Impaired ECM Remodeling and Macrophage Activity Define Necrosis and Regeneration Following Damage in Aged Skeletal Muscle

Abstract

Regenerative capacity of skeletal muscle declines with age, the cause of which remains largely unknown. We investigated extracellular matrix (ECM) proteins and their regulators during early regeneration timepoints to define a link between aberrant ECM remodeling, and impaired aged muscle regeneration. The regeneration process was compared in young (three month old) and aged (18 month old) C56BL/6J mice at 3, 5, and 7 days following cardiotoxin-induced damage to the tibialis anterior muscle. Immunohistochemical analyses were performed to assess regenerative capacity, ECM remodeling, and the macrophage response in relation to plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-9 (MMP-9), and ECM protein expression. The regeneration process was impaired in aged muscle. Greater intracellular and extramyocellular PAI-1 expression was found in aged muscle. Collagen I was found to accumulate in necrotic regions, while macrophage infiltration was delayed in regenerating regions of aged muscle. Young muscle expressed higher levels of MMP-9 early in the regeneration process that primarily colocalized with macrophages, but this expression was reduced in aged muscle. Our results indicate that ECM remodeling is impaired at early time points following muscle damage, likely a result of elevated expression of the major inhibitor of ECM breakdown, PAI-1, and consequent suppression of the macrophage, MMP-9, and myogenic responses.

Keywords: aging; extracellular matrix; macrophage; plasminogen activator inhibitor-1; regeneration; skeletal muscle.

Figure 1

Plasminogen system. Plasminogen is the inactive substrate of the plasminogen system that is activated into plasmin primarily by urokinase plasminogen activator (uPA) in skeletal muscle. Plasmin can directly degrade fibrin into fibrin degradation products (FDPs) and activate MMPs. The MMPs work to degrade connective tissue in the ECM and also activate additional MMPs in a positive feedback loop. PAI-1 functions as the upstream inhibitor of the plasminogen system by inhibiting uPA and thus preventing the activation of plasminogen. Adapted from Higazi, A.A.-R [32].

Figure 2

Regenerative capacity of aged skeletal muscle is impaired. (A) hematoxylin and eosin (H&E) cryosections of the TA muscle throughout the regeneration time course (3–7 days). Control (undamaged) TA (B) mean cross-sectional area (p = 0.003) and (C) muscle mass were significantly greater in the aged group (p < 0.001). * denotes a significant difference detected by two-tailed t-test. (D) Damaged TA muscle mass was different between young and aged groups and across recovery time points. * denotes a significant main effect of age (p = 0.001). † denotes a significant main effect of recovery time point following damage (p < 0.001). (E) Regenerating myofibers were identified via embryonic myosin heavy chain (eMHC; red) and 4,6-diamidino-2-phenylindole (DAPI; blue) at five and seven days following damage. (F) A significant interaction between age and recovery time points was observed (p = 0.02). Regenerating myofiber cross-sectional area was found to increase from five to seven days in young mice (simple main effects post-hoc analysis: p < 0.001), and there was a significant difference at seven days between young and aged muscle (simple main effects post-hoc analysis: p = 0.001). (G) Relative regenerating myofiber area was significantly greater in young muscle compared to aged muscle at five and seven days following damage. * denotes a significant main effect of age (p < 0.001). † denotes a significant main effect of recovery time point following damage (p = 0.013). n = 4–5 per group. Data presented are means ± standard deviation. Scale bar represents 50 μm.

Figure 3

Necrosis is increased in aged muscle regeneration. (A) Representative images of whole muscle sections undergoing regeneration at their respective time points. (B) A significant interaction between age and recovery time points was identified (p < 0.001). Necrotic regions were found to be identical three days following damage in young and aged muscle, however, necrotic area decreased significantly in young muscle at five days following damage (simple main effects post-hoc analysis: p < 0.001 in all instances). The * indicates significant differences detected by a simple main effects post-hoc analysis (p < 0.001). n = 4–5 per group. Data presented are means ± standard deviation. Scale bar represents 500μm.

Figure 4

Collagen I expression is greater in the necrotic regions of aged muscle. (A) Immunostaining of collagen I (green) and DAPI (blue) at each time point following damage. Arrows denote regions of dense collagen I persistent at 5 days post-CTX in aged muscle. (B) Collagen I positive area in the undamaged contralateral leg was observed to be greater in aged muscle compared to young (t-test: p = 0.006; indicated by *). (C) Percent positive area of collagen I was greater in the necrotic region of aged muscle at three and five days following damage. * denotes a significant main effect of age (p = 0.002). (D) A significant interaction between age and recovery time points was observed (p = 0.034). Simple main effect post-hoc analysis demonstrates a significantly greater accumulation of collagen I in the regenerating region of aged muscle at seven days following damage (p = 0.024). * indicates significant differences detected by a simple main effects post-hoc analysis (p = 0.024). n = 4–5 per group. Data presented are means ± standard deviation. Scale bar represents 50 μm.

