BDNF is a mediator of glycolytic fiber-type specification in mouse skeletal muscle

Abstract

Brain-derived neurotrophic factor (BDNF) influences the differentiation, plasticity, and survival of central neurons and likewise, affects the development of the neuromuscular system. Besides its neuronal origin, BDNF is also a member of the myokine family. However, the role of skeletal muscle-derived BDNF in regulating neuromuscular physiology in vivo remains unclear. Using gain- and loss-of-function animal models, we show that muscle-specific ablation of BDNF shifts the proportion of muscle fibers from type IIB to IIX, concomitant with elevated slow muscle-type gene expression. Furthermore, BDNF deletion reduces motor end plate volume without affecting neuromuscular junction (NMJ) integrity. These morphological changes are associated with slow muscle function and a greater resistance to contraction-induced fatigue. Conversely, BDNF overexpression promotes a fast muscle-type gene program and elevates glycolytic fiber number. These findings indicate that BDNF is required for fiber-type specification and provide insights into its potential modulation as a therapeutic target in muscle diseases.

Keywords: endurance exercise; myokine; neuromuscular junction; neurotrophic factor; oxidative fiber.

Fig. 1.

BDNF MKO mice show altered spontaneous gait behavior and locomotion. (A) Bdnf gene expression in CTRL and BDNF MKO tissues. Expression values were determined by qPCR and normalized to Hprt. Data are shown as the average fold change ± SEM (n = 3 to 9 per genotype per tissue) relative to the expression in CTRL set to 1. CB, cerebellum; DIA, diaphragm; FB, forebrain; H, heart. (B) Histology of CTRL and MKO TA muscles as determined by hematoxylin and eosin staining. (Scale bar: 100 µm.) (C) Total gross locomotor activity (n = 15 per genotype, average of a 10-d period) and (D) gait locomotor parameters (n = 8 per genotype) of CTRL and MKO animals. Results are expressed as mean ± SEM. Unpaired Student’s t test (A and D) and 2-way ANOVA followed by Sidak’s multiple comparisons (C and D). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Muscle-specific BDNF deletion reduces motor end plate size in the EDL muscle. (A) Gene expression in CTRL and BDNF MKO GAS muscles. Expression values were determined by qPCR and normalized to Hprt. Data are shown as the average fold change ± SEM (n = 12 per genotype) relative to the expression in CTRL set to 1. (B) Confocal microscopy images illustrating the apposition of both pre- and postsynaptic markers and the motor neuron innervation of EDL NMJs from CTRL and MKO mice. (Scale bar: 20 µm.) Quantification of (C) pre- and postsynapse apposition, (D) NMJ innervation, and (E) NMJ fragmentation. (F) NMJ volume distribution from CTRL and MKO EDL muscles (Materials and Methods has the number of NMJ analyzed per muscle per genotype). Results are expressed as percentage (mean ± SEM; n = 4 per genotype). Unpaired Student’s t test (A, C, and E) and 2-way ANOVA followed by Sidak’s multiple comparisons (D and F). *P < 0.05; ***P < 0.001.

Fig. 3.

Lack of BDNF promotes slow muscle contraction and enhances fatigue resistance. (A) In situ TA absolute muscle force frequency relationship as evoked by electrical sciatic nerve stimulation from CTRL (n = 4) and MKO (n = 5) mice. (B) Representative traces of twitch force from both genotypes. (C) Time-to-peak tension and (D) half-relaxation time. (E) CSA-normalized maximal twitch and tetanic forces. (F) Average curves showing the force decline during a 4-min muscle fatigue protocol and of muscle force recovery up to 3 min after fatigue. (G) Representative EMG traces from CTRL (Upper) and MKO (Lower) GAS muscle on 50-Hz stimulation of the sciatic nerve. (H) Average decrement in the amplitude of GAS CMAPs from 1st to 4th stimulation (5- to 50-Hz stimulation: n = 9 to 10 per genotype; 100-Hz stimulation: n = 6 per genotype). Results are expressed as percentage. Unpaired Student’s t test (C–E) and 2-way ANOVA followed by Sidak’s multiple comparisons (A, F, and H). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

BDNF MKO mice show improved running endurance capacity. (A) RER and (B) O₂ consumption as a function of speed in CTRL and MKO animals during endurance exercise challenge. Note that data in A and B are only depicted until a speed of 22 m/min (i.e., 60% of maximum speed). (C) Blood lactate and (D) glucose levels at rest and within 1 min after exhaustion. (E) Maximal O₂ consumption at exhaustion. (F) Maximal speed and (G) total distance reached at exhaustion. Results are expressed as mean ± SEM (n = 9 per genotype except in A, B, and E, where data from 1 CTRL and 1 MKO mouse could not be included due to O₂ artifacts during run acquisition). Unpaired Student’s t test (E–G) and 2-way ANOVA followed by Sidak’s multiple comparisons (C and D). *P < 0.05; **P < 0.01; ^#Significant difference (P < 0.05) between experimental conditions.

