TrkB signaling contributes to transdiaphragmatic pressure generation in aged mice

Abstract

Ventilatory deficits are common in old age and may result from neuromuscular dysfunction. Signaling via the tropomyosin-related kinase receptor B (TrkB) regulates neuromuscular transmission and, in young mice, is important for the generation of transdiaphragmatic pressure (Pdi). Loss of TrkB signaling worsened neuromuscular transmission failure and reduced maximal Pdi, and these effects are similar to those observed in old age. Administration of TrkB agonists such as 7,8-dihydroxyflavone (7,8-DHF) improves neuromuscular transmission in young and old mice (18 mo; 75% survival). We hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors. Old male and female TrkB^F616A mice, with a mutation that induces 1NMPP1-mediated TrkB kinase inhibition, were randomly assigned to systemic treatment with vehicle, 7,8-DHF, or 1NMPP1 1 h before experiments. Pdi was measured during eupneic breathing (room air), hypoxia-hypercapnia (10% O₂/5% CO₂), tracheal occlusion, spontaneous deep breaths (“sighs”), and bilateral phrenic nerve stimulation (Pdi_max). There were no differences in the Pdi amplitude across treatments during ventilatory behaviors (eupnea, hypoxia-hypercapnia, occlusion, or sigh). As expected, Pdi increased from eupnea and hypoxia-hypercapnia (∼7 cm H₂O) to occlusion and sighs (∼25 cm H₂O), with no differences across treatments. Pdi_max was ∼50 cm H₂O in the vehicle and 7,8-DHF groups and ∼40 cm H₂O in the 1NMPP1 group (F_8,74 = 2; P = 0.02). Our results indicate that TrkB signaling is necessary for generating maximal forces by the diaphragm muscle in old mice and are consistent with aging effects of TrkB signaling on neuromuscular transmission.

NEW & NOTEWORTHY TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkB^F616A mice susceptible to 1NMPP1-induced inhibition of TrkB kinase activity, maximal Pdi generated by bilateral phrenic nerve stimulation was ∼20% lower after 1NMPP1 compared with vehicle-treated mice. Treatment with the TrkB agonist 7,8-dihydroxyflavone did not affect Pdi generation when compared with age-matched mice. Inhibition of TrkB kinase activity did not affect the forces generated during lower force behaviors in old age.

INTRODUCTION

Signaling through the tropomyosin-related kinase receptor subtype B (TrkB) at the diaphragm muscle (DIAm) is important to sustaining neuromuscular transmission (1, 2) and generating maximal forces (3). Neurotrophins, such as brain-derived neurotrophic factor (BDNF), and TrkB agonists, such as 7,8-dihydroxyflavone (7,8-DHF), acutely enhance DIAm neuromuscular transmission in rodents (2, 4, 5). Indeed, TrkB agonists (including BDNF and 7,8-DHF) increase spontaneous and evoked neurotransmitter release in amphibian neuromuscular junctions (6, 7). In contrast, inhibition of TrkB signaling impairs neuromuscular transmission (8). Previous studies have used a chemical-genetic approach to ascertain the functional and structural effects of disrupting TrkB kinase activity at the neuromuscular junction (3, 4, 8, 9). The TrkB^F616A mice used in these studies harbor a point mutation that renders TrkB kinase activity susceptible to rapid and selective inhibition by the phosphoprotein phosphatase 1 (PP1) derivative 1NMPP1 (10). Inhibition of TrkB kinase activity worsened neuromuscular transmission failure (NMTF) at the DIAm following repetitive nerve stimulation (4, 8) and impaired maximal Pdi generation (Pdi_max) elicited by bilateral phrenic nerve stimulation (3).

