Myofiber damage precedes macrophage infiltration after in vivo injury in dysferlin-deficient A/J mouse skeletal muscle

Abstract

Mutations in the dysferlin gene (DYSF) lead to human muscular dystrophies known as dysferlinopathies. The dysferlin-deficient A/J mouse develops a mild myopathy after 6 months of age, and when younger models the subclinical phase of the human disease. We subjected the tibialis anterior muscle of 3- to 4-month-old A/J mice to in vivo large-strain injury (LSI) from lengthening contractions and studied the progression of torque loss, myofiber damage, and inflammation afterward. We report that myofiber damage in A/J mice occurs before inflammatory cell infiltration. Peak edema and inflammation, monitored by magnetic resonance imaging and by immunofluorescence labeling of neutrophils and macrophages, respectively, develop 24 to 72 hours after LSI, well after the appearance of damaged myofibers. Cytokine profiles 72 hours after injury are consistent with extensive macrophage infiltration. Dysferlin-sufficient A/WySnJ mice show much less myofiber damage and inflammation and lesser cytokine levels after LSI than do A/J mice. Partial suppression of macrophage infiltration by systemic administration of clodronate-incorporated liposomes fails to suppress LSI-induced damage or to accelerate torque recovery in A/J mice. The findings from our studies suggest that, although macrophage infiltration is prominent in dysferlin-deficient A/J muscle after LSI, it is the consequence and not the cause of progressive myofiber damage.

Figure 1

Loss and recovery of contractile torque after large-strain injury. The data indicate that dysferlin-deficient muscle is not more susceptible to initial injury compared with dysferlin-sufficient muscle. Partial recovery occurs over 3 to 6 hours in both dysferlin-sufficient A/WySnJ and dysferlin-deficient A/J muscle, after which A/J muscle (gray bars) loses contractile torque whereas A/WySnJ muscle (white bars) continues to recover. ^∗P < 0.05 versus approximately 10 minutes; ^†P < 0.05 versus 3 hours; ^‡P < 0.05 versus preceding time point other than approximately 10 minutes or 3 hours; and ^§P < 0.05 between strains.

Figure 2

T₂-weighted magnetic resonance (MR) images showing the progression of injury over time. T₂-weighted MR imaging of the hind limbs of the same dysferlin-deficient A/J mouse and dysferlin-sufficient A/WySnJ mouse studied longitudinally after injury shows that muscle damage progresses over several days in A/J muscle. An increased T₂ signal after injury, which corresponds to increased edema, can be appreciated by an increase in the whiteness of the muscle. The tibia and the tibialis anterior muscle of the injured (left) and uninjured (right) hind limbs are encircled in pink and yellow, respectively, in the uppermost panels. Arrows indicate tibialis anterior muscle in all panels.

Figure 3

Quantitative analysis of T₂-weighted magnetic resonance images. Data obtained from images of tibialis anterior muscle such as those shown in Figure 2, analyzed as described in Materials and Methods, show that injured dysferlin-deficient A/J muscle (gray bars) has a greater T₂ signal than does injured dysferlin-sufficient A/WySnJ muscle (white bars) soon after large-strain injury. At 6 hours after injury, the T₂ signal intensity is decreased in A/J muscle and is statistically indistinguishable from that in A/WySnJ muscle. At 24 and 48 hours, however, the signal in A/J muscle is significantly greater than that in A/WySnJ muscle. By 7 and 14 days after injury, the T₂ signal is decreased in A/J muscle, suggesting that edema resolves by this time. ^∗P < 0.05 versus approximately 30 minutes; ^†P < 0.05 versus 6 hours; and ^‡P < 0.05 between strains.

Figure 4

Progression of myofiber damage and mononuclear cell infiltration after injury. Hematoxylin and eosin staining of tibialis anterior muscle cross sections reveals that after injury, damaged fibers (blue arrows) appear over several hours and days in dysferlin-deficient A/J muscle. Over time, as myofiber damage becomes more widespread, damaged areas are infiltrated by mononuclear cells (yellow arrows). Myofiber damage and cellular infiltration are low in dysferlin-sufficient A/WySnJ muscle.

