Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays

Abstract

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 (kcat; 8–9 s−1) and actin concentration for half maximal activity (KATPase; 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.

New paradigms in actomyosin energy transduction: Critical evaluation of non‐traditional models for orthophosphate release

“…We specifically proposed that the double mutation R243E/E466R in cardiac myosin II could be useful [89] and preferred over the single R243E mutation (disrupting the secondary Pi-site) because the double mutation is expected to preserve other aspects of actomyosin function. [89] We have found that both the R243E and R243E/E466R mutations are properly transfected into C2C12 cells by a new virus free method [90] that would allow convenient screening of a wide range of mutants. Both mutants also express well (Figure S2).…”

“…We have found that both the R243E and R243E/E466R mutations are properly transfected into C2C12 cells by a new virus free method [ 90 ] that would allow convenient screening of a wide range of mutants. Both mutants also express well (Figure S2).…”

Feasibility study for the use of gene electrotransfer and cell electrofusion as a single-step technique for the generation of activated cancer cell vaccines

“…Dim fluorescence of GFP in GFP hybrids is most likely the consequence of large cytoplasmic volume and high number of nuclei in a single cytoplasm which are interrupting normal cell function required for proper expression of GFP. The situation might be specific for artificialy obtained polynucleated cells as such situation was not observed in naturally polynucleated muscle cells (Velayuthan et al, 2023).…”

New paradigms in actomyosin energy transduction: critical evaluation of non-traditional models for orthophosphate release

“…We have found that both the R243E and R243E/E466R mutations are properly transfected into C2C12 cells by a new virus free method [76] that would allow convenient screening of a range of mutants. Both mutants also express well in these cells (Fig.…”