The DIAm must generate a wide range of forces to accomplish a variety of behaviors throughout the life span (11, 12). In old age, dysfunction of the phrenic motor system is evident by worse NMTF after repetitive nerve stimulation (4, 13, 14), impairment in Pdi_max generation (14, 15), DIAm fiber denervation (9), sarcopenia (16), and loss of phrenic motor neurons (17). Indeed, NMTF worsens in early old age (18 mo of age; 90% survival) when compared with young adults (6 mo of age; 100% survival), and this effect persists into old age (24 mo of age; 75% survival). In contrast, changes in specific force generation of the DIAm are only evident by 24 mo of age in mice (16, 18), indicating earlier neuromuscular dysfunction that may depend on TrkB signaling.

The role of TrkB signaling may vary across age groups. In a previous study (18), TrkB agonists improved neuromuscular transmission in mice at 6 mo and 18 mo, but not at 24 mo of age. In contrast, inhibition of TrkB kinase activity worsened NMTF in mice at 6 mo, but not at 18 mo or 24 mo of age, compared with age-matched groups. However, NMTF reflects primarily the fatigue of the more fatigable motor units (13, 19, 20), and there is evidence of aging-related atrophy of more fatigable, higher force-generating motor units comprising type IIx and/or IIb DIAm fibers (14). Thus, the effect of TrkB signaling on NMTF in old age may be underestimated in studies using phrenic nerve-DIAm preparations. Pdi measurements can be measured across behaviors that require different levels of force generation by the DIAm and reflect recruitment of DIAm motor units with different functional properties (15, 21–23). The present study evaluated the effects of TrkB signaling on the phrenic motor system in old age; we hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors and exerts minimal effects on ventilation.

METHODS

Animals

Adult male (n = 12) and female (n = 12) TrkB^F616A mice at 19–21 mo of age were used in these experiments. Animals were group caged by sex and maintained in a 12-h light cycle with free access to food and water at Mayo Clinic housing facilities. All protocols were approved by the Institutional Animal Care and Use Committee, in compliance with the National Institute of Health guidelines.

Experimental Treatment

TrkB^F616A mice were assigned in randomized, masked fashion to receive a single 5 μL intraperitoneal injection of vehicle (0.3% DMSO; Sigma-Aldrich, St Louis, MO), 7,8-DHF (10 μm; Tocris, Bristol, UK), or 1NMPP1 (10 mM; Millipore Sigma, Burlington, MA). Previous studies using similar doses and timing of 7,8-DHF showed evidence of rapid effects on TrkB dimerization and phosphorylation (0.5–4 h) (24), as well as neuromuscular transmission (1 h) (5). The dosage and timing of 1NMPP1 is also equivalent with previous work documenting inhibition of TrkB kinase activity in TrkB^F616A mice following 1 h administration (25, 26), as well as functional effects of 1 h 1NMPP1 treatment on neuromuscular transmission (4, 8, 27) and Pdi generation (3). In a previous study, acute 1NMPP1 treatment reduced TrkB phosphorylation by 13-fold in brain protein homogenates in TrkB^F616A mice (27). In the present study, vehicle, 7,8-DHF, and 1NMPP1 treatments were administered via intraperitoneal injection 1 h before experimental procedures, with group allocation revealed only after data analyses were completed.

Transdiaphragmatic Pressure Measurements

Experimental procedures were performed as described previously (3, 16, 21, 22, 28–30). Briefly, mice were lightly anesthetized with fentanyl (0.3 mg/kg), diazepam (5 mg/kg), and droperidol (15 mg/kg) (3, 21). Adequate anesthetic depth was monitored by heart rate, respiratory rate, and deep tissue stimulation. Heart rate and O₂ saturation were monitored with a thigh pulse-oximeter (MouseOx Plus, Starr Life Sciences Corp, Oakmont, PA). Supplemental O₂ was administered when saturation fell below 80% and anesthetic agents were redozed (one-third of the initial dose) as needed.

