4-AMINOPYRIDINE ATTENUATES MUSCLE ATROPHY AFTER SCIATIC NERVE CRUSH INJURY IN MICE

Abstract

Introduction

We recently demonstrated the beneficial effects of 4-aminopyridine (4-AP), a potassium channel blocker, in enhancing remyelination and recovery of nerve conduction velocity and motor function after sciatic nerve crush injury in mice. Although muscle atrophy occurs very rapidly after nerve injury, the effect of 4-AP on muscle atrophy and intrinsic muscle contractile function is largely unknown.

Methods

Mice were assigned to sciatic nerve crush injury and no-injury groups and were followed for 3, 7, and 14 days with/without 4-AP or saline treatment. Morphological, functional, and transcriptional properties of skeletal muscle were assessed.

Results

In addition to improving in vivo function, 4-AP significantly reduced muscle atrophy with increased muscle fiber diameter and contractile force. Reduced muscle atrophy was associated with attenuated expression of atrophy-related genes and increased expression of proliferating stem cells.

Discussion

These findings provide new insights into the potential therapeutic benefits of 4-AP against nerve injury-induced muscle atrophy and dysfunction.

Traumatic peripheral nerve injury (TPNI) represents a major clinical and public health problem that often leads to significant functional impairment and permanent disability. Functional impairment after TPNI could be due a loss in axonal continuity, neuronal cell death, nerve demyelination, conduction defects, and/or muscle denervation. Acute TPNI occurs in 2%−3% of civilians, and more than 50,000 peripheral nerve repair procedures are performed for TPNI annually in the United States alone.^1–7 Traumatic peripheral nerve injuries are also common and increasingly prevalent in combatrelated extremity injuries.^8–10 Despite modern diagnostic procedures and advanced microsurgical techniques, most patients with TPNIs do not regain full motor or sensory function.

Although innervation of skeletal muscle is essential for the maintenance of muscle size, structure, and contractile function,¹¹ denervation results in contractile deficits and rapid muscle-fiber atrophy within the first 2 weeks.^12–14 Muscle atrophy is associated with increased atrophy genes (atrogenes) including muscle ring finger 1 (MuRF-1,) muscle atrophy F-box (or atrogin-1), and specific signaling pathways.^11,15–19 Myogenin is upregulated in skeletal muscle soon after denervation and promotes the expression of MuRF-1 and atrogin-1.¹¹ Forkhead box O (FoxO) transcription factors are also activated and promote muscle atrophy in several pathophysiological conditions, including denevation.^15,18,19 However, adult skeletal muscle has a remarkable regenerative capacity because of its paired box transcription factor 7 (Pax7)-expressing resident stem cells (satellite cells [SC]).^20–22 Prevention of muscle atrophy could potentially improve the functional outcome of TPNI, but few studies have focused on the prevention of muscle atrophy after peripheral nerve damage.^14,23–26

Recently, we discovered that treatment with the potassium channel blocker 4-aminopyridine (4-AP), which is clinically approved to provide symptomatic treatment in patients with multiple sclerosis,^27,28 has the unexpected effect of promoting recovery of function in an established mouse model of peripheral nerve crush injury.²⁹ Beginning 4-AP treatment shortly after injury enhances global functional recovery, promotes remyelination, and improves nerve conduction velocity;²⁹ however, it remained unknown whether 4-AP treatment in this setting has any effect on skeletal muscle loss and contractile activity.

Therefore, this study was designed to explore the possible beneficial effects of 4-AP treatment in muscle atrophy, intrinsic muscle function, and muscle regeneration after acute sciatic nerve crush injury. We hypothesized that if electrical stimulation (ES) can prevent some amount of nerve injury-induced muscle atrophy and dysfnnction,^30–32 then a clinically approved pharmacological agent that excites tissue through modulation of K⁺ channels could also prove beneficial.

MATERIALS AND METHODS

The experimental design and animal protocols were approved by the university committee on animal research at The University of Rochester, NY and The Pennsylvania State University College of Medicine, PA. Ten-week-old female C57BL/6J mice (Jackson Laboratories, Bar Harbor, Maine) weighing 20–25 g were used in this study.

