A role for nitric oxide in muscle repair: nitric oxide-mediated activation of muscle satellite cells

Abstract

Muscle satellite cells are quiescent precursors interposed between myofibers and a sheath of external lamina. Although their activation and recruitment to cycle enable muscle repair and adaptation, the activation signal is not known. Evidence is presented that nitric oxide (NO) mediates satellite cell activation, including morphological hypertrophy and decreased adhesion in the fiber-lamina complex. Activation in vivo occurred within 1 min after injury. Cell isolation and histology showed that pharmacological inhibition of nitric oxide synthase (NOS) activity prevented the immediate injury-induced myogenic cell release and delayed the hypertrophy of satellite cells in that muscle. Transient activation of satellite cells in contralateral muscles 10 min later suggested that a circulating factor may interact with NO-mediated signaling. Interestingly, satellite cell activation in muscles of mdx dystrophic mice and NOS-I knockout mice quantitatively resembled NOS-inhibited release of normal cells, in agreement with reports of displaced and reduced NOS expression in dystrophin-deficient mdx muscle and the complete loss of NOS-I expression in knockout mice. Brief NOS inhibition in normal and mdx mice during injury produced subtle alterations in subsequent repair, including apoptosis in myotube nuclei and myotube formation inside laminar sheaths. Longer NOS inhibition delayed and restricted the extent of repair and resulted in fiber branching. A model proposes the hypothesis that NO release mediates satellite cell activation, possibly via shear-induced rapid increases in NOS activity that produce "NO transients."

Figure 1

Representative graphs from one experiment each on normal control (C57BL/6) (A–H) and mdx mice (I–L). Panels show the time course of changes in muscle weight to body weight (mg/g) (A–D, I, and J) and cell yield (cells/muscle × 10⁵) (E–H, K, and L) in three muscles (RTA [♦], LTA [▪], and RSOL [▴]) for groups of mice treated 30 min before injury with saline (A, E, I, and K), l-NAME (B, F, J, and L), l-Arg (C and G), or l-NAME plus l-Arg (D and H). Data from the same animals are represented for muscle weight and cell yield. In normal mice, the immediate increase in RTA yield in saline-treated animals was absent after l-NAME treatment, and the transient increase in LTA yield at 10 min was reduced by l-NAME treatment. In mdx mice, there was no immediate increase in RTA yield above the LTA basal level after injury, whereas at 10 min RTA yield was increased. l-NAME treatment in mdx mice prevented the increased RTA yield at 10 min.

Figure 2

Time course of cell yield (cells/muscle) expressed as the ratio of RTA to LTA (mean ± SEM) for normal mice (C57BL/6 and B6129SF, three experiments, ♦), normal mice treated with l-NAME (C57BL/6, three experiments, ▴) and “NOS mutant” mice including mdx and B6129S-Nos1^tm1Plh (NOS-I knockout mice, three experiments, ▪). Satellite cell activation (cell yield ratio of RTA to LTA) in normal mice begins at 0 min and is significantly greater than in mice with NOS inhibition as a result of pharmacological treatment (by l-NAME), a primary gene defect (NOS-I knockout mice), or secondary to dystrophin deficiency (mdx mice).

Figure 3

Representative effects of crush injury in normal muscle at 0 min (A and B) and 10 min (C–E) after injury and after saline (A, B, and E) or l-NAME (C and D) pretreatment. (A) LTA section shows normal undamaged muscle. (B) RTA section at 0 min after crush injury. (C) At low magnification, a dark band of hypercontraction in fiber segments (to the left) and extravasated blood cells between fibers appear across the muscle belly at 10 min in a saline-treated animal. Fibers are thin and retracted to the right of the hypercontracted region. (D) Two delta lesions in a fiber after l-NAME treatment and 10 min after crush. (E) Higher-magnification view of muscle 10 min after injury showing extravasated blood cells between hypercontracted and retracted fiber segments and segments with early sarcomere disruption. Original magnification in A, B, D, and E ×132; in C, ×33.

Figure 4

Satellite cell changes in vivo are delayed by NOS inhibition in normal mice treated with saline (A–H) or l-NAME (I–P). (A) M-cadherin outlines a large satellite cell at 0 min after injury. (B) Large m-cadherin⁺ satellite cell on the external lamina 10 min after injury. (C) H&E-stained satellite cells (arrows) in low-magnification RTA fibers at 0 min. (D) At high magnification, hypertrophic satellite cells on fibers in RSOL at 10 min. (E and F) Large satellite cell shows colocalized (yellow) staining for HGF/SF (Texas Red) and c-met (FITC) at 0 min (E) and 10 min (F). (G) Two resin sections (stained with toluidine blue) show large satellite cells (between arrowheads) at 0 min in RTA. (H) At 10 min in RTA, satellite cells (arrows) with granulated cytoplasm and euchromatic nuclei are partially lifting off adjacent fibers. (I) After l-NAME treatment, m-cadherin stains an attenuated satellite cell at 0 min in RTA. (J) Satellite cells are not prominent by H&E staining of RTA at 0 min. (K) Thin strips of cytoplasm and a contoured nucleus are probable satellite cells (at arrowhead) at the fiber periphery in resin sections. (L) At high magnification, a myonucleus in a resin section from RTA 10 min after injury shows a folded upper membrane near the contracted fibrils. (M) Ten minutes after injury, large m-cadherin⁺ satellite cells are adjacent to an unstained fiber. (N) At 0 min after injury, c-met (FITC) in satellite cells is not colocalized with HGF/SF (red). (O) A large satellite cell at 10 min after injury shows colocalization (yellow) of c-met (FITC) and HGF/SF (red) fluorescence. (P) A hypertrophic satellite cell (between arrows) is partly separated from an RTA fiber 10 min after injury. Original magnification, ×330 except in C and J (×132).

