Fig. 2: Acetylcholine analog prevents cellular alterations in cultured myofibers. Primary cultures of myofibers from flexor digitorum brevis (FDB) muscles were used.

a The permeability of the sarcolemma was measured in time-lapse experiments of ethidium (Etd⁺) uptake performed after 0, 24, 48, and 72 h of culture. Myofibers were cultured under control conditions (white), or treated with 500 µM ATP (blue) or 50 ng/mL + NGF/50 ng/mL BDNF (NGF + BDNF; yellow), or 200 nM Cbc (red). b Etd⁺ uptake rate of myofibers. N = 4 independent experiments; six myofibers were recorded in each experiment, each value is the mean ± SEM. ***p < 0.001 compared with myofibers at 0 h of culture; n.s. non-significant difference, by ANOVA with Bonferroni post hoc test. c Resting membrane potential (RMP) was evaluated at 0, 24, 48, and 72 h of culture. Myofibers were cultured under control conditions, or treated with ATP, NGF/BDNF, or Cbc. N = 5 independent experiments with at least twenty myofibers recorded in each independent experiment. *p < 0.05, compared with myofibers at 0 h of culture by ANOVA with Bonferroni post hoc test. d Upper panel, intracellular Ca²⁺ signal (340/380), lower panel, intracellular Na⁺ signal was recorded at 0 and 48 h of culture using FURA-2 or SBFI, respectively. The colored scale to the right of the panels depicts the color shifts from blue to green as the dye is bound to Ca²⁺ or Na⁺. Parallel cultures were treated at time 0 with 200 nM carbachol (Cbc) and 48 h later the Ca²⁺ and Na⁺ signal was evaluated^. Parallel cultures were treated at time 0 with 200 nM carbachol (Cbc) and 48 h later the Ca²⁺ and Na⁺ signal was evaluated^. Scale bar: 50 µm.