Pdi was calculated from the difference between esophageal (Pes) and gastric (Pgas) pressures. After tracheal cannulation (19 G), two pressure transducers (MikroTip catheter transducer, 3.5 F, No. 8405249, SPR-524; Millar Instruments, Houston, TX) were placed in the esophagus and stomach. Esophageal (Pes) and gastric (Pgas) pressures were recorded and digitized (400 Hz) with PowerLab 4/35 (ADInstruments, Colorado Springs, CO) and visualized in real-time with LabChart 8. Accurate positioning of the catheters was confirmed by correct deflections of the Pes and Pgas across motor behaviors, followed by postmortem evaluation. A custom-made restriction device was used for abdominal binding to obtain near isometric conditions for DIAm activation. The Pdi signal was band-pass filtered between 0.3 Hz and 30 Hz. Data were exported, downsampled to 100 Hz, and analyzed using a custom-designed semiautomated script in MATLAB (MathWorks, Natick, MA) (3, 15, 22, 28, 31–33). Animals were allowed to reach regular and rhythmic breathing at rest, commonly for ∼30 min. Based on previous studies showing consistent values for resting Pdi (8 ± 2 cm H₂O; means ± SD) in lightly anesthetized mice (3, 22, 33), animals with a eupneic Pdi value outside this means ± 2 SD were not included in the analyses as they may reflect technical issues during the experiment including variances in electrode placement, anesthetic depth, or animal health. Accordingly, Pdi measurements were obtained during: 1) quiet breathing of room air (eupnea); 2) breathing of a hypoxic-hypercapnic gas mixture of 10% O₂ and 5% CO₂ (hypoxia-hypercapnia) for 2 min; 3) breathing efforts against an occluded airway (occlusion) for 15 sec; and 4) maximal Pdi elicited by bilateral phrenic nerve stimulation (Pdi_max). Supramaximal stimulation frequency at 150 Hz was used, and stimulation current was adjusted until maximal Pdi response was elicited, as previously described (3, 22). Deep breaths (“sighs”) were defined as large and spontaneous inspiratory events of at least two times normal eupneic Pdi, followed by an apneic period with elimination of at least one full breath (3, 29, 32, 34). The animals were allowed to rest for at least 2 min between behaviors to allow Pdi amplitude to return to eupneic baseline.

After measuring Pdi across the ventilatory behaviors, the phrenic nerves were exposed and isolated bilaterally by microdissection of the ventral cervical region, before stimulation. Straight parallel bipolar electrodes (FHC, Cat. No. 30211, Bowdoin, ME) and a Grass stimulator (Grass S88, Grass Telefactor, Warwick, RI) were employed to stimulate the phrenic nerves with 0.02-ms duration pulses at a frequency of 150 Hz, in 330-ms duration trains repeated every second, for 3–5 trains. During supramaximal stimulation, deflections in Pes and Pgas were verified to be in the appropriate direction with the duration of the Pdi response matching the stimulation period, and measurements were examined for obvious signs of movement artifact in the individual Pes and Pgas signals or the resulting Pdi signal (22).

Behaviors were analyzed for a 1- to 2-min period during eupnea, the complete 2 min during hypoxia-hypercapnia, every sigh recorded during eupnea and the hypoxia-hypercapnia exposure (up to a total of 5), 2–5 maximal breaths during occlusion, and 1–5 maximal stimulation events for Pdi_max (3, 15, 22, 28, 29, 31, 32). Baseline heart rate before placement of transducers, requirements of extra anesthetic dose, requirements of supplemental O₂, and surgery duration time were also recorded to monitor for adequacy of anesthetic depth. No differences in these parameters were evident across groups.