Figure 5

Quantitative analyses of damaged fibers, sarcolemmal damage, and infiltration by neutrophils and macrophages after injury. Images such as those in Figures 4, 6, and 7 were analyzed quantitatively, as described in Materials and Methods. A: After injury, myofiber damage slowly increases in dysferlin-deficient A/J muscle over several hours and days, with a sharp increase occurring between 12 and 24 hours and a further increase over 24 to 48 hours after injury. Myofiber damage in dysferlin-sufficient A/WySnJ muscle is minimal. B: After injury, the number of mouse IgG-positive (Ms-IgG⁺) fibers, a measure of sarcolemmal damage, increases over several hours and days in dysferlin-deficient A/J muscle. An increase of roughly 2.5-fold in Ms-IgG⁺ fibers occurs between 6 and 12 hours and then again between 24 and 48 hours after injury. Compared with A/J muscle, very few A/WySnJ myofibers become permeable to Ms-IgG after injury. C: Low levels of neutrophil infiltration are seen at early time points in both A/WySnJ and A/J muscle, with the latter being significantly greater. Macrophage infiltration is low in A/WySnJ muscle, but greater in A/J muscle, with a sharp increase occurring between 24 and 48 hours and a further increase occurring between 48 and 72 hours. ^∗P < 0.05 versus uninjured; ^†P < 0.05 versus preceding time point other than uninjured; and ^‡P < 0.05 between strains.

Figure 6

Progression of sarcolemmal damage after injury. Immunofluorescence labeling of tibialis anterior muscle cross sections reveals that after injury, the number of fibers that are positive for mouse IgG-positive (Ms-IgG⁺) entry (a marker of sarcolemmal damage, arrows) increases slowly over several hours and days in dysferlin-deficient A/J, similar to the appearance of damaged fibers in hematoxylin and eosin–stained sections. Compared with A/J muscle, very few fibers in A/WySnJ muscle are positive for Ms-IgG. Myofiber boundaries are labeled green, with wheat germ agglutinin (WGA) conjugated to Alexa 488 (Life Technologies Corporation, Carlsbad, CA), and nuclei are labeled blue with DAPI.

Figure 7

Progression of neutrophil and macrophage infiltration after injury. Frozen sections of muscle collected at different times after injury were immunolabeled with antibodies to neutrophils or macrophages. Dystrophin was also labeled to outline the boundaries of healthy myofibers. Low levels of neutrophil infiltration occur in dysferlin-deficient A/J muscle at early times after injury. Macrophage infiltration is more pronounced, however, and becomes very prominent at 48 and 72 hours after large-strain injury. Neutrophils and macrophages are labeled red, and nuclei and myofiber boundaries are labeled blue (DAPI and dystrophin, respectively). Ly6G⁺, positive for neutrophil marker (MCA2387; AbD Serotec, Kidlington, UK).

Figure 8

Changes in cytokine levels after injury. Homogenates of injured and uninjured tibialis anterior muscle collected from dysferlin-sufficient A/WySnJ and dysferlin-deficient A/J mice 72 hours after large-strain injury (LSI) were profiled for 11 cytokines or chemokines that mediate inflammation. The data suggest that molecules related to monocyte/macrophage infiltration, Il-1A, tumor necrosis factor (Tnf)-α, monocyte chemotactic protein (Mcp)-1, and macrophage colony-stimulating factor (M-Csf) are elevated in A/J muscle (gray bars) compared with levels in A/WySnJ muscle (white bars) at 72 hours after LSI. ^∗P < 0.05 between strains. Mig, monokine induced by γ-interferon; Mip, macrophage inflammatory protein; Rantes, regulated on activation normal T cell expressed and secreted; Vegf, vascular endothelial growth factor.

Figure 9

Effect of macrophage depletion by clodronate on the response to injury. We used liposomes loaded with clodronate injected IP to reduce the number of circulating monocytes and thus the macrophages that can invade muscle in mice subjected to large-strain injury (LSI). As macrophage infiltration increases steeply between 24 and 48 hours after LSI in dysferlin-deficient A/J muscle, we injected animals with 15 μL/g body weight of clodronate-incorporated liposomes 24 hours after LSI. A and B: Treatment of A/J mice with clodronate-incorporated liposomes neither affects preinjury torque nor promotes faster recovery of function at 72 hours after LSI. C–F: Treatment of A/J mice with clodronate-incorporated liposomes does not reduce the number of damaged fibers (C and D; hematoxylin and eosin staining), although it significantly reduces the number of infiltrating macrophages (E and F; immunofluorescence). Macrophages, nuclei, and myofiber boundaries are labeled with antibodies to CD68 (red), DAPI (blue), and antibodies to dystrophin (green), respectively. ^∗P < 0.05. PBS, phosphate-buffered saline; Nmm, Newton millimeter