Degradation of Proteins in Human Skeletal Muscle. Characterization of the Biopsy System and Its Application to Patients Undergoing Abdominal Surgery.

Abstract

In order to study the degradation of skeletal muscle proteins during basal conditions and following surgery of moderate and major severity, we examined the proteinase activities in skeletal muscle tissue. Proteinase activities were analyzed in biopsies (30 to 60mg) taken from skeletal muscle of healthy human volunteers and from patients undergoing abdominal surgery. Acid and neutral proteinase activities toward the added substrate, hemoglobin, and autolytic degradation of intrinsic proteins were measured by analysis of the tyrosine released during incubation. Acid proteinase activity in the presence of added substrate was inhibited by pepstatin to 99% and autolytic degradation to 75%, indicating the presence of the lysosomal enzyme cathepsin D (EC 3.4.23.5) in human skeletal muscle. Storage of the biopsies prior to analysis should not exceed 3 weeks at -80°C. Reproducibility of the assay for specimens from a single individual was high, with an error of ±3%. Among the proteins degraded intrinsically at acid pH myosin heavy chain was analyzed by immunoblotting. The protein was one of the substrates of the pepstatin-insensitive pathways of protein degradation at acid pH. As neutral proteinases were lower in activity than acid proteinases, the studies on patients focused on acid proteinase activities. The analysis was applied to biopsies taken from patients undergoing abdominal surgery. Following cholecystectomy, classified as moderately severe surgery, the rate of acid proteinase activity per mg wet weight of muscle decreased significantly (p<0.05) at 24h and was marginally significantly decreased at 72h after surgery (p=0.075). Per mg of protein the values were 94.5±7.5% and 96.5±5.0% for 24 and 72h, respectively. Per mg of DNA a significant decrease (p<0.05) was noted at 24h after surgery. Autolytic degradation decreased (p<0.05) at 24h after moderately severe surgical trauma. Following major abdominal surgery acid proteinases without or with added substrate increased in activity. In the absence of added substrate the peak was at 6h, and in its presence, at 24h, following surgery. The activity of the acid proteinases appears to be a useful tool when protein metabolic events are studied in biopsy specimens of human skeletal muscle.