Statistical Analyses

All statistical analyses were performed using standard statistical software (JMP Pro 11, SAS Institute Inc., Cary, NC). Data were assessed for normal distribution using D’Agostino and Pearson omnibus tests. Pdi amplitude and ventilatory parameters across experimental groups and motor behaviors were evaluated using a mixed linear model with behavior (eupnea, hypoxia-hypercapnia, occlusion, sigh, stimulation), treatment group (vehicle, 7,8-DHF, or 1NMPP1), and their interaction as fixed effects, and animal as a random effect. Based on previous reports of Pdi in mice (3, 4, 22), we estimated that six animals per group would be sufficient to detect a 25% change in maximal Pdi following treatments, with 80% power and α = 0.05. A lack of sex-based differences in aging effects on the phrenic motor system was previously reported for both Pdi (15) and NMTF (35). Accordingly, sex was considered in the experimental design only to ensure balanced allocation into the various groups, and previously undetected sex effects were evaluated by a model that included behavior, treatment, and sex as fixed effects. When appropriate, post hoc analyses were conducted using Tukey–Kramer honestly significant difference (HSD) test. Statistical significance was established at P < 0.05. All experimental data in the text of the manuscript are presented as means ± 95% confidence interval (CI) across behaviors, unless otherwise specified.

RESULTS

Animals

All mice (4 male and 4 female in each treatment group) were treated with vehicle, 7,8-DHF, or 1NMPP1 for 1 h. The final analyses included seven vehicle-treated (4 male, 3 female), seven 7,8-DHF-treated (4 male, 3 female), and eight 1NMPP1-treated mice (4 male, 4 female) after exclusion of two mice using a priori defined criteria for eupneic Pdi (see methods). A significant difference in body weight across sex was observed (male: 36 ± 3 g, female: 28 ± 3 g; F_1,16 = 14; P < 0.01).

Transdiaphragmatic Pressure Measurements

Pdi was successfully recorded during all behaviors in all included vehicle (n = 7), 7,8-DHF (n = 7), or 1NMPP1-treated animals (n = 8). Continuous monitoring of heart rate, respiratory frequency, and O₂ saturation, as well as regular assessment of the corneal reflex and deep pain response were used to maintain anesthetic depth across animals. Data are presented with male and female animals combined because mixed linear models for Pdi did not show an effect of sex (F_1.17 < 0.1; P = 0.99). Representative Pdi tracings across all behaviors and Pdi_max are presented in Fig. 1.

Figure 1.

Representative transdiaphragmatic pressure (Pdi) tracings from a single, aged (19–21 mo old) TrkB^F616A mouse from each treatment group during eupnea (breathing room air), hypoxia-hypercapnia (10% O₂-5% CO₂), breathing against an occluded airway, spontaneous deep breaths (sighs), and maximal Pdi generated by bilateral phrenic nerve stimulation at 150 Hz (Pdi_max). TrkB^F616A mice were treated intraperitoneally with 5 µL of vehicle (0.3% DMSO), 7,8-dihydroxyflavone (7,8-DHF; 10 µM), or 1NMPP1 (10 mM). Note expected differences in Pdi across behaviors. Across treatment groups, Pdi during eupneic breathing of room air and hypoxia-hypercapnia, as well as during occlusion and sighs are comparable. Pdi_max, maximal Pdi generation.

The mixed pool of motor units comprising the DIAm allows for generation of a wide range of forces across multiples species, including rodents and humans (3, 11, 12, 36). Ventilatory behaviors require lower levels force compared with sighs or breathing against an occluded airway (3, 14, 22). As expected, there was a range of Pdi amplitudes (Fig. 2). There was a significant effect on Pdi amplitude of behavior (F_4,74 = 164; P < 0.01) and treatment (F_2,20 = 6; P < 0.01). In addition, there was a behavior × treatment interaction (F_8,74 = 2; P = 0.02). Across treatment groups, there were no differences in Pdi amplitude during eupnea (6 ± 2 cm H₂O), hypoxia-hypercapnia (9 ± 1 cm H₂O), occlusion (23 ± 3 cm H₂O), or sighs (26 ± 4 cm H₂O). Pdi amplitude during occlusion and sighs was ∼4 times higher than the amplitude during eupnea across treatment groups. Pdi_max was significantly reduced by ∼40% in the 1NMPP1 treatment group (37 ± 3 cm H₂O) compared with both the vehicle control (53 ± 9 cm H₂O; P < 0.01) and the 7,8-DHF (60 ± 5 cm H₂O; P < 0.01) treatment groups. There was no difference in Pdi_max between the 7,8-DHF and the vehicle treatment groups (P = 0.56).

