Isolation of Sarcolemmal Plasma Membranes by Mechanically Skinning Rat Skeletal Muscle Fibers for Phospholipid Analysis

Abstract

Membrane phospholipid (PL) composition has been shown to affect cellular function by altering membrane physical structure. The sarcolemma plasma membrane (SLpm) is integral to skeletal muscle function and health. Previous studies assessing SLpm PL composition have demonstrated contamination from transverse (t)-tubule, sarcoplasmic reticulum, and nuclear membranes. This study assessed the possibility of isolating SL by mechanically skinning skeletal muscle fiber segments for the analysis of SLpm PL composition. Mechanically skinned SLpm from rat extensor digitorum longus (EDL) muscle fibers underwent Western blot analysis to assess contamination from t-tubule, sarcoplasmic reticulum, nuclear and mitochondrial membranes. The results indicate that isolated SLpm had minimal nuclear and mitochondrial membrane contamination and was void of contamination from sarcoplasmic reticulum and t-tubule membranes. After performing both high-performance thin layer chromatography and gas chromatography, we found that the SLpm obtained by mechanical skinning had higher sphingomyelin and total fatty acid saturation and lower phosphatidylcholine when compared to previous literature. Thus, by avoiding the use of various chemical treatments and membrane fractionation, we present data that may truly represent the SLpm and future studies can use this technique to assess potential changes under various perturbations and disease conditions such as insulin resistance and muscular dystrophy.

Higher PLIN5 but not PLIN3 content in isolated skeletal muscle mitochondria following acute in vivo contraction in rat hindlimb

“…In addition, an appropriate marker for sarcolemma has yet to be identified (Fajardo et al. ). However, this is likely not a concern for our study because we were not able to detect any PLIN3 or PLIN5 content in isolated sarcolemmal cuffs either at rest or with contraction.…”

“…Sarcolemmal cuffs were isolated as previously described (Fajardo et al. ). Briefly, fibers were isolated using microdissecting forceps and visualized with a dissecting microscope (Nikon SMZ645 with a Nikon 3002752 objective and Nikon C‐W10 9 A/22 eyepiece).…”

“…Curved platinum electrical wires were attached to the sciatic nerve for stimulation with the left leg being stimulated, whereas the right remained as a resting control. Upon completion of the stimulated contraction, the plantaris muscle (n = 5) was removed and prepared for mechanical sarcolemma isolation (Fajardo et al 2013). Red gastrocnemius muscles were then excised and divided as follows; the belly of the muscle was cut and set in embedding compound (Cryomatrix, Pittsburgh, PA) and cooled in methyl-butane for 90 sec before being stored at À80°C.…”

“…As a precaution, sarcolemmal cuffs were collected from the plantaris muscle to determine if PLIN3 and/or PLIN5 content were present in the sarcolemma and/or if it changed with contraction as seen with PLIN1B and insulin stimulation in human adipocytes (Aboulaich et al 2006). Sarcolemmal cuffs were isolated as previously described (Fajardo et al 2013). Briefly, fibers were isolated using microdissecting forceps and visualized with a dissecting microscope (Nikon SMZ645 with a Nikon 3002752 objective and Nikon C-W10 9 A/22 eyepiece).…”

Co-Expression of SERCA Isoforms, Phospholamban and Sarcolipin in Human Skeletal Muscle Fibers

“…Due to the small volume used to store single fibers (each in 12µl total volume), protein concentration prior to Western blot analysis was not determined, and no analysis of other MHCII isoforms was performed. Tricine SDS-PAGE electrophoresis was performed on gradient gels for efficient separation of all proteins on one gel [26]. Specifically, a 13% separating gel was overlaid with a layer of 6.6% separating gel and topped with 4% stacking gel.…”