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

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.

Stimulation of calcineurin Aα activity attenuates muscle pathophysiology in mdx dystrophic mice

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin Aα transgene (CnAα) was overexpressed in skeletal muscles of mdx ( mdx CnAα*) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnAα* and mdx mice was examined in vitro. Hindlimb muscles from mdx CnAα* mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnAα* than mdx mice. In the diaphragm, despite a slower phenotype and a ∼35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnAα* compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity.

Differential response of macrophage subpopulations to soleus muscle reloading after rat hindlimb suspension

The hypothesis that distinct populations of macrophages are associated with muscle necrosis and regeneration was examined in Wistar rat soleus muscle after 10 days of hindlimb suspension and 2, 4, and 7 days after the resumption of weight bearing. Necrosis was identified using histological features, such as muscle fiber infiltration, and regeneration was identified using immunohistochemical techniques for developmental myosin heavy chain (dMHC). Light-microscopic observations show that necrotic fibers in 2-day reloaded soleus muscle were invaded by ED1+ and Ia+ macrophages. The number of invaded fibers in muscles reloaded for 2 days increased to 2.8/mm2 compared with 0.2/mm2 in age-matched normal muscle but returned to control values by the 4th day of resumed weight bearing. In the interstitial spaces of 2-day recovery muscle, ED1+ and Ia+ macrophages numbered 369 and 332/mm2, respectively, compared with 12 and 72/mm2, respectively, in control soleus. After 7 days of reloading, the number of ED1+ cells was similar to that of control. Ia+ macrophages decreased to 240/mm2 at 4 days but after 7 days rose above control values to 429/mm2. ED2+ macrophages in 4- and 7-day reloaded soleus increased 70–80% in the interstitial spaces compared with control but were not observed to infiltrate necrotic muscle fibers at any time points. Immunohistochemistry and immunoblots using a monoclonal anti-dMHC antibody demonstrate a greater proportion of myofibers expressing dMHC isoforms after 4 and 7 days of reloading. These findings indicate that macrophage subpopulations are associated with distinct stages during the recovery process from hindlimb suspension: ED1+ macrophages are associated with muscle necrosis, whereas ED2+ cells are associated with muscle regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)