Mouse Model of Peripheral Nerve Crush Injury and 4-AP Treatment

Sciatic nerve crush injury was performed as previously described.³³ Briefly, after intraperitoneal ketamine (60 mg/kg)/xylazine (4 mg/kg) anesthesia, the sciatic nerve was bluntly exposed through the iliotibial band, and crush injury was performed 5 mm proximal to the tibial and peroneal divisions of the sciatic nerve on the right leg by using a smooth forceps with a metal calibration ring to standardize pressure for 30 s. Uninjured limbs were contralateral normal limbs without injury. Control limbs were from animals with no injury on either side.

Mice were given intraperitoneal injections of soluble 4-AP (Sigma-Aldrich, St Louis, Missouri) at a dose of 10 μg or saline starting immediately after crush injury and continued for 3, 7, and 14 days. Before each corresponding day’s dose, mice underwent gait analysis. At the time of humane killing, leg muscles (tibialis anterior [TA], extensor digitorum longus [EDL], gastrocnemius-soleus [GS]) were harvested for in vitro and ex vivo studies.

Sciatic Functional Index as Determined by Walking Track Analysis

Walking track analysis was performed as previously described.^33–35 Briefly, gait was measured by using 3 variables of footprints: (1) toe spread (TS, first through fifth toes), (2) total print length (PL), and (3) intermediate toe spread (IT, second, third and fourth toes) and the formula sciatic functional index (SFI) = −38.3 ([EPL – NPL] / NPL) + 109.5 ([ETS – NTS] / NTS) + 13.3 ([EIT – NIT] / NIT) − 8.8, where E for experimental (injured) and N for normal (contralateral uninjured) sides.³⁵

Ex Vivo Muscle Contraction Measurement

Isolated EDL muscle contraction was measured by using an ASI muscle contraction system (Aurora Scientific, Aurora, Ontario, Canada) as previously described.^36,37 Briefly, EDL muscles were mounted between 2 platinum electrodes and continuously perfused with oxygenated Ringer solution in the chamber. Muscle optimal length was determined by using a series of 1-HZ stimulations. Stimulus output was set at 120% of the voltage that elicited maximal force.³⁷ Muscles were first equilibrated by using three 500-ms, 150-HZ tetani at 1-min intervals and then subjected to a force frequency. Maximum muscle contractile force was measured at stimulation frequencies ranging from 25 to 250 HZ. To obtain specific force values, absolute force was normalized to muscle cross-sectional area determined by EDL weight and length.³

Gene Expression Levels by Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from GS muscles by using TRIzol reagent according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from RNA by using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, California). All primers were synthesized by Integrated DNA Technologies (Coralville, Iowa). One microgram of DNA was used as the template for real-time polymerase chain reaction (RT-PCR) analysis. Quantitative PCR reactions were performed with SYBR Green Fast Mix in a RotorGene RT-PCR machine (Corbett Research, Carlsbad, California). Changes in the gene expressions of myogenin, MuRF-1, and FoxO transcription factors (FoxO1, FoxO3, and FoxO4) were measured, and all data were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). A list of primers and their corresponding sequences can be found in Supporting Information Table 1.

Immunofluorescence Analysis

Frozen and OCT-embedded experimental (sciatic nerve injury) and contralateral (uninjured) TA or EDL muscles from each test group were sequentially cross-sectioned at 10 μm and stored at −80° C. Tissue sections were incubated with 0.2% Triton X-100 for 10 min and blocked in 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA) > for 30 min at room temperature. After overnight incubation with primary antibody at 4°C, the fluorescent-labeled secondary antibody was incubated for 1 h at room temperature. All slides were mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, Alabama) to detect nuclei. Pax7⁺ and Ki67⁺ cells were counted in the same total area (10 mm²) of cross-sections from 3 mice per group. Minimal Feret’s diameter (MFD) and fiber distributions were quantitated from myofiber cross-sectional area in ImageJ (https://imagej.nih.gov).

Western Blot Analysis

Gastrocnemius muscles obtained from control (CTL) and 7-day postinjury mice were homogenized in radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich) supplemented with phosphatase and protease inhibitor cocktail (Sigma-Aldrich) with zirconium oxide beads (0.5 mm) and Bullet Blender Storm 24 (BBY24M; Next Advance, Troy, New York). Protein concentration was quantitated by bicinchoninic acid assay protein assay kit (Sigma-Aldrich). Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, Massachusetts). Membrane were blocked with 5% bovine serum albumin phosphate-buffered saline Tween-20 and then probed with primary anti-myogenin and anti-GAPDH antibodies overnight at 4°C. After having been washed, membranes were incubated with the appropriate infrared-labeled secondary antibodies (1:20,000) for 1 h. Immunoreactivity was detected by using an Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, Nebraska). Image J was used for densitometry. The experiments were repeated at least 3 times.

