Background

Skeletal muscle is the largest human organ by mass and is essential for autonomous locomotion (Janssen et al., 2000). It is also the target of several degenerative genetic diseases collectively termed muscular dystrophies and myopathies. Recent improvements in genome sequencing have rapidly expanded the list of genes and gene variants causing muscle diseases, many of which have unknown or unconfirmed functions (Lek and MacArthur, 2014; Biancalana and Laporte, 2015; Angelini et al., 2018; Fichna et al., 2018). Besides the obvious desire to understand these novel disease-causing genes for therapeutic development, an exploration of their expression patterns and function, including cellular trafficking and localization, binding partners, and responses to various environmental stressors, is required to expand the current understanding of muscle cell biology. In vitro systems are convenient for the study of new gene products, as they consist of a single or defined mixture of cell types, are usually amenable to genetic manipulation (both knockout and overexpression), and deliver results quickly relative to in vivo systems. However, mature skeletal muscle fibers differ substantially from myotubes cultured in vitro in terms of size, structure, and three-dimensional organization (Dessauge et al., 2021). For example, nuclei regularly cluster in cultured myotubes but are separated into myonuclear domains in vivo. Furthermore, muscle in vivo consists primarily of contractile units (sarcomeres), which are interwoven with highly organized mitochondrial, t-tubule, and sarcoplasmic reticular systems, but typical muscle cell culture systems do not develop well-organized sarcomeres (Denes et al., 2019). These factors combine to influence expression, localization, and behavior of proteins in skeletal muscle fibers, which can differ substantively from what is observed in vitro (Deshmukh et al., 2015).

Historically, animal models (knockout or transgenic) have been the gold standard for probing protein function in vivo but are associated with substantial production costs and long characterization times. Another disadvantage of engineered mice is that a separate mouse is required for each protein of interest, and studying the behavior of several interacting proteins becomes daunting. One would like to use common in vitro tools, like fluorescent and biochemical tags, in vivo. Labeled proteins can be expressed via vectors in skeletal muscle, provided that the vector is able to enter the cell. Adeno-associated viral vectors can freely transduce muscle following intramuscular injection but are labor intensive to produce (Gregorevic et al., 2004; Qiao et al., 2011). Other viral vectors (e.g., lentivirus or retrovirus) can transduce stem cells in vitro, which can then be transplanted into muscle, but this is technically impractical in most cases (Li et al., 2005; Ousterout et al., 2015). A simpler approach is to transfect cells in vivo with plasmid DNA. This is efficiently accomplished through electroporation, a physical method for reversibly permeabilizing the cell membrane with strong electric field. Here, we describe a method for expressing tagged proteins in skeletal muscle fibers of the flexor digitorum brevis (FDB) muscle of the foot through direct intramuscular injection of plasmid DNA followed by electroporation. While this method is theoretically applicable to most skeletal muscles, the FDB possesses several advantages: it is small, accessible, and easily dissociable to generate a suspension of single muscle fibers for downstream analyses. This protocol provides a simple system for evaluating protein functions in skeletal muscle in vivo (Demonbreun and McNally, 2015; Foltz et al., 2021) with supplies that are attainable for most research labs.