MACROPHAGE INFILTRATION AND ACTIVATION IN OLD AND YOUNG

MACROPHAGE INFILTRATION AND ACTIVATION IN OLD AND YOUNG SKELETAL MUSCLE FOLLOWING LENGTHENING CONTRACTIONS Jamie P. Kaluhiokalani, Jacob R. Sorensen, and Robert D. Hyldahl Department of Exercise Sciences, Brigham Young University, Provo, UT Introduction Results • Human skeletal muscle is capable of robust regeneration following damage. • Muscle repair following injury is dependent on the timely activity of pro-inflammatory (M 1) and anti-inflammatory, pro-regenerative (M 2) macrophages. Following acute muscle injury, healthy skeletal muscle experiences a rapid and vigorous inflammatory response. Figure 2: A. Figure 3: B. Group x time: p=0. 037 * # Time: p=0. 002 Group: p=0. 0005 # # B. A. • Macrophages clear necrotic tissue and facilitate satellite cell behavior through the secretion of cytokines and growth factors. • It is known that aged muscle experiences poor regenerative potential following injury, but the cause is not well-understood. C. Figure 3: Macrophage percentages. (A) Young and old subjects experienced a significant increase in M 1 macrophages 24 hr post-exercise. However, the percentage of M 1 macrophages was greater in the young. (B) At 24 hr post-exercise, the percentage of M 2 macrophages declined in both groups, but was significantly higher in the old. # indicates significant difference from pre. * indicates significance between groups. Significance set at p<0. 05. Purpose: The purpose of this study was to investigate macrophage behavior in old and young people in the context of muscle damage. Hypothesis: We hypothesized that 1) old skeletal muscle would have a greater proportion of M 2 like macrophages at baseline and in the early stages of muscle repair compared to the young, and 2) old myoblasts will demonstrate increased proliferation and differentiation potential when cultured in young macrophage conditioned medium. Rationale: The immune system, which deteriorations with age, has an important role in promoting skeletal muscle regeneration. This suggests that aged immune cells, like macrophages, may contribute to the gradual decline of skeletal muscle regenerative capacity. Figure 6: Figure 2: Macrophage Stain. (A) Macrophages were found in extracellular matrix surrounding muscle cells and (B) increased by 24 hr post-exercise, peaking at 72 hours. (C) M 2 macrophages increased at 72 hr post-exercise, with a greater overall appearnce in the old. # indicates a significant difference from the pre-exercise timepoint. Significance set at p<0. 05. Young Male Young Female Old Male Figure 5: Figure 4: A. C. B. Group x Time: p= 0. 046 B. Figure 1: Design Assessment of Muscle Damage Muscle Biopsies Pre-Exercise 24 Hours 72 Hours Post Exercise: 30 sets of 10 maximal isokinetic lengthening contractions were performed at 60° sec-1 on a Biodex dynamometer. Biopsies: Percutaneous needle biopsies were taken from the m. vastus lateralis preexercise, and again at 3, 24, and 72 hours post-exercise. Immunohistochemistry: Slides were stained for pro-inflammatory (CD 68/CD 11 b/dystrophin/DAPI) or anti-inflammatory (CD 68/CD 206/dystrophin/DAPI) macrophage markers or satellite cells(Pax 7/DAPI). Cell Culture Study: 5 young and 5 old subjects had a 70 m. L blood draw and a percutaneous muscle biopsy to isolate monocyte and myoblast cells respectively. Following 6 days of incubation, monocytes matured into macrophages and were used to generate macrophage conditioned medium (CM). Myoblast Proliferation and Differentiation: Proliferation and differentiation of old human primary myoblasts were measured 24 and 72 hr following incubation with or without macrophage conditioned medium (CM) from young and old individuals. Analysis: A mixed model repeated measures analysis was performed with a Tukeys HSD post-hoc for significant main effects. Significance set at p<0. 05. B. Young CM Old CM Methods Subjects: 11 young (22. 7± 2. 25 years) and 8 old (70. 9± 7. 5 years), completed a bout of lengthening contractions on a randomized leg. The study design and time course of measurements are presented below. A. D. Figure 4: Satellite cell proliferation in young and old subjects. (A) Satellite cell content in young and old subjects 72 hr postexercise. (B) The expression of Pax 7+ cells increased in young subjects, but not old, 72 hours post-exercise. * indicates significant difference between groups. Significance set at p<0. 05. Figure 5: Myoblast Proliferation. (A) Old myoblasts cultivated in macrophage CM increased proliferative potential relative to unconditioned medium. Young macrophage CM was especially effective. (B) Ed. U stain protocol used to observe human primary myoblast proliferation. # indicates significant difference from the negative control. * indicates significant difference between groups. Figure 6: Myoblast Differentiation. (A) Differentiation of myoblasts isolated from old subjects using control, young, or old macrophage CM. Old myoblasts treated with macrophage CM, especially young CM, demonstrated a greater capacity to differentiate compared to medium alone as indicated by (B) differentiation index, (C) myotube area, and (D) Myogenin expression. # indicates significance from the pre-exercise time point. * indicates significant difference between groups. Significance set at p<0. 05. Conclusions • Macrophage infiltration was similar between groups, but pro-inflammatory (M 1) macrophages were 25% less prevalent in the old group 24 hr post-exercise. There was also a greater overall proportion of anti-inflammatory (M 2) macrophage accumulation in the early phases of muscle repair. • Studies have shown that M 1 macrophages are an important factor in satellite cell proliferation. Interestingly, we observed a poor M 1 response in old subjects, coupled with unresponsive satellite cell proliferation. • Proliferation was 30% higher in old myoblasts treated with young macrophage CM. Differentiation was greater in the young CM for some conditions, but not all, relative to the old CM. However, old human primary myoblasts treated with macrophage CM, showed greater potential to differentiate compared to medium alone.