Additional Materials Used

Antibodies used were anti-Pax7 (mouse IgG1, dilution 1:100, catalog No. AB_528428; Developmental Studies Hybridoma Bank, Iowa City, Iowa), antibody of laminin (rat or rabbit, dilution 1:1,500, catalog No. L0663 or L939; Sigma-Aldrich), anti-Ki67 (dilution 1:400, catalog No. 9129S; Cell Signaling Technology, Danvers, Massachusetts), anti-myogenin (F5D, dilution 1:100, sc-12732; Santa Cruz Bio-technology, Dallas, Texas), anti-GAPDH (dilution 1:2,500, ab9485; Abcam, Cambridge, Massachusetts), AffiniPure Fab fragment goat anti-mouse IgG (H + L, AFFGAI, 0.1 mg/ml; catalog No. 115–007-003; Jackson ImmunoResearch), AlexaFluor 594-conjugated goat anti-mouse IgG (H + L, dilution 1:1,500, catalog No. A-11032,; Life Technologies, Grand Island, New York), AlexaFluor 488-conjugated goat anti-mouse IgM (dilution 1:1,500, catalog No. A-21042; Life Technologies), and AlexaFluor 647-conjugated goat anti-rabbit or anti-mouse antibodies (dilution 1:1,500, catalog No. A-21244 or A-21235; Life Technologies). All reagents that are not specifically documented were purchased from Sigma-Aldrich.

Data Analysis

All results are presented as mean ± SEM, and all experiments were repeated at least 3 times. Data were analyzed either by two-tailed Student’s t test for paired data from the same experiment and unpaired data from different experiments or by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test or by two-way ANOVA followed by Bonferroni post hoc test for multiple comparisons. Statistical analysis was performed in PRISM 6 (GraphPad Software, San Diego, California), and P <0.05 was considered significant.

RESULTS

4-Aminopyridine Treatment Improved SFI

Once-daily administration of 10 μg 4-AP consistently and significantly improved SFI at both 3 and 7 days postinjury compared with saline-treated mice with identical injuries (Supp. Fig. 1).

4-Aminopyridine Treatment Improved Muscle Mass and Histomorphometry

Uninjured untreated EDL muscle mass was not different from uninjured 4-AP-treated counterparts at all time points (3, 7, and 14 days). Uninjured untreated versus uninjured 4-AP-treated EDL muscle mass (in milligrams) at days 3, 7 and 14 was 8.7 ± 0.52 versus 8.89 ± 0.81, 8.76 ± 0.13 versus 8.72 ± 0.23, and 8.66 ± 0.18 versus 8.58 ± 0.14, respectively. At day 3, mean EDL muscle mass (in milligrams) in uninjured saline and injured saline muscles was 8.7 ± 0.52 versus 8.9 ± 0.26, showing no loss of mass in the injured limb early on. EDL muscle mass (in milligrams) in the uninjured saline treated group at days and 7 and 14 was 8.72 ± 0.10, a combined value of both days because these animals had no injury. EDL muscle (in milligrams) of uninjured, saline treated animals was significantly less atrophied compared with injured groups (injured treated with saline day 7, 7.12 ± 0.26; day 14, 7.02 ± 0.14) but not when compared with injured 4-AP-treated groups (day 7, 8.62 ± 0.67; day 14, 7.63 ± 0.09; Fig. 1B). When expressed as a percentage of uninjured saline group, EDL muscle mass in injured saline groups at day 7 and day 14 postinjury was significantly reduced to 81.7% ± 2.9% and 80.6% ± 1.5%, respectively, whereas EDL muscle mass in injured 4-AP animals at days 7 and 14 postinjury was 98.9% ± 7.7% and 87.5% ± 1.1% of the uninjured saline group, respectively. Thus, compared with the uninjured saline group, EDL muscle mass was significantly protected in the 4-AP treatment group by 18% and 7% at days 7 and 14, respectively. Measurements of the muscle fiber size, in tissue stained with anti-laminin antibodies, confirmed extensive muscle atrophy in saline-treated injured limbs (Fig. 1C,D). 4-Aminopyridine treatment significantly protected muscle fiber area in injured limb (~1.8-fold) compared with the saline-treated injured group (P < 0.01), without an effect on uninjured muscle fiber size (Fig. 1D).

