Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation

Abstract

Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ∼80% from pre-EP levels. Within 3 h, torque recovered to ∼50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ∼25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ∼20% and ∼70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.

Myofiber Damage Precedes Macrophage Infiltration after in Vivo Injury in Dysferlin-Deficient A/J Mouse Skeletal Muscle

“…17, 25 We stained 8-mm cross sections of unfixed, snap-frozen TA muscle with Harris modified hematoxylin (HHS 16; SigmaAldrich Corporation, St. Louis, MO) and aqueous eosin-Y (HT110216; Sigma-Aldrich) and observed the sections under a light microscope (Zeiss Axioskop, Â20 objective; Carl Zeiss, Oberkochen, Germany). We studied 9 or 10 unique visual fields per muscle in three muscles per time point (uninjured, at approximately 10 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours).…”

“…Sections were collected onto slides (15-188-48; Fisher Scientific, Pittsburgh, PA), fixed for 10 minutes with 4% paraformaldehyde in phosphatebuffered saline and labeled as described earlier except that primary antibodies were omitted. 18,25 We sectioned and labeled two muscles per time point and studied six visual fields per muscle (Zeiss Axioskop, Â20 objective; Carl Zeiss). We then combined the numbers of mouse IgG-positive (MsIgG þ ) fibers from each of the fields to yield a sample size of n Z 12 fields per time point.…”

“…18,25 Briefly, 16-mm cross sections were labeled with either of these antibodies in addition to antibodies to dystrophin to mark the boundaries of myofibers and with DAPI to label nuclei. Slides were subsequently incubated with biotinylated goat anti-rat secondary antibodies (A10517; Life Technologies), which were in turn labeled with streptavidin conjugated to Alexa 568 dye (S-11226; Life Technologies).…”

DNA Electroporation, Isolation and Imaging of Myofibers

“…However, to allow more complete recovery after electroporation, we prefer isolation and study of myofibers 7–14 days after electroporation 10 . Expression can be assayed as long as 4 weeks after electroporation.…”

“…Transgenesis can be employed to establish expression of tagged proteins, but this method is costly and time consuming. In vivo electroporation of mouse muscle has emerged as a preferred method for its speed and reliability 6–10 .…”