Changes in muscle force recovery and myofiber satellite cell incorporation by modulating nitric oxide signaling in vivo during muscle repair after lengthening contractions

• Published in: The FASEB journal Vol. 36 Suppl 1
• Main Authors: Chuong, Timothy H, Mattson, Megan K, Do, Christina H, Shen, Yiyi, Stevens, Nicole E, Nogueira, Leonardo
• Format: Journal Article
• Language: English
• Published: United States 01.05.2022
• ISSN: 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.R5904

During muscle repair, nitric oxide (NO) has an important role in the activation and proliferation of muscle satellite cells (MuSC). Our group has shown that increasing intracellular S-nitrosothiol signaling in vitro by the inhibition of S-nitrosoglutathione reductase (GSNOR), potentiates the proliferation of muscle progenitor cells. However, it is not known whether S-nitrosothiols interfere with muscle regeneration following injury. We hypothesize that inhibiting GSNOR activity during the proliferative phase of MuSC accelerates force recovery after lengthening contractions. We investigated whether providing excess NO (with ISDN) or inhibiting inducible NO synthase (with L-NIL) or inhibiting GSNOR (with SPL-334) during the first 3 days of recovery from lengthening contractions (MuSC proliferation phase) in vivo would affect muscle re-gain of strength and MuSC fusion to damaged myofibers. Procedures were approved by UCSD-IACUC (protocol# S00250) and followed APS guidelines for animal research. Experiments were performed in 5-6 months old Pax7CreERTdTomato mice (n=20 mice, 10 male and 10 female). Mice were treated tamoxifen (2 mg/mouse/day, i.p., for 5 days), and 7 days later the right leg was subjected to a lengthening contraction protocol (LCP). Contralateral leg was used as a non-injured control. LCP consisted of contracting the anterior crural muscles via electrical stimulation of the peroneal nerve for 200 ms (0.1 ms pulses, 150 Hz pulse-frequency), implementing active plantar flexion in the last 60 ms of stimulation (1,000º.s-1), every 12 s for 30 min (150 contractions). After LCP, mice were separated in 5 groups of 4 mice each and in the first 3 days of recovery from LCP, animals were treated with either DMSO (50 µL/day), or ISDN (20 mg/Kg/day in DMSO), or SPL-334 ([GSNORi] 1mg.Kg/day in DMSO) or L-NIL (0.6 mg/mL in drinking water) or untreated. After 5 days of recovery from LCP, extensor digitorum longus (EDL) muscles from both legs were isolated for contractility (300 ms trains, 0.5 ms pulses, 1-200 Hz, 95% O , 5% CO , 22ºC), tibialis anterior (TA) muscles were fixed for immunohistology, and serum was collected for NO byproducts (nitrate + nitrite) measurements. The LCP resulted in a decrease of the initial torque by ~72% which was not different between the groups of mice. Maximum force of the EDL from LCP-subjected legs was significantly smaller vs control legs for untreated (20±5%), DMSO (7±11%), ISDN (47±10%), and L-NIL (27±7%) (P<0.05 vs. control, two-way ANOVA), but not for GSNORi (-7±5%; P>0.05). TA muscles were fixed, frozen in OCT compound, sliced, then labeled with anti-laminin antibody and DAPI. Satellite cell incorporation into myofibers was determined by the appearance of endogenous TdTomato fluorescence in myofibers. TA and EDL muscles from LCP legs showed a higher quantity of immunofluorescent myofibers compared to the control legs. Plasma NO byproducts were not different between groups. The data suggest that either excessive NO (ISDN) or low NO (L-NIL) impair recovery of force after injury but blocking GSNOR may accelerate the re-gain of strength after a physiologically relevant model of muscle injury.