Figure 2.

Summary results of transdiaphragmatic pressure (Pdi) generated during eupnea, hypoxia-hypercapnia (10% O₂-5% CO₂), tracheal occlusion, spontaneous deep breaths (sighs), and bilateral phrenic nerve stimulation at 150 Hz (Pdi_max) in TrkB^F616A mice treated with vehicle (n = 7), 7,8-dihydroxyflavone (7,8-DHF; n = 7) or 1NMPP1 (n = 8) for 1 h. Bars show means ± 95% confidence interval (CI). Unique symbols are matched across behaviors per animal in each treatment group. 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_4,74 = 164; P < 0.01) and treatment (F_2,20 = 6; P < 0.01), with a behavior × treatment interaction (F_8,74 = 2; P = 0.02). *Significantly different the other groups in post hoc Tukey–Kramer honestly significant difference (HSD) tests (P < 0.05). Note that the treatment effects are limited to Pdi_max across treatment groups and that there were no significant differences in any of the lower force, ventilatory behaviors. Unique symbols are matched across behaviors per animal in each treatment group.

Ventilatory Parameters

Possible acute effects of altering TrkB signaling on ventilatory parameters (i.e., respiratory rate, inspiratory time, and duty cycle) were evaluated and are shown in Table 1. All data reflect males and females combined because the mixed linear models for ventilatory parameters did not show evidence of an effect of sex on any of these parameters (F_1,16 < 1; P > 0.33). There was a significant effect on respiratory rate of behavior (F_1,19 = 75; P < 0.01), but not treatment (F_2,19 = 2; P = 0.13), and there was no behavior × treatment interaction (F_2,19 = 1; P = 0.57). Respiratory rate was significantly higher during hypoxia-hypercapnia than during eupnea by ∼40%. Overall, respiratory rate increased to 140 ± 11 min⁻¹ during hypoxia-hypercapnia compared with 105 ± 12 min⁻¹ during eupnea. There was a significant effect on inspiratory duration of behavior (F_1,19 = 27; P < 0.01), but not treatment (F_2,19 < 1; P = 0.96) and there was no behavior × treatment interaction (F_2,19 < 1; P = 0.89). Inspiratory duration decreased to 197 ± 12 ms during hypoxia-hypercapnia compared with 223 ± 13 ms during eupnea. Accordingly, there was a significant effect on duty cycle of behavior (F_1,19 = 25; P < 0.01), but not treatment (F_2,19 = 9; P = 0.53) and there was no behavior × treatment interaction (F_2,19 < 1; P = 0.68). Duty cycle increased to ∼45% during hypoxia-hypercapnia compared with ∼40% during eupnea.

Table 1.

Ventilatory parameters are unaffected by altering TrkB signaling

	Vehicle	7,8-DHF	1NMPP1
Respiratory rate, min−1*
Eupnea	120 ± 16	93 ± 22	102 ± 26
Hypoxia-hypercapnia	150 ± 17	128 ± 24	141 ± 21
Inspiratory duration, ms*
Eupnea	223 ± 27	222 ± 23	223 ± 27
Hypoxia-hypercapnia	201 ± 26	194 ± 12	195 ± 26
Duty cycle, %*
Eupnea	45 ± 6	35 ± 7	37 ± 7
Hypoxia-hypercapnia	50 ± 10	41 ± 7	46 ± 7

Data analyzed using a mixed linear model with animal as a random effect and presented as means ± 95% confidence interval (CI). *Behavior effect was evident across parameters (P < 0.001; see results), but no effect of treatment. 7,8-DHF, 7,8-dihydroxyflavone; TrkB, tropomyosin-related kinase receptor B.

