Expression levels of sarcolemmal membrane repair proteins following prolonged exercise training in mice

Abstract

Membrane repair is a conserved cellular process, where intracellular vesicles translocate to sites of plasma membrane injury to actively reseal membrane disruptions. Such membrane disruptions commonly occur in the course of normal physiology, particularly in skeletal muscles due to repeated contraction producing small tears in the sarcolemmal membrane. Here, we investigated whether prolonged exercise could produce adaptive changes in expression levels of proteins associated with the membrane repair process, including mitsugumin 53/tripartite motif-containing protein 72 (MG53/TRIM72), dysferlin and caveolin-3 (cav3). Mice were exercised using a treadmill running protocol and protein levels were measured by immunoblotting. The specificity of the antibodies used was established by immunoblot testing of various tissue lysates from both mice and rats. We found that MG53/TRIM72 immunostaining on isolated mouse skeletal muscle fibers showed protein localization at sites of membrane disruption created by the isolation of these muscle fibers. However, no significant changes in the expression levels of the tested membrane repair proteins were observed following prolonged treadmill running for eight weeks (30 to 80 min/day). These findings suggest that any compensation occurring in the membrane repair process in skeletal muscle following prolonged exercise does not affect the expression levels of these three key membrane repair proteins.

Fig. 1

MG53 expression levels vary between different murine anatomical muscles [A) Whole tissue lysates were isolated from the indicated mouse tissues and then used for immunoblotting. MG53/TRIM72 bands of appropriate molecular weight (arrow) were observed in lysates from various anatomical muscles. Numbers provided (left) indicate the position of molecular weight markers; and (B) Whole tissue lysates of rat samples were used for immunoblotting for MG53/TRIM72 expression. A MG53/TRIM72 band of appropriate molecular weight (arrow) appears in rat skeletal (Gastroc) and cardiac muscle. Numbers provided (left) indicate molecular weight markers. Immunoblots for actin levels in tissue lysates were also provided as a control for loading]

Fig. 2

MG53 protein appears at sites of membrane disruption in isolated skeletal muscle fibers [(A) FDB muscle fibers were isolated from C57Bl/6 mice and immunostained for the localization of MG53/TRIM72 protein (right). When observing the sealed end of the isolated muscle fibers (left) concentration of MG53 protein was observed (arrow). Scale bar represents 10 µm; and (B) During FDB fiber isolation, there were membrane disruptions created in some fibers that could be observed in bright field microscopy (left). Such disruptions showed concentrations of MG53/TRIM72 immunostaining (right) at sites of membrane damage (arrowheads) as well as the resealed end of the fibers (arrows). Scale bar represents 10 µm]

Fig. 3

Expression levels of cell membrane repair associated proteins do not change following prolonged exercise [(A) Whole tissue lysates of soleus muscles from exercised (E) and sedentary (S) mice were used for immunoblot analysis of the protein levels of MG53/TRIM72, dysferlin and cav3. Both actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels in these tissue lysates were used for a loading control; (B, C) Densitometry measurements were used to determine the relative concentration of the proteins of interest in the tissue lysates. A bar graph represents the data for each group (n = 4 mice per group) relative to expression levels of GAPDH (B) or actin (C). Analysis using Student’s t-test did not show any statistically significant differences between the exercised and sedentary groups for the proteins tested]