Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression

Abstract

Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (approximately 15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.

Fig. 1.

Membrane blebbing require extracellular calcium. A: C₂C₁₂ myotube with membrane blebs (dark) indicated by arrows. These blebs were formed after 14 s of exposure to hypotonic solution (50% dilution of media by water). B: C₂C₁₂ myotubes 14 s after exposure to the same hypotonic solution as in A, but with EGTA added to chelate extracellular calcium, no membrane blebs formed. This figure can be viewed in real time in Supplemental Movie 1. (The online version of this article contains supplemental data.)

Fig. 2.

Membrane blebbing requires dysferlin. A: primary C57 mouse myotube after 10 s of exposure to hypotonic solution (50% dilution of media by water) demonstrating membrane blebs. Membrane blebs are indicated by arrows. (Again note that contrast was adjusted to optimize visualization of blebs. In this case, the blebs appear light.) B: primary A/J mouse myotube exposed for 10 s to hypotonic solution. No blebbing is seen. C: primary A/J mouse myotube that is expressing full-length human dysferlin (infection with adenovirus expressing dysferlin) exposed for 10 s to hypotonic solution. In this case, blebbing is easily visualized, demonstrating that dysferlin is sufficient to rescue blebbing function.

Fig. 3.

Detection of dysferlin expression in myotubes by immunofluoresence. A: shown are C57 and A/J myotubes stained with either anti-myosin (red) or anti-dysferlin (green) antibodies. Note that A/J myotubes express no detectable dysferlin, unless infected with the recombinant adenovirus that expresses full-length human dysferlin (Ad-DYSF). B: shown are myotubes from an unaffected human and from a Miyoshi patient. Note that, as in the A/J mouse, the Miyoshi myotubes do not express dysferlin, unless infected with recombinant adenovirus. In the case of the Miyoshi myotubes, treatment with PTC124 also results in dysferlin expression. All scale bars represent 20 μm.

Fig. 4.

Western blots to assess dysferlin expression. A: comparison of dysferlin expression in mouse and human myotubes. Note, as in Fig. 3, no dysferlin expression can be detected in A/J and Miyoshi myotubes, unless they are infected with recombinant adenovirus expressing full-length human dysferlin (Ad-DYSF). B: treatment of Miyoshi myotubes with PTC124 results in ∼15% of the levels of dysferlin, as found in unaffected myotubes (based on densitometry). Normalization was performed using the levels of α-sarcoglycan expressed in unaffected and Miyoshi myotubes.

Fig. 5.

Membrane bleb formation in human myotubes requires dysferlin. A: shown is a primary human (unaffected) myotube after 10 s of exposure to hypotonic solution (25% dilution of media by water) demonstrating membranes blebs. Membrane blebs are indicated by arrows. B: primary myotube from a Miyoshi patient exposed for 10 s to hypotonic solution. No blebbing is seen. C: primary Miyoshi myotube that is expressing full-length human dysferlin (infection with adenovirus expressing dysferlin) exposed for 10 s to hypotonic solution. In this case, blebbing is easily visualized, demonstrating that dysferlin is sufficient to rescue blebbing function in Miyoshi myotubes. D: membrane blebs are seen in the myotubes derived from the Miyoshi patient carrying a R1905X mutation following PTC124 treatment. This rescue of blebbing by treatment with PTC124 demonstrates that the dysferlin produced via nonsense suppression is functional.