DISCUSSION

This study presents evidence that selective and rapid inhibition of TrkB kinase activity, using 1NMPP1 in TrkB^616A mice (8, 10, 27) of old age (19–21 mo; <90% survival), impairs the ability of the DIAm to generate maximal forces but not to generate the lower forces required for ventilatory behaviors. In contrast, acute treatment with the TrkB agonist 7,8-DHF does not affect Pdi generation across behaviors, when compared with the vehicle-treated, age-matched group. These results indicate that TrkB signaling contributes to Pdi_max generation in aged mice and highlight the importance of TrkB signaling in maintaining neuromuscular function in vivo. It is worth noting that in a previous study evaluating the effects of aging and TrkB signaling on neuromuscular transmission failure in TrkBF^616A mice, there was no evidence of an effect of 1NMPP1 on NMTF in mice at 18 or 24 mo of age. In addition, there was reduced NMTF with 7,8-DHF in the 18-mo-old, but not 24-mo-old mice. Although overall changes in NMTF were assessed after 2 min of repetitive stimulation (4), there were no differences across treatment groups in the first 15 s of stimulation, consistent with NMTF following repetitive stimulation (for 2 min) reflecting primarily the fatigue of the more fatigable motor units (13, 19, 37). Thus, the importance of TrkB signaling in old age may be underestimated by studies relying exclusively on NMTF. Indeed, the results of the present study show that TrkB kinase activity remains crucial in maintaining maximal DIAm force generation in aged mice.

TrkB Signaling Is Important in Maintaining Neuromuscular Function in Old Age

Previous studies using acute administration of TrkB agonists, such as BDNF, neurotrophin-4, or 7,8-DHF, highlight the importance of TrkB signaling in maintaining neuromuscular transmission at the adult neuromuscular junction (2, 5, 38–40). Indeed, TrkB activation reduces NMTF after repetitive stimulation (4) and increases acetylcholine release at the presynaptic terminals of individual neuromuscular junctions across rodent models (39). In agreement, acute inhibition of TrkB signaling with short-term use of 1NMPP1 in TrkBF^616A mice or K252a worsens NMTF (2, 4, 8) and impairs Pdi_max generation (3) in similar rodent models. Importantly, the effects of TrkB signaling vary with age across several organ systems (41), including the phrenic motor system (4). In the present study, acute inhibition of TrkB signaling in aged mice impaired Pdi_max generation without affecting the Pdi generated during ventilatory behaviors, consistent with previous results in young mice at 6 mo of age (3). On the other hand, acute treatment with 7,8-DHF did not have an effect on Pdi generation across behaviors, suggesting that endogenous TrkB signaling maintains DIAm force generation across ventilatory behaviors in vivo. In contrast, in vitro inhibition of TrkB signaling worsens NMTF in 6-mo-old but not in the aged mice at 18 mo or 24 mo of age, whereas TrkB activation reduces NMTF after repetitive stimulation in mice both at 6 mo and 18 mo of age (4). Of note, this study showed no differences across treatment groups in NMTF during the first 15 s of stimulation in any age group. Taken together, these results suggest differences between the in vivo and in vitro approaches. Other studies evaluating neurotrophin effects in the phrenic motor system found a lack of neurotrophin effects on the contractile properties of the DIAm across age groups (4, 8).

