ACL injury reduces satellite cell abundance and promotes fibrogenic cell expansion within skeletal muscle

Abstract

Anterior cruciate ligament (ACL) injuries are associated with significant loss of strength in knee extensor muscles that persists despite physical therapy. The underlying mechanisms responsible for this protracted muscle weakness are poorly understood; however, we recently showed significant myofiber atrophy and altered muscle phenotype following ACL injury. We sought to further explore perturbations in skeletal muscle morphology and progenitor cell activity following an ACL injury. Muscle biopsies were obtained from the injured and non-injured vastus lateralis of young adults (n = 10) following ACL injury, and histochemical/immunohistochemical analyses were undertaken to determine collagen content, abundance of connective tissue fibroblasts, fibrogenic/adipogenic progenitor (FAP) cells, satellite cells, in addition to indices of muscle fiber denervation and myonuclear apoptosis. The injured limb showed elevated collagen content (p < 0.05), in addition to a greater abundance of fibroblasts and FAPs (p < 0.05) in the injured limb. Fibroblast content was correlated with increased accumulation of extracellular matrix in the injured limb as well. A higher frequency of interstitial nuclei were positive for phospho-SMAD3 in the injured limb (p < 0.05), providing some evidence for activation of a fibrogenic program through transforming growth factor β following an ACL injury. The injured limb also displayed reduced satellite cell abundance, increased fiber denervation and DNA damage associated with apoptosis (p < 0.05), indicating alterations within the muscle itself after the ligament injury. Injury of the ACL induces a myriad of negative outcomes within knee extensor muscles, which likely compromise the restorative capacity and plasticity of skeletal muscle, impeding rehabilitative efforts. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1876-1885, 2017.

Keywords: CD56; FAP; anterior cruciate ligament; collagen; fibroblast.

Figure 1. Increased muscle collagen accumulation following ACL injury

(A–B) Representative images showing collagen (red) staining in the non-injured (A) and injured (B) limb skeletal muscle. Scale bar = 50µm. C) Quantification of muscle collagen content presented as mean percentage of total muscle area ± SEM. N = 10 subjects. NI = non-injured; I = injured. * Significantly different from the non-injured limb (p < 0.05).

Figure 2. ACL injury induces increased connective tissue fibroblast abundance in skeletal muscle

(A–B) Representative immunohistochemical images demonstrating Tcf4+ connective tissue fibroblasts (white arrowheads, red), muscle connective tissue (green), and DAPI (blue) in the non-injured (A) and injured (B) limb skeletal muscle. Scale bar = 50µm. (C) Quantification of Tcf4+ fibroblasts presented as mean number of fibroblasts per muscle area (mm²) ± SEM. (D) Correlation of the number of fibroblasts per mm² with percent area of muscle connective tissue in the injured limb. N = 10 subjects. NI = non-injured; I = injured. * Significantly different from the non-injured limb (p < 0.05).

Figure 3. Elevated number of fibrogenic/adipogenic progenitor (FAP) cells in the muscle following an ACL injury

(A–B) Representative immunohistochemical images demonstrating PDGFRα+ FAPs (white arrowheads, red), muscle connective tissue (green), and DAPI (blue) in the non-injured (A) and injured (B) limb skeletal muscle. Scale bar = 50µm. (C) Quantification of PDGFRα+ FAPs presented as mean number of FAPs per muscle area (mm²) ± SEM. (D) Correlation of the number of FAPs per mm² with percent area of muscle connective tissue in the injured limb. N = 9 subjects. NI = non-injured; I = injured. * Significantly different from the non-injured limb (p < 0.05).

Figure 4. PDGFRα+ and Tcf4+ co-localization is affected by ACL injury

Representative images of PDGFRα (A), Tcf4 (B) and DAPI (C) staining in ACL injured muscle. (D) Merged immunohistochemical image demonstrating a PDGFRα+ / Tcf4+ cell (white arrow) and a PDGFRα− / Tcf4+ cell (yellow arrow). Scale bar = 50µm. (E) Quantification presented as mean relative frequency of cells staining for both PDGFRα and Tcf4 versus Tcf4 alone ± SEM. * Significantly different from the non-injured limb (p < 0.05); † Significant effect of PDGFRα-staining status (p < 0.05).

Figure 5. Increased frequency of p-SMAD3+ nuclei in the skeletal muscle following ACL injury

(A) Representative immunohistochemical image demonstrating p-SMAD3 (red), dystrophin (green), and DAPI (blue) in the non-injured limb skeletal muscle. A white arrowhead denotes a p-SMAD3− myonucleus, a yellow arrowhead denotes a p-SMAD3+ myonucleus, a white arrow denotes a p-SMAD3− interstitial nucleus and a yellow arrow denotes a p-SMAD3+ interstitial nucleus. Scale bar = 20µm. (B) Quantification of the frequency of p-SMAD3 myonuclei, expressed as mean percentage of p-SMAD3+ myonuclei ± SEM. (C) Quantification of the frequency of p-SMAD3 interstitial nuclei, expressed as mean percentage of p-SMAD3+ interstitial nuclei ± SEM. N = 10 subjects. NI = non-injured; I = injured. * Significantly different from the non-injured limb (p < 0.05).

Figure 6. Myonuclear apoptosis increases following ACL injury

Representative images of dystrophin (A), TUNEL (B) and DAPI (C) staining in ACL injured muscle. (D) Merged immunohistochemical image demonstrating a TUNEL+ myonucleus (white arrow). Scale bar = 50µm. (E) Quantification presented as mean percentage of fibers with a TUNEL+ myonucleus ± SEM. (F) Quantification of individual subject TUNEL+ myonuclei fiber frequency data. N = 10 subjects. NI = non-injured; I = injured. * Significantly different from the non-injured limb (p < 0.05).

Figure 7. Reduced satellite cell abundance and increased frequency of muscle fiber denervation following ACL injury

(A–D) Representative images of myosin heavy chain type 1 (A), laminin (B), CD56/Neural Cell Adhesion Molecule (NCAM) (C) and DAPI (D) staining in ACL injured muscle. (E) Merged immunohistochemical image demonstrating a CD56+ satellite cell (yellow arrowhead) and a NCAM+ muscle fiber (yellow arrow). Scale bar = 50µm. (F) Quantification of fiber type-specific and pooled CD56+ satellite cell content, expressed as mean CD56+ satellite cells per fiber ± SEM. (G) Quantification of fiber type-specific and pooled NCAM+ fibers, expressed as mean percentage of fibers positive for NCAM ± SEM. N = 10 subjects. NI = non-injured; I = injured. * Significantly different from the non-injured limb (p < 0.05); † Significant effect of fiber type (p < 0.05).