FIGURE 1.

In vivo 4-AP treatment prevenís skeletal muscle atrophy after sciatic nerve crush injury. (A) Representative images of EDL muscles. (B) Weight of EDL muscles (n = 4–6/group). Gray bars for Sal and solid black bars for 4-AP-treated groups. ***P < 0.001 vs. uninjured saline-treated group, ^##P < 0.01 vs. respective saline-treated group. (C) Representative images of laminin IF staining of TA and EDL muscles at different days after injury. Each image represents 9 images from 3 different mice. (D) Quantitation of fiber area in muscle cross-sections (n = 3/group). Open bars for CTL (without injury and treatment), gray bars for Sal, and solid black bars for 4-AP-treated groups. *P <0.05, **P <0.01 vs. CTL, ^##P < 0.01 versus respective saline-treated group. Scale bars = 1 cm in A; 100 μm in C. 4-AP, 4-aminopyridine; CTL, control; EDL, extensor digitorum longus; IF, immunofluorescence; Sal, saline-treated; TA, tibialis anterior. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2 illustrates hematoxylin and eosin (H&E) staining and quantitative analysis of MFD and muscle fiber distribution of TA muscles in CTL, saline, and 4-AP groups. Representative images (Fig. 2A) show more intramyofiber spacing with inflammatory infiltration and few centrally located myocyte nuclei in injured saline-treated compared with CTL animals, whereas injured 4-AP-treated animals had more centralized nuclei with minimal intramyofiber spacing along with inflammatory infiltration compared with the injured saline-treatment group. MFD in CTL and saline groups was 35.85 ± 0.24 μm and 30.43 ± 0.22 μm, respectively, whereas 4-AP-treated mice had an MFD of 32.43 ± 0.27 μm (Fig. 2B). The analysis of muscle fiber distribution revealed a shift toward a higher number of smaller myofibers in the saline group compared with CTL or 4-AP groups, with significantly more myofibers sized 25–30 μm, whereas myofibers in the 4-AP group were more uniformly distributed (Fig. 2C).

FIGURE 2.

Effect of in vivo 4-AP treatment on muscle histology and quantitative measurements of muscle fibers in CTL, injured Sal, and injured 4-AP groups. (A) Representative images of TA cross-section stained with H&E; each image (×20) represents 9 images from 3 different mice. Arrows indicate centrally located myocyte nuclei. (B) Mean MFD (in μm); n = 3/group. ***P < 0.001 vs. CTL, ^###P < 0.001 vs. injured saline group. (C) Fiber size distribution determined from MFD as percentage of total fiber number; n = 3/group. *P < 0.05, ***P < 0.001 vs. CTL, ^###P < 0.001 vs. injured saline group. Scale bar =100 μm for all images in A. 4-AP, 4-aminopyridine; CTL, control; TA, tibialis anterior; H&E, hematoxylin and eosin; MFD, minimal Feret’s diameter; Sal, saline-treated.

4-Aminopyridine Treatment Improves Ex Vivo Muscle Force After Crush Injury

Graphs in Figure 3A–C illustrate that, in the uninjured group, there was no effect of 4-AP treatment on the EDL muscle specific force and force frequency relationship (FFR) at any time point and that the effect of 4-AP treatment in these mice was identical to that of saline. In the crush injury group (Fig. 3B,D,F), 4-AP treatment tended to increase the specific force of EDL muscles at day 3 compared with the saline group, and the force was significantly higher at all frequencies with 4-AP treatment compared with the saline-treated group at days 7 and 14 postinjury.

FIGURE 3.

Effect of in vivo 4-AP treatment on the ex vivo contractile function in EDL muscles. Mean specific force in EDL muscles of Sal (solid circles) and 4-AP-treated (open squares) animals. (A,C,E) Uninjured groups; n = 3–4/group. (B,D,F) Injured groups; n = 3–6/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective Sal group. 4-AP, 4-aminopyridine; EDL, extensor digitorum longus; Sal, saline-treated.