The forces generated by the DIAm require the recruitment of motor units, with slow-twitch (type S) and fast-twitch fatigue resistant (type FR) motor units being recruited primarily in ventilatory behaviors (eupnea and hypoxia-hypercapnia) and fast-twitch fatigue intermediate (type FInt) and fast-twitch fast fatigable (type FF) motor units being recruited to accomplish nonventilatory and maximal force behaviors (22, 29, 30, 32, 42). NMTF with repetitive stimulation models reflect primarily the fatigue of type FF motor units compared with type S and type FR motor units (13, 19, 20) and, with repetitive stimulation, further fatigue may not be evident if only fatigue resistant units remain. Thus, aging effects that are primarily on the most fatigable units, including loss of motor neurons comprising type FInt and FF units (13, 43), increased susceptibility of type FInt and type FF units to NMTF (17) and increased contribution of fatigue-resistant units to force generation (42, 44) likely underestimated the contribution of TrkB signaling to force generation. Motor behaviors that require the recruitment of the more fatigable motor units (i.e., Pdi_max) are accordingly more dependent on intact neuromuscular transmission. It is important to point out that Pdi_max represents only the initial stimulation trains, whereas NMTF compares the change in force elicited by nerve stimulation to muscle stimulation after 2 min of repetitive stimulation. There is evidence suggesting that in conditions with prominent NMTF, the failure is present in the initial train and it displays progression with repetitive stimulation (13). Indeed, in a study using the spa mouse model that displays early hypertonia and motor unit dysfunction, significantly worsened NMTF during the first stimulation train was evident when compared with the initial train in wild-type mice (45). These differences between the in vivo and in vitro approaches suggest that TrkB kinase activity is essential in maintaining maximal force generation during the initial stimulation trains. Understanding aging and TrkB signaling effects during fatiguing protocols is of interest to future studies.

DIAm Force Generation Is Impaired in Old Age

The DIAm comprises a mixed pool of motor units that allows for generation of a wide range of forces required for several ventilatory and nonventilatory behaviors across species, including mice (3, 11, 12, 36). Previous studies looking at various aspects of DIAm neuromuscular function have observed age-related impairments in neuromuscular function. These effects on the phrenic motor system likely start at the motor neuron level and culminate with reduced DIAm force-generation capacity. Aged rodents display: loss of 25% of phrenic motor neurons (17), impairment of ∼30% in neuromuscular transmission (4), denervation of 20% of DIAm fibers (9, 17), atrophy of type IIx and/or IIb DIAm muscle fibers (comprising type FInt and type FF units), and reduction (∼30%) in maximum specific force (16), when compared with young adult animals (6 mo of age; 100% survival).

Ventilatory behaviors can be accomplished with only a fraction of the maximal forces generated by the DIAm. Indeed, Pdi was ∼10%–20% of maximal during eupneic breathing and hypoxia-hypercapnia and ∼50% Pdi_max during occlusion and sighs, values comparable to previous reports in mice (3, 14, 22). Taken together, these finding suggest that there is a period of vulnerability to age-related effects between 18 mo and 24 mo of age in rodents, likely at the motor neuron level. In the present study, vehicle-treated TrkB^F616A mice between 19 mo and 21 mo of age displayed Pdi_max of 53 ± 10 cmH₂O, a ∼20% reduction compared with the previously reported 65–70 cm H₂O in young mice (3, 22).

Conclusions

The present study shows that inhibition of TrkB kinase activity in old mice reduces the capacity of the DIAm to generate maximal forces by ∼20%, exacerbating age-related reductions in Pdi. In contrast, there is no evidence of effects of aging or TrkB kinase inhibition on the ability of the DIAm to generate the forces necessary for ventilatory behaviors. As Pdi generation reflects the combined effort of all recruited motor units, contributions to reduced Pdi may include phrenic motor neuron loss, impairments in neuromuscular transmission, and sarcopenia (44, 46, 47). Motor neuron loss and sarcopenia appear to be late events (present only at 24 mo of age in rodents; 75% survival). Neuromuscular transmission is impaired early in the aging process and depends on TrkB signaling, and thus related interventions may serve to mitigate neuromuscular dysfunction in old age.

GRANTS

This work was supported by National Institutes of Health Grants R01 AG057052 and R01 AG044615 and the Mayo Clinic.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

M.P.-C., H.M.G., G.C.S., and C.B.M. conceived and designed research; M.P.-C. and H.M.G. performed experiments; M.P.-C. analyzed data; M.P.-C. and C.B.M. interpreted results of experiments; M.P.-C. prepared figures; M.P.-C. drafted manuscript; M.P.-C., H.M.G., G.C.S., and C.B.M. edited and revised manuscript; G.C.S. and C.B.M. approved final version of manuscript.