Extensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries

Abstract

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.

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

“…Short-term treatment with diltiazem before in vivo injury significantly decreased the number of fibers displaying disrupted DHPR morphology shortly after LSI and significantly increased recovery of torque 3 d later. As previously reported, the downstream consequences of LSI in dysferlin-deficient muscle include considerable necrosis and inflammation, followed by muscle regeneration (18)(19)(20). Diltiazem treatment decreased the number of necrotic fibers and the severity of the inflammatory response 3 d after physiological injury and decreased the number of centrally nucleated fibers 14 d after injury.…”

“…In vivo measurements of torque and large-strain injury of the hind-limb ankle dorsiflexor muscle group were performed as described (19,43). Briefly, injury was induced by a series of 20 lengthening contractions performed by stretching the tetanically stimulated left-ankle dorsiflexors through an arc of 90°-180°of plantarflexion at 1,200°/sec.…”

“…To count necrotic myofibers, frozen cross-sections (8 μm) of the injured (left) and uninjured (right) TA muscles were stained with hematoxylin and eosin (H&E), as described (19). The percentage of necrotic myofibers in each muscle was calculated by taking the average across 12 unique fields (20× objective lens, Zeiss Axioskop; Carl Zeiss, Inc.).…”

“…Previously, we reported that impaired recovery of contractile torque following large-strain injury (LSI) in dysferlin-null mice was concurrent with an increase in necrosis and macrophage infiltration (19). As diltiazem protected dysferlin-null myofibers from injury by osmotic shock in vitro, we tested whether systemic treatment of dysferlin-null mice with diltiazem improved the response of muscle injured by LSI in vivo.…”

“…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). Consistent with this localization and the potential for a functional role in this specialized compartment, dysferlin-deficient murine muscle demonstrates altered transverse tubule (t-tubule) structure (15) as well as increased oxidative stress (16,17), inflammation, and necrosis (18)(19)(20) after injury.…”