Impairment of caveolae formation and T-system disorganization in human muscular dystrophy with caveolin-3 deficiency

Abstract

Caveolin-3, a muscle specific caveolin-related protein, is the principal structural protein of caveolar membranes. We have recently identified an autosomal dominant form of limb girdle muscular dystrophy (LGMD-1C) that is due to caveolin-3 deficiency and caveolin-3 gene mutations. Here, we studied by electron microscopy, including freeze-fracture and lanthanum staining, the distribution of caveolae and the organization of the T-tubule system in caveolin-3 deficient human muscle fibers. We found a severe impairment of caveolae formation at the muscle cell surface, demonstrating that caveolin-3 is essential for the formation and organization of caveolae in muscle fibers. In addition, we also detected a striking disorganization of the T-system openings at the sub-sarcolemmal level in LGMD-1C muscle fibers. These observations provide new perspectives in our understanding of the role of caveolin-3 in muscle and of the pathogenesis of muscle weakness in caveolin-3 deficient muscle.

Figure 1.

Immunofluorescent localization of caveolin-3 in the muscle fibers of normal and LGMD-1C patients. A: By high-resolution immunofluorescence, in longitudinal sections, caveolin-3 shows a network-like organization at the cell surface, with main bands running along the longitudinal axis of the fibers, interconnected by transverse bands. This pattern is reminiscent of the network organization of caveolae at the cell surface, as seen by freeze-fracture in normal muscle fibers (see Figure 3A ▶ ). B: By immunofluorescence in normal muscle, in cross sections, caveolin-3 reveals a uniform staining pattern at the sarcolemma. C: In LGMD-1C patients, note that there is a severe deficiency of caveolin-3 at the cell surface. A: Original magnification, ×1200; B: Original magnification, ×40; C: Original magnification, ×40.

Figure 2.

Transmission electron microscopic analysis of muscle fibers from normal and LGMD-1C patients. By routine transmission electron microscopy, caveolae in normal muscle fibers appear as flask shaped vesicles localized subjacent to the subsarcolemmal membrane (A, arrows). In LGMD-1C muscle fibers (patient 1), very few caveolae were found near to the cell surface (B, C). Furthermore, in LGMD-1C fibers, we observed large vacuolated structures close to the cell surface. These vacuolated structures appeared membranous and were generally empty, or containing amorphous material. (B, C, arrows). Some of these structures revealed continuity with the membrane (B, arrowhead). A–C: Original magnification, ×12,500.

Figure 3.

Freeze-fracture analysis of muscle fibers from normal and LGMD-1C patients. In freeze-fracture preparations of normal muscle cell plasma membrane, caveolae appeared as small invaginations or indentations of the plasma membrane (A). The distribution of caveolae at the cell surface was not homogeneous; caveolae were present in ordered arrays or rows that may correspond to the horizontal and vertical band-like structures we observed by immunofluorescence microscopy (see Figure 1A ▶ ). In striking contrast, in LGMD-1C muscle fibers, we observed very few scattered caveolae at the cell surface (B, patient 1 and C, patient 2, arrows). A–C: Original magnification, ×12,500.

Figure 4.

Lanthanum nitrate staining of muscle fibers from normal and LGMD-1C patients. After lanthanum nitrate treatment, normal control muscle samples showed dense staining of the T-tubules, which were generally observed between the I- and A-band. Lanthanum penetration was able to stain the first few sarcomeres close to the membrane (A). In LGMC-1C muscle fibers, we confirmed that only very few caveolae are found near to the cell surface, in contrast with the large number of caveolae that are present in adjacent non-muscle cells (B). In LGMD-1C muscle fibers, note the vacuolated structures that are penetrated by lanthanum and show a lanthanum-positive membrane (C, arrowsheads). Some of these structures revealed continuity with the cell membrane (C, D, arrows). Furthermore, some vacuolated structures were associated with honeycomb structures, typical of abnormal proliferation of the T-tubule system (D, arrowheads). A: Original magnification, ×7,000; B: Original magnification, ×12,500; C: Original magnification, ×7,000; D: Original magnification, ×10,000.