Inhibition of TrkB kinase activity impairs transdiaphragmatic pressure generation

Abstract

Signaling via the tropomyosin-related kinase receptor subtype B (TrkB) regulates neuromuscular transmission, and inhibition of TrkB kinase activity by 1NMPP1 in TrkB^F616A mice worsens neuromuscular transmission failure (NMTF). We hypothesized that acute inhibition of TrkB kinase activity will impair the ability of the diaphragm muscle to produce maximal transdiaphragmatic pressure (Pdi) without impacting the ability to generate forces associated with ventilation, consistent with the greater susceptibility to NMTF in motor units responsible for higher-force nonventilatory behaviors. Adult male and female TrkB^F616A mice were injected with 1NMPP1 (n = 8) or vehicle (DMSO; n = 8) 1 h before Pdi measurements during eupneic breathing, hypoxia/hypercapnia (10% O₂/5% CO₂), tracheal occlusion, spontaneous deep breaths ("sighs") and during maximal activation elicited by bilateral phrenic nerve stimulation. In the vehicle-treated group, Pdi increased from ~10 cmH₂O during eupnea and hypoxia/hypercapnia, to ~35 cmH₂O during sighs and tracheal occlusion, and to ~65 cm H₂O during maximal stimulation. There was no effect of acute 1NMPP1 treatment on Pdi generated during most behaviors, except during maximal stimulation (~30% reduction; P < 0.05). This reduction in maximal Pdi is generally similar to the worsening of NMTF previously reported with TrkB kinase inhibition in rodents. Accordingly, impaired TrkB signaling limits the range of motor behaviors accomplished by the diaphragm muscle and may contribute to neuromuscular dysfunction, primarily by impacting fatigable, higher force-generating motor units.NEW & NOTEWORTHY TrkB signaling plays an important role in maintaining neuromuscular function in the diaphragm muscle and may be necessary to accomplish the various motor behaviors ranging from ventilation to expulsive, behaviors requiring near-maximal forces. This study shows that inhibition of TrkB kinase activity impairs maximal pressure generation by the diaphragm muscle, but the ability to generate the lower pressures required for ventilatory behaviors is not impacted.

Keywords: diaphragm muscle; motor unit; neuromuscular junction; neuromuscular transmission; neurotrophins.

Fig. 1.

Representative transdiaphragmatic pressure (Pdi) tracings from adult 6 mo old TrkB^F616A mice during eupnea (breathing room air), hypoxia-hypercapnia (HH; 10% O₂-5% CO₂), spontaneous deep breaths (sighs), and sustained tracheal occlusion, as well as maximal Pdi generated by bilateral phrenic nerve stimulation at 150 Hz. TrkB^F616A mice were treated with vehicle or 1NMPP1 for 1 h.

Fig. 2.

Summary results of transdiaphragmatic pressures (Pdi) generated during eupnea, hypoxia-hypercapnia (10% O₂-5% CO₂), sighs, tracheal occlusion and maximal stimulation in TrkB^F616A mice treated with vehicle (n = 6) or 1NMPP1 (n = 7) for 1 h. Data were analyzed using a mixed linear model with animal as a random effect (see Methods). There was a significant effect on Pdi amplitude of behavior (F_4,44 = 76, P < 0.001), treatment group (F_1,11 = 8, P = 0.015), and their interaction (F_4,44 = 5, P = 0.002). Boxplot shows the median, first and third quartiles (box), minimum and maximum values (whiskers). *Significantly different compared with vehicle-treated group in post hoc Tukey-Kramer HSD tests.

Fig. 3.

The diaphragm muscle accomplishes several behaviors requiring a wide range of force generation. A: in a model of motor unit recruitment, orderly recruitment of type slow (S) and fast-twitch fatigue resistant (FR) motor units generates the forces necessary for eupnea, hypoxia-hypercapnia, and even sigh or airway occlusion in mice. Fatigable type FInt and FF units are recruited only during behaviors that require higher forces such as during expulsive behaviors (e.g., coughing) or maximal stimulation. B: force development differs across motor units, with tetanic stimulation of type S and FR motor units (active at lower stimulation frequencies; left panel) resulting in substantially lower forces than stimulation of type FInt or FF motor units (active at higher frequencies; right panel) (2, 64). Inhibition of TrkB kinase activity with 1NMPP1 treatment in TrkB^F616A mice results in neuromuscular transmission failure predominantly at the higher stimulation frequencies of type FInt and FF units (30–50 Hz versus 10–20 Hz at type S and FR units). Accordingly, inhibiting TrkB kinase activity worsens overall force development (B) and Pdi generation (A) in adult lightly anesthetized mice.