Effect of 4-AP Treatment on the Expression of Muscle Atrophy Genes and Transcription Factors

Quantitative RT-PCR analysis of muscle atrophy genes (Fig. 4) revealed significantly increased expression of myogenin and MuRF-1 in the saline-treated group at days 3 and 7 postinjury, respectively, and 4-AP treatment totally abolished this increased expression (Fig. 4A,B). FoxO1 and FoxO3 expression were also significantly higher in the saline-treated group initially after 3 days of injury (Fig. 4C,D), but FoxO1 returned to baseline within 7 days, and FoxO3 remained elevated for 14 days. 4-Aminopyridine treatment had no effect on the initial increased levels of FoxO1 and FoxO3 at day 3, but 4-AP treatment reduced both FoxO1 and FoxO3 to a critically low level after 7 days (Fig. 4C,D).

FIGURE 4.

Effect of in vivo 4-AP treatment on the relative expression levels of skeletal muscle atrophy genes and transcription factors. Gene expression levels of myogenin (A), MuRF-1 (B), FoxO1 (C) and FoxO3 (D) in GS muscles normalized to GAPDH and shown as fold increase from the CTL. (A,B,D) n = 3/group; (C) n = 3–7/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CTL; *P < 0.05, ^###P < 0.001 vs. respective saline-treated group. 4-AP, 4-aminopyridine; CTL, control; FoxO, forkhead box O transcription factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GS, gastrocnemius-soleus; MuRF-1, muscle ring finger-1.

Western Blot Analysis of Myogenin Protein and the Effect of 4-AP Treatment

Western blot analysis revealed that the expression level of myogenin in gastrocnemius muscles of injured limb after 7 days of injury was significantly increased (~3-fold) in the saline group compared with the CTL group, and it was reversed by 4-AP treatment to the CTL level (Fig. 5).

FIGURE 5.

Effect of in vivo 4-AP treatment on the expression profiles of skeletal muscle myogenin protein in CTL, injured Sal, and injured 4-AP groups. (A) Representative Western blots for CTL, injured Sal, and injured 4-AP groups. (B) Bar graphs illustrate the relative expression levels of myogenin protein; n = 3–4/group. *P < 0.05 vs. CTL; ^#P < 0.05 vs. injured saline group. 4-AP, 4-aminopyridine; CTL, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Sal, saline-treated.

Effect of 4-AP Treatment on the Cellular and Molecular Markers of Muscle Regeneration

Graphs in Figure 6 provide visualization of expression levels of Pax7-expressing SCs and cell proliferation marker Ki67³⁹ in relation to crush injury-induced muscle atrophy. Results of immunofluorescence analysis of the cross-sections of TA and EDL muscles revealed that 4-AP treatment significantly increased the number of Pax7⁺ cells in the injured muscles compared with the saline group; the increase was 1.9-fold at day 14 postinjury (Fig. 6D). The number of proliferating cells (Ki67⁺ cells) was also significantly higher (1.4-fold) at day 14 with 4-AP treatment compared with saline (Fig. 6E). In contrast, 4-AP treatment had no effect on these markers in the contralateral uninjured leg muscles. Supplementary Figure 2 illustrates how TA muscle cross-sections define the sarcolemma of myofiber with laminin staining and shows the location of Pax7⁺ cells.

FIGURE 6.

In vivo 4-AP treatment increases Pax7- and Ki67-expressing SCs in TA and EDL muscles of injured limb. (A) Representative Pax7/Ki67/DAPI IF images in CTL muscles at day 0; each image represents 9 images from 3 different mice. (B) Representative images of Pax7 (red), Ki67 (green), and DAPI (blue) in 4-AP-treated group at day 14 postinjury; each image represents 9 images from 3 different mice. (C) Representative Pax7/Ki67/DAPI IF images in Sal and 4-AP-treated groups at days 7 and 14 postinjury. Frozen muscle cross-sections were incubated simultaneously with 2 different primary antibodies (Pax7 mouse antibody and Ki67 rabbit antibody) and 2 different secondary antibodies (AlexaFluor 594-conjugated goat anti-mouse IgG and AlexaFluor 488-conjugated goat anti-rabbit IgM). In addition to individual red (Pax7) or green (Ki67) color, combination of red and green colors resulted in yellow cells; each image represents 9 images from 3 different mice. (D,E) The pooled number of Pax7⁺ (D) and Ki67⁺ (E) cells in uninjured and injured groups with or without treatment; n = 3/group. *P < 0.05, ***P < 0.001 vs. CTL; ^#P < 0.05 vs. respective saline-treated group. Scale bars = 100 μm in A; 25 μm in B; 100 μm in C. 4-AP, 4-aminopyridine; CTL, control; DAPI, 4′,6′-diamidino-2 phenylindole; EDL, extensor digitorum longus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IF, immunofluorescence; Pax7, paired box transcription factor 7; SC, satellite cell; TA, tibialis anterior.

