Growth associated protein 43 is expressed in skeletal muscle fibers and is localized in proximity of mitochondria and calcium release units

Abstract

The neuronal Growth Associated Protein 43 (GAP43), also known as B-50 or neuromodulin, is involved in mechanisms controlling pathfinding and branching of neurons during development and regeneration. For many years this protein was classified as neuron-specific, but recent evidences suggest that a) GAP43 is expressed in the nervous system not only in neurons, but also in glial cells, and b) probably it is present also in other tissues. In particular, its expression was revealed in muscles from patients affected by various myopathies, indicating that GAP43 can no-longer considered only as a neuron-specific molecule. We have investigated the expression and subcellular localization of GAP43 in mouse satellite cells, myotubes, and adult muscle (extensor digitorum longus or EDL) using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy. Our in vitro results indicated that GAP43 is indeed expressed in both myoblasts and differentiating myotubes, and its cellular localization changes dramatically during maturation: in myoblasts the localization appeared to be mostly nuclear, whereas with differentiation the protein started to display a sarcomeric-like pattern. In adult fibers, GAP43 expression was evident with the protein labeling forming (in longitudinal views) a double cross striation reminiscent of the staining pattern of other organelles, such as calcium release units (CRUs) and mitochondria. Double immuno-staining and experiments done in EDL muscles fixed at different sarcomere lengths, allowed us to determine the localization, from the sarcomere Z-line, of GAP43 positive foci, falling between that of CRUs and of mitochondria. Staining of cross sections added a detail to the puzzle: GAP43 labeling formed a reticular pattern surrounding individual myofibrils, but excluding contractile elements. This work leads the way to further investigation about the possible physiological and structural role of GAP43 protein in adult fiber function and disease.

Figure 1. GAP43 is expressed at all stages of skeletal muscle maturation.

Representative western blot of samples obtained from: A) proliferating myoblasts; B) differentiated myotubes; and C) adult extensor digitorum longus (EDL, 4 months of age). On the right, a preparation obtained from mouse brain (D- Brain) is used as positive control. All specimens (i.e. satellite cells, myotubes, EDL and brain) show the same immuno-reaction at ∼43KDa, when probed with the mGAP43 antibody.

Figure 2. Localization of GAP43 changes in myoblasts and myotubes from nuclear to sarcomeric.

Light and confocal microscopy of proliferating myoblasts (A) and differentiated myotubes (C) stained with the mGAP43 antibody (B and D) reveal changes in localization during the differentiation process. A-B) GAP43 is mainly localized in the nucleus in un-differentiated cells (arrowhead), even if bright spots are also visible in the cytoplasm. C-D) The nuclear localization typical of proliferating myoblasts disappears with differentiation, i.e. it is replaced by a striated pattern (double arrows). E-G) Double labeling of myotubes with anti-GAP43 (green, E) and anti-triadin (red, F) antibodies shows that GAP43 double cross striation does not superimpose with triadin, a protein marking the position of CRUs (G, merged image). Bars: 10 µm.

Figure 3. In adult fibers, GAP43 is localized between Z-lines and CRUs, close to the band occupied by mitochondria.

A-C) In adult EDL fibers, both CRUs (marked with an anti-RYR antiboby) and mitochondria (marked with an anti-TOM20 antibody) staining form double rows on both sides of the Z-lines (the position of which is marked by arrowheads in merged figures), with RYR flanking TOM20 staining. D-I) Also GAP43 staining produces a double cross-striation (D and G), which seems to be partially co-localized with that of RYR (F) but outside that of mitochondria (I). Bar: 10 µm.

Figure 4. GAP43 is localized around the myofibrils and alternates to mitochondria.

A-C) Immuno-fluorescence of transverse sections stained with anti-GAP43 antibody, shows a reticular pattern surrounding myofibrils, quite similar to that formed by mitochondria (marked by TOM20). In some points red and green foci are contiguous, clearly distinct (inset in C). In A and B arrowheads indicate only TOM20 (red) staining, while arrows only GAP43 (green) staining. D) Electron micrograph of an EDL muscle in cross section. Both mitochondria and sarcoplasmic reticulum (SR) encircling myofibrils forming a network. In D arrowheads indicate only mitochondria presence, while arrows only SR. Bars: A-D,10 µm; D, 0.500 µm.

Figure 5. Fluorescence image profile and co-localization analyses.

Graphs represent fluorescence intensity profiles calculated on images obtained from samples co-immunostained for; A) RYR and mitochondria; B); GAP43 and RYR; and C) GAP43 and mitochondria. The RyR fluorescence peak appears closer to that of GAP43 than mitochondria’s one in which the line profile is slight shifted in respect to that of GAP43 (B-C). D) Graph of the degree of co-localization calculated by Pearson’s coefficient in samples stained as in A-C, shows the different degree of co-localization between GAP43 and the other structure/organelles around the Z-line (i.e. mitochondria and CRUs).

Figure 6. Immuno-fluorescence and electron microscopy (EM) of EDL fibers at different sarcomere lengths.

Immuno-fluorescence with antibodies against GAP43 (A and B) and a-actinin (C and D), marking the position of Z-lines, and EM images (E and F) of EDL fibers prepared at two different length: rest (∼1.9µm) and stretched (∼2.5µm) were used to generate the data included in Tables I and II of Figure 7. Arrowheads in the EM micrographs (E and F) point to CRUs, white arrows point to mitochondria and black arrows mark the position of Z-disks. Bars: A-F, 10 µm; G-H, 0.500 µm.

Figure 7. Quantitative analyses of images presented in Figure 6 .

Data in columns A and B (Fluorescence measurements) and in columns C and D (E.M measurements) contain respectively average measurements collected on immuno-fluorescence images (indicated by A1 and B1 in the left cartoon) and on electron microscopy micrographs (indicated by C1–C2 and D1–D2 in the right cartoon). Analysis of these data indicates that the position of GAP43 is between CRUs and mitochondria (see text for more details).

Figure 8. Model proposing GAP43 positioning in skeletal muscle.

In the proposed model the fiber is seen along its longitudinal axis (as in figure 3). The SR is reproduced in yellow, while mitochondria on both sides of the Z–lines, are represented in green. The white structures represent the T-tubules and the blue spots are RYRs. Orange blobs represent GAP43.