Fig. 5.

Lack of BDNF leads to a type IIB to IIX transition in glycolytic muscles. (A) Representative fluorescence microscopy images illustrating the fiber-type composition in the TA-EDL muscle of CTRL and MKO animals. Corresponding color legend for fiber types: type I = red, type IIA = blue, type IIX = unstained (black), type IIB = green, and laminin = white. (Scale bar: 200 µm.) (B) Quantification of fiber-type content in (Left) TA and (Right) EDL muscles (n = 5 per genotype). Note that EDL and TA muscles were analyzed separately. (C) Representative SDH staining of TA-EDL muscles from different CTRL and MKO animals. (Scale bar: 500 µm.) (D) CSA based on minimal Feret’s diameter of (Left) TA and (Right) EDL myofibers (n = 5 per genotype). Results are expressed as percentage (mean ± SEM). (E and F) Gene expression in CTRL and BDNF MKO GAS muscles. Expression values were determined by qPCR and normalized to Hprt. Data are shown as the average fold change ± SEM (n = 12 per genotype) relative to the expression in CTRL set to 1. Unpaired Student’s t test (E and F) and 2-way ANOVA followed by Sidak’s multiple comparisons (B and D). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

BDNF influences skeletal muscle fiber-type specification. (A) Expression of BDNF protein in EV- and BDNF-electroporated TA muscles (n = 5 per condition) as determined by western blot. Note that muscle protein extracts from MKO animals were used as negative controls. (B and C) Gene expression in EV- and BDNF-electroporated TA muscles. Expression values were determined by qPCR and normalized to Hprt. Data are shown as the average fold change ± SEM (n = 5 per condition) relative to the expression in EV set to 1. (D) CSA based on minimal Feret’s diameter of TA myofibers (n = 5 per genotype). (E) Fiber-type composition in small (Left) vs. large (Right) TA fibers (n = 5 per genotype). Results are expressed as percentage (mean ± SEM). Unpaired Student’s t test (B and C) and 2-way ANOVA followed by Sidak’s multiple comparisons (E). *P < 0.05; **P < 0.01.

Fig. 7.

The 24-mo-old BDNF MKO mice show higher muscle mass, grip strength, and oxidative fiber number. (A) Gene expression in young (n = 6 per genotype) and old (n = 7 to 9 per genotype) CTRL and BDNF MKO GAS muscles. Expression values were determined by qPCR and normalized to Hprt. Data are shown as the average foldchange ± SEM relative to the expression in young CTRL set to 1. (B) Balance beam (CTRL n = 9, MKO n = 11), (C) rotarod (CTRL n = 9, MKO n = 12), and (D) treadmill running data (CTRL n = 8, MKO n = 9). (E) Absolute and body mass-normalized forelimb muscle strength as determined by grip test (CTRL n = 9, MKO n = 12). Note that experiments were performed from the age of 23 mo and that some mice from this specific cohort spontaneously died between the start of our behavioral investigation and their euthanasia. (F) Normalized lean and fat mass and (G) absolute vs. normalized muscle mass from 24-mo-old CTRL (n = 7) and MKO (n = 9) animals just before euthanasia. QUAD, quadricep. (H) Representative SDH staining of TA muscles from different old CTRL and MKO animals. Note that sections with the same number originate from the same slide. (Scale bar: 500 µm.). (I) Evaluation of TA muscle fiber composition of old CTRL and MKO animals (n = 6 per genotype from randomly chosen muscles). Results are expressed as mean ± SEM. Unpaired Student’s t test (B–E) and 2-way ANOVA followed by Sidak’s multiple comparisons (A, F, G, and I). *P < 0.05; **P < 0.01; ***P < 0.001; ^##Significant difference (P < 0.01) between conditions.

Fig. 8.

Proposed model by which BDNF signaling regulates neuromuscular physiology. (1) BDNF could act as an autocrine factor to influence the expression of transcriptional regulators involved in fast-twitch muscle-specific gene expression or the expression of synaptic proteins involved in AChR clustering. (2) As a paracrine factor, BDNF could regulate the differentiation of satellite cells into slow vs. fast myofibers. (3) BDNF might also affect myofiber identity and motor end plate structure indirectly (e.g., by modulating the activity of the TrkB receptors present in nerve terminals of motor neurons).