DISCUSSION

We demonstrate that, in addition to improving in vivo global motor function as early as day 3, 4-AP treatment significantly reduces muscle atrophy of the injured limb with increased muscle fiber size and improves ex vivo intrinsic contractile force of muscle 7 days postinjury. Our most important finding is that the reduced muscle mass with 4-AP treatment occurred in the presence of more centralized myonuclei, increased MFD, attenuated expression of atrophy-related genes (myogenin, MuRF-1, FoxO1, FoxO3), and increased expression of Pax7⁺ SCs and proliferating Ki67⁺ cells. These findings provide new insights into the beneficial effects of 4-AP in nerve injury-induced muscle atrophy and dysfunction and open a new window for additional investigation.

Contractile deficits from nerve injury accompany a decrease in muscle fiber size, and the final functional outcome of TPNI depends partially on the extent of atrophy of target muscle.¹² We hypothesized that if ES can improve muscle atrophy and function after nerve injury,^{23–26,30,31} then an entirely different approach of cell excitation with pharmacological modulation of membrane K⁺ channels could also prove beneficial in muscle contraction. K⁺ channel blockers are known to prolong action potential duration, increase Ca²⁺ influx, augment force, and lengthen the duration of isometric contraction.⁴⁰ In line with our hypothesis, we found that 4-AP treatment significantly protected muscle mass and muscle fiber size. In addition, a higher number of centrally located myofiber nuclei were revealed by H&E staining as well as a higher number of smaller fibers (MFD), which are characteristics for newly formed muscle fibers during repeated cycles of regeneration and degeneration. Moreover, muscle fiber distribution analysis revealed a more even distribution of myofibers in the 4-AP group compared with the saline group. 4-Aminopyridine treatment also significantly increased contractile force compared with injured saline-treated group. The force that a muscle can generate is determined by muscle size, fiber type, and excitation-contraction (E-C) coupling. Although the mechanism of 4-AP-induced beneficial effects on muscle contractile force remains unknown, evaluation of FFR curves indirectly indicates that improved intracellular Ca²⁺ handling and E-C coupling might play an important role.^41–43 We observed that, in saline-treated mice, FFR curves for crush-injured mice were depressed and shifted to the right compared with those of the uninjured group, suggesting an impairment of E-C coupling and lower twitch-to-tetanus ratio.⁴⁴ In contrast, FFR curves for 4-AP treated crush injury mice were shifted to the left compared with the injured saline-treated group at days 3, 7, and 14, and they were comparable to the FFR curves of the saline-treated uninjured group at days 7 and 14 postinjury. These findings lead us to suggest that 4-AP treatment may improve E-C coupling and intracellular Ca²⁺ handling in injured muscles and, thus, can induce an enduring improvement in ex vivo contractile function in the affected muscle in addition to our recent demonstration of 4-AP-induced improved in vivo nerve conduction velocity and motor function.²⁹

Several gene transcription factors that control the expression of atrophy genes in the evolution of muscle atrophy have been identified.^11,15–17 Myogenin is upregulated in skeletal muscle after nerve injury and regulates the expression of atrogenes that promote muscle proteolysis and atrophy.^11,45 The role of these specific components of muscle atrophy has been directly demonstrated in genetically modified animals.^11,16 It has also been reported that the expression level of muscle atrophy genes after nerve injury increases initially and then remains at higher levels for a short time before decreasing^44,46 and that denervation-induced muscle atrophy peaks at day 7.^16,46,47 We also observed maximum muscle atrophy at day 7, evidenced by significantly reduced muscle weight and size, and 4-AP treatment critically abolished the normal increases in the relative expression levels of myogenin and MuRF-1 between 3 and 7 days of crush injury. In line with RT-PCR data, results of Western blot analysis in muscle homogenates confirmed the role of 4-AP in restoring myogenin levels in affected muscle after sciatic nerve crush injury. The upstream FoxO transcription factors FoxO1 and FoxO3 are involved in the regulation of muscle mass and atrophy.^{15,19,48–50} We found that both FoxO1 and FoxO3 were significantly upregulated within 3 days of crush injury, but the significant effect of 4-AP treatment on the FoxO expression levels was evident only at day 7, and it was more pronounced on FoxO3. Although FoxO3 activation alone has been reported to be sufficient to cause significant muscle atrophy in vivo,^15,19 FoxO members are redundant in their function, and both FoxO1 and FoxO3 participate in muscle atrophy.^50,51 Thus, we were able to show that 4-AP treatment can abolish the upregulation of transcription factors associated with muscle atrophy inTPNI.

