Unmasking Potential Intracellular Roles For Dysferlin through Improved Immunolabeling Methods

Abstract

Mutations in the DYSF gene that severely reduce the levels of the protein dysferlin are implicated in muscle-wasting syndromes known as dysferlinopathies. Although studies of its function in skeletal muscle have focused on its potential role in repairing the plasma membrane, dysferlin has also been found, albeit inconsistently, in the sarcoplasm of muscle fibers. The aim of this article is to study the localization of dysferlin in skeletal muscle through optimized immunolabeling methods. We studied the localization of dysferlin in control rat skeletal muscle using several different methods of tissue collection and subsequent immunolabeling. We then applied our optimized immunolabeling methods on human cadaveric muscle, control and dystrophic human muscle biopsies, and control and dysferlin-deficient mouse muscle. Our data suggest that dysferlin is present in a reticulum of the sarcoplasm, similar but not identical to those containing the dihydropyridine receptors and distinct from the distribution of the sarcolemmal protein dystrophin. Our data illustrate the importance of tissue fixation and antigen unmasking for proper immunolocalization of dysferlin. They suggest that dysferlin has an important function in the internal membrane systems of skeletal muscle, involved in calcium homeostasis and excitation-contraction coupling.

Dysferlin stabilizes stress-induced Ca 2+ signaling in the transverse tubule membrane

“…Dysferlin was also found to coimmunoprecipitate with DHPR from developing myotubes and was hypothesized to be important for the development of the t-tubule (11,15). Other studies found dysferlin at the level of the A-I junction of stretched muscle (14), and we previously localized dysferlin to that region in mature muscle (10). Our current data (cf.…”

“…Recently, we demonstrated the localization of dysferlin at the A-I junction in mature muscle fibers (10). These results agree with earlier reports associating dysferlin with the dihydropyridine receptor (DHPR, L-type Ca 2+ channel), Ahnak, caveolin 3, and several other proteins involved in Ca 2+ -based signaling and the function of transverse (t-) tubules (11)(12)(13)(14).…”

“…Although originally identified as a protein of the sarcolemma (3, 30, 31), dysferlin has also been shown to be present in the interior of myofibers, particularly in dystrophic muscle (10,32,33). Dysferlin was also found to coimmunoprecipitate with DHPR from developing myotubes and was hypothesized to be important for the development of the t-tubule (11,15).…”

“…We followed the protocols in Roche et al (10). Animals were perfusion-fixed with ice-cold 4% (wt/vol) paraformaldehyde in PBS.…”

“…10. Disruption of t-tubules was quantified by counting the number of fibers that showed disrupted DHPR labeling and expressing it as a percentage of the total number of fibers counted in each visual field.…”