Figure 5

l-NAME treatment for 6 d reduces normal muscle regeneration. (A) At low magnification (H&E), normal muscle repair after saline pretreatment includes a small necrotic crushed region (to the right), a region of adjacent mononuclear cells and myotubes (arrows), and surviving fiber segments (to the left). (B) New myotubes in the adjacent region contain many central nuclei and eosinophilic sarcoplasm after 6 d of regeneration. (C) New myotubes (arrows) are also present among surviving fibers. (D) After continuous l-NAME treatment for 6 d, the necrotic area (to the left) and the adjacent region of mononuclear cells are enlarged, and a few myotubes (arrow) are present. (E) Among mononuclear cells in the adjacent region, new myotubes (arrows) are thin and contain immature, basophilic cytoplasm. (F) Very few myotubes are found between surviving fiber segments at the ends of the RTA. Original magnification in A and D, ×13; in B, C, E, and F, ×132.

Figure 6

A single l-NAME injection before injury affects myogenic repair in normal muscle. (A) At low magnification (H&E), the RTA 6 d after injury shows a large necrotic region (to the right), an adjacent area of mononuclear cells and small new myotubes (arrows), and surviving fiber segments (to the left). (B) At high magnification, a myotube (arrows) extends between mononuclear cells and a fiber segment. (C) A very thin intensely eosinophilic myotube originates immediately beside a surviving fiber segment. (D) At higher magnification, the same myotube has formed from the satellite cell position, apparently inside the external lamina. (E) An eosinophilic satellite cell (arrow) is elongated into a thin myotube. (F) A column of apparently unfused centrally nucleated cells with granular cytoplasm makes up a myotube. (G) A BrdU⁺ nucleus adjacent to a new myotube. (H and I) Thin new myotube segments are positive for devMHC (Texas Red fluorescence) whether they extend from a larger myotube (H) or are located among mononuclear cells near the crush (I). (J) A crimson satellite cell (arrow) on an EDL fiber. (K) A large satellite cell (arrow) with crimson cytoplasm on a SOL fiber. (L) M-cadherin is present between a satellite cell (arrow) and a small new myotube (arrowheads). (M) M-cadherin staining is intense on satellite cells located on the four intrafusal muscle fibers in a spindle complex. Original magnification in A, ×13; in C, ×33; in B, E, and M, ×132; in D and F–L, ×330.

Figure 7

A single treatment with l-NAME 30 min before injury affects dystrophic muscle regeneration. (A) Low magnification (H&E) shows a large crush region (just to the left), an adjacent region of new myotubes (arrows), and surviving fiber segments (to the right). (B) Many large new myotubes adjacent to the crush. (C) Elongated mononuclear cells and myotubes are m-cadherin⁺. (D) An elongated crimson cell is binucleate and located in the satellite position on a surviving fiber segment. (E) A new myotube extends from a surviving segment and contains devMHC (Texas Red fluorescence). (F) A BrdU⁺ nucleus next to new myotubes with unstained central nuclei. (G) A new myotube contains apoptotic BrdU⁺ nuclear fragments. (H) Large c-met⁺ satellite cells (FITC) on HGF/SF⁺ fibers (Texas Red) in LTA. (I) A large satellite cell (arrow) with granular cytoplasm (H&E) on an LEDL fiber has less prominent margins than in undamaged normal muscles (see Figure 5, J and K). Original magnification in A, ×13; in B, ×130; in C–I, ×330.

Figure 8

A model for the process of shear-induced, NO-mediated events that activate satellite cells after skeletal muscle injury. (A) In undamaged muscle with normal contraction and relaxation, thin quiescent satellite cells are demarcated by m-cadherin and contain few organelles. They are interposed between the overlying external lamina and the sarcolemma of a subjacent fiber and are subject to pulsatile NO released from NOS-Iμ that is anchored to syntrophin. Normally, NO diffuses cylindrically out from the fiber to act on cells and enzymes in the interstitium or is neutralized by red cell hemoglobin in the vessels that wrap each fiber. (B) After sarcolemmal injury, depolarization is not followed by repolarization. A single large contraction produces intense shear between the fiber membrane and the external lamina. Shear induces a bolus release of NO that diffuses down its concentration gradient through the satellite cells hugging the fiber. (C) Satellite cells become activated and begin to enlarge as organelles such as mitochondria hypertrophy. HGF/SF from the damaged fiber is activated and shifts to the c-met receptor on satellite cells. Fibrils hypercontract and damaged segments retract within the external lamina, maintaining shear and NO release and activating satellite cells along the fiber length. The adhesiveness of m-cadherin decreases, and the damaged fiber releases proteins, including HGF/SF, to the interstitium. A released factor, such as HGF/SF, enters the circulation and can transiently activate distant satellite cells on undamaged muscles, although normal pulsatile NO release will mostly attenuate that response. Capillaries dilate and blood cells extravasate into the interstitium. (D) Fiber segments fully retract and satellite cells become motile precursors as HGF/SF binds to c-met. The external lamina remains as a scaffold for the satellite cells, now surrounded by less adhesive m-cadherin. The precursors may leave the fiber as the sequential expression of early immediate genes, muscle regulatory genes, proliferating cell nuclear antigen, and later DNA synthesis begin before proliferation.