Adult SCs are normally quiescent, but, in response to degenerative stimuli, they become rapidly activated, enter the cell cycle, proliferate, and generate myoblasts that either form new myofibers or repair damaged segments of existing myofibers.^52–54 Satellite cells specifically express transcription factor Pax7, and Pax7 is frequently used as a reliable and specific marker for identifying the pool of quiescent and adult SCs.^22,55 Extensive analysis with genetically modified mice has confirmed that Pax7⁺ cells are essential for acute injury-induced skeletal muscle regeneration.^20–22,54 Moreover, induced SC depletion is reported to exacerbate skeletal myofiber atrophy after neuromuscular disruption in mice.³⁶ Ki67 protein is present in low levels in quiescent cells but is increased in proliferating cells, and several studies have used Ki67 reactivity as a specific marker for cell proliferation.³⁹ In our mouse model, 4-AP treatment significantly increased the number of Pax7⁺ and Ki67⁺ cells, implying that an increased number of adult muscle SCs after injury in 4-AP-treated mice may be an important contributing factor to reduced muscle atrophy compared with saline-treated mice.

Our study has some limitations. First, we quantitated the relative gene expressions but not the protein levels for all muscle atrophy gene markers. Second, we did not investigate the molecular and cellular mechanisms of 4-AP treatment-induced improvement of ex vivo muscle function. Finally, we studied the crush injury and not the permanent denervation model.

In previous work, we demonstrated that 4-AP treatment beginning shortly after nerve crush injury enhanced functional recovery and promoted remyelination and nerve conduction velocity.²⁹ In that study, SFI improved within 3 days, and nerve conduction velocity began to show improvement within 7 days of injury. With consistent SFI recovery in the present study, we observed several important 4-AP treatment-related effects on muscle at day 7 post-injury downstream of the same nerve injury. Although we do not have definite evidence of a muscle-specific mechanism for these changes, which are protective and regenerative, our results provide strong evidence that both nerve and muscle recovery are integral components of 4-AP-induced overall functional recovery after crush injury. Skeletal muscle function is also intimately related to neuromuscular junction (NMJ), and NMJ consists of presynaptic nerve terminal, postsynaptic muscle fiber rich in acetylcholine receptors, and terminal Schwann cells (TSC; or perisynaptic Schwann cells). Terminal Schwann cells (nonmyelinating SCs) have been reported to be an integral and essential component of NMJ, and several studies have reported that TSCs play an important role in the development, function, maintenance, and regulation ofNMJs.^36,56–63 Terminal Schwann cells are dynamic after acute nerve injury, and cytoplasmic processes from TSCs serve as platforms for axonal growth and nerve regeneration. Terminal Schwann cells also modulate activity-dependent neurotransmitter release and neurotransmission. It would be interesting to investigate the time course of 4-AP effect on NMJ properties after TPNIs and, thus, the role of TSCs in nerve regeneration and functional recovery.

In conclusion, this study demonstrates the therapeutic potential of 4-AP treatment for the prevention of early neurogenic muscle atrophy and intrinsic muscle dysfunction secondary to TPNI. We have extended our research of 4-AP beyond its known effects on global functional recovery, nerve myelination, and conduction velocity.²⁹ Additional studies in the TPNI model should determine important mechanistic insights in 4-AP-induced neuromuscular protection.

Abbreviations

4-AP

4-aminopyridine

CTL

control

E-C

excitation-contraction

EDL

extensor digitorum longus

ES

electrical stimulation

FFR

force frequency relationship

FoxO

forkhead box O transcription factor

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GS

gastrocnemius-soleus

H&E

hematoxylin and eosin

IT

intermediate toe spread

MFD

minimal Feret’s diameter

MuRF-1

muscle ring finger-1

NMJ

neuromuscular junction

Pax7

paired box transcription factor 7

PL

print length

RT-PCR

real-time polymerase chain reaction

SC

satellite cell

SFI

sciatic functional index

TA

tibialis anterior

TPNI

traumatic peripheral nerve injury

TS

toe spread

TSC

terminal Schwann cell