutations.
Enables safer in vitro study of myosin mutations; leaves open translation to disease models or therapies.
Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5–1.7 µm/s; 25 °C) in the in vitro motility assay as well as maximum actin activated catalytic activity (k cat ; 8–9 s −1 ) and actin concentration for half maximal activity (K ATPase ; 70–80 µM) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.
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Velayuthan et al. (2023) studied this question. Virus-free transfection (JetPrime) was evaluated on Actin filament gliding velocity. A virus-free transfection method using JetPrime in C2C12 cells successfully produced functional human cardiac myosin with gliding velocities of 1.5-1.8 µm/s, similar to virus-based methods.
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