Figure 5

Collagen IV expression following cardiotoxin (CTX)-induced muscle damage in young and aged skeletal muscle. (A) Immunostaining of collagen IV (green) and DAPI (blue) at each time point following damage. (B) Collagen IV positive area in the undamaged contralateral leg was not significantly difference in young and aged muscle (t-test: p = 0.83). No significant differences in collagen IV percent positive area were detected in (C) necrotic and (D) regenerating regions. (E) Magnified of collagen IV area in young and aged muscle. Arrows denote regions of thicker collagen IV layers at five days post-CTX in aged muscle. (F) A significant interaction between age and recovery time point was observed (p = 0.015). Simple main effects post-hoc analysis found aged muscle to have a significantly greater collagen IV layer thickness compared to young at five and seven days following damage (p < 0.001 in both instances). * indicates significant differences detected by a simple main effects post-hoc analysis (p < 0.001). n = 4–5 per group. Data presented are means ± standard deviation. Scale bar represents 50 μm.

Figure 6

Fibronectin content is acutely greater in the necrotic region of young muscle. (A) Immunostaining of fibronectin (green) and DAPI (blue) at each time point following damage. Arrows denote regions of broad fibronectin at three days post-CTX in young muscle. (B) Fibronectin positive area in the undamaged control TA was not significantly different between young and aged (t-test: p = 0.256). (C) Fibronectin content increases over time from three to five days following damage in the necrotic region. Additionally, fibronectin was greater in the young groups during these acute time points. * denotes a significant main effect of age (p = 0.002). † denotes a significant main effect of recovery time point following damage (p < 0.001). (D) No statistically significant changes were observed in fibronectin content in the regenerating region of young and aged muscle. Note that the seven-day time point in the necrotic region and the three-day time point in the regenerating regions were not used in the statistical analysis due to insufficient instances of those regions depending on age and time point. n = 4–5 per group. Data presented are means ± standard deviation. Scale bar represents 50μm.

Figure 7

Macrophage responses to skeletal muscle differ between young and aged skeletal muscle. (A) Immunostaining of F4/80 (red) and DAPI (blue) used to identify macrophages. White arrows indicate F4/80+ cells (macrophages). White frame indicates region of higher magnification shown in neighbouring inset. Scale bars both represent 50 μm. (B) Macrophage density was greater in the necrotic region of aged muscle and remains steady between three to five days following damage. * denotes significant main effect of age (p = 0.025). (C) A significant interaction between age and recovery time point following damage was observed (p < 0.001). Simple main effect post-hoc analysis revealed a significant difference in macrophage density at five days following damage, with young muscle having greater density (p < 0.001). However, at seven days following damage, macrophage density in aged muscle was greater than young (p < 0.001). A significant drop in macrophage density between five and seven days in young muscle was observed (p = 0.001), while the opposite was observed in aged muscle (p < 0.001). The * indicates significant differences detected by a simple main effects post-hoc analysis (p < 0.05). n = 4–5 per group. Data presented are means ± standard deviation.

Figure 8

MMP-9 expression was elevated acutely following damage in young muscle. (A) Immunostaining of MMP-9 (green), F4/80 (red), and DAPI (blue). Arrows indicate F4/80+/MMP-9+ cells and arrowheads indicate F4/80-/MMP-9+ cells five days post-CTX. (B) A significant interaction between age and recovery time point in MMP-9+ area following damage was detected within the necrotic regions (p = 0.004). Simple main effect post-hoc analyses demonstrated a significant greater MMP-9 expression within young muscle at three days following damage (p < 0.001). MMP-9 expression declined significantly from three to five days following damage (p < 0.001). (C) No significant differences in MMP-9-positive area were observed in the regenerating regions of young and aged muscle (p > 0.05). (D) A significant interaction between age and recovery time point on MMP-9+/F4/80+ cells were detected following damage (p = 0.004). A simple main effects post-hoc analysis showed significantly greater macrophage-specific (F4/80+) MMP-9 expression in the necrotic regions of young muscle five days following damage (p = 0.009). Additionally, macrophage-specific MMP-9 expression increased significantly between three and five days in young, however, the opposite was observed in aged muscle (p = 0.026 and p = 0.031, respectively). (E) A significant main effect of age was found in the regenerating regions, with young muscle displaying a greater percentage of macrophage-specific MMP-9 expression compared to aged muscle (p = 0.008). * in (B) and (D) indicates significance from the simple main effects post-hoc analyses (p < 0.05). * in (E) indicates a main effect of age. n = 4–5 per group. Data presented are means ± standard deviation. Scale bar represents 100 μm.

Figure 9

PAI-1 expression is greater in aged skeletal muscle. (A) Immunostaining of PAI-1 (green) and DAPI (blue) at five days following damage. (B) A significant interaction between age and recovery time point following damage was observed in the necrotic regions (p = 0.044). Simple main effect post-hoc analyses demonstrated a significant greater extramyocellular PAI-1 expression within aged muscle at three and five days following damage (p = 0.005 and p < 0.001, respectively). Extramyocellular PAI-1 within the necrotic regions of young muscle declined significantly between three and five days following damage (p = 0.015). * denotes significant differences between groups identified by the simple main effects post-hoc analyses (p < 0.05). (C) No significant differences in extramyocellular PAI-1 were observed in the regenerating region of young and aged muscle (p > 0.05). (D) Brightness analysis of PAI-1 within regenerating myofibers showed significantly greater PAI-1 signal within aged regenerating myofibers compared to young (* denotes main effect of age: p = 0.029). n = 4–5 per group. Data presented are means ± standard deviation. Scale bar represents 50 μm.