Functional impact of diaphragm muscle sarcopenia in both male and female mice

Abstract

To perform a range of ventilatory and nonventilatory behaviors, the diaphragm muscle (DIAm) must be able to generate sufficient forces throughout the lifespan. We hypothesized that sarcopenia impacts DIAm force generation and thus limits performance of expulsive, higher force, nonventilatory behaviors. Male and female mice (n = 79) at 6 and 24 mo of age (100 vs. 70–75% survival, respectively) were used to examine transdiaphragmatic pressure (Pdi) generation across motor behaviors in vivo and in vitro DIAm specific force. We found a significant effect of age on maximum Pdi (20–41% decline during tracheal occlusion and bilateral phrenic nerve stimulation), maximum DIAm specific force (30% decline), and DIAm fatigue resistance (15% increase). There were no differences between sexes in these age effects on DIAm performance. These results support our hypothesis that sarcopenia primarily impacts higher force, nonventilatory motor behaviors of the DIAm. Such functional limitations may have negative implications in the ability of the DIAm to generate forces needed for airway clearance in old age and thereby contribute to age-related respiratory complications.

throughout the lifespan, the diaphragm muscle (DIAm) must be able to produce and sustain significant forces (transdiaphragmatic pressure, Pdi) to accomplish a range of ventilatory and nonventilatory behaviors. In human studies, maximum Pdi [determined by the maximal sniff (47) or Mueller maneuver (57)] was found to be 13–25% lower in old (65–83 years of age) compared with young (19–40 years of age) adults. However, the examination of maximal voluntary Pdi in people does not reflect the whole range of the behaviors the DIAm can accomplish. In male subjects, it was reported that age-related respiratory muscle dysfunction limits activities of daily life and thus the quality of life for aging patients (3, 4). Furthermore, old age is also associated with greater susceptibility to respiratory complications (13, 32) and related mortality (30).

We have recently found that in male mice with increasing age, there is sarcopenia, such that the DIAm is both weaker (reduced force per cross-sectional area, specific force) and atrophic by 24 mo of age (23, 24). The atrophy was evident mostly in the type IIx and/or IIb muscle fibers, the largest fibers and those with the greatest specific force (19). Indeed, these fibers represent those comprised in type fatigue intermediate (FInt) and fatigable (FF) motor units, which are necessary for maximal efforts (6). In other pathophysiological conditions, there is also selective atrophy of type IIx and/or IIb muscle fibers. For example, selective atrophy of type IIx and/or IIb DIAm fibers and reduced specific force is evident in hypothyroidism (17), chronic obstructive pulmonary disease (45), undernutrition (35), cervical spinal cord injury (38), and denervation (18, 40, 44). This selective effect on type IIx and/or IIb fibers likely impacts the performance of higher force motor DIAm behaviors. Recruitment of DIAm motor units follows an orderly fashion from slow-twitch (type S) and fatigue-resistant fast-twitch (FR) through more fatigable type FInt and FF motor units. Recruitment of type S and FR DIAm motor units is required for ventilatory behaviors (i.e., eupnea and hypoxia-hypercapnia), whereas higher force nonventilatory behaviors require recruitment of type FInt and FF units (15, 39, 41, 50, 51, 53, 54, 56). Accordingly, we hypothesize that sarcopenia impacts DIAm force generation and thus limits performance of expulsive, higher force, nonventilatory behaviors.

Physiological differences based on sex have been studied in various systems, with and without examining the complication of aging. Sex differences in the cardiovascular system have been well established, even when accounting for age (27). However, in other physiological systems such as the musculoskeletal system much less is known (28). In regards to skeletal muscle, older women have a smaller range of type II fiber cross-sectional areas in the quadriceps muscle than men (7). Furthermore, sarcopenia-related weakness occurs in men and women at varying rates; specifically, there is a greater decline in force in women around the time of menopause, although this sex difference in muscle weakness disappears at much older ages (46). In the present study, sex-related differences in DIAm sarcopenia were also evaluated.

MATERIALS AND METHODS

Animals.

Both male and female mice (C57Bl/6 × 129) were bred and maintained at the Mayo Clinic under specific pathogen-free conditions. Mice were group housed by sex, maintained on a 12-h light-dark schedule, and given free access to food and water. Mice were examined at 6 (young) and 24 (old) mo of age, representing ∼100 and 70–75% survival, respectively. Survival data were calculated from our colony in addition to published values (14, 59). Old female mice were used at a time point that is past the time of ovarian failure (22).

Experimental considerations for the use of animals in gerontological research were taken into advisement for the design of the present study (43). Subsets of mice were used for Pdi and DIAm specific force analysis. At the terminal experiment, mice were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) ip and euthanized by exsanguination. All animal care guidelines and experimental protocols were approved by the Institutional Animal Care and Use Committee at the Mayo Clinic, in compliance with National Institutes of Health and the American Physiological Society guidelines.

Pdi measurements.

A recently adapted method for measuring Pdi in mice with the use of solid-state pressure catheters was used throughout this study (26), in accordance with our previously published method (20, 39, 41, 53, 54, 56). Briefly, two 3.5-Fr Millar solid-state pressure catheters (SPR-524; Millar Instruments, Houston, TX) were inserted through the mouth and positioned to span the thoracic and abdominal surfaces of the DIAm. Measurements of Pdi were conducted during the following conditions (listed in order of recording), with adequate time between motor behaviors to return to eupneic Pdi pressures: 1) spontaneous breathing of room air (eupnea) for 5 min, 2) spontaneous deep breaths (sighs) that occur every 45–60 s, 3) spontaneous breathing stimulated by exposure to hypoxia (10% O₂)-hypercapnia (5% CO₂) for 5 min, 4) spontaneous breathing efforts against sustained tracheal occlusion for 15 s, 5) responses evoked by bilateral phrenic nerve stimulation (0.5-ms-duration pulses at 150 Hz in 300-ms trains repeated each second) by use of straight bipolar electrodes (FHC, Bowdoin, ME), and 6) reflex sneezing induced by chemical stimulation of the nasal airway by intranasal infusion of 10 μl of 30 μM capsaicin. Nine animals per group were tested, although not all animals completed all behaviors (specifically sneezing and bilateral phrenic nerve stimulation); at least three per group were completed in all behaviors. All measurements of Pdi were conducted while the mice were anesthetized (ketamine and xylazine) and spontaneously breathing.

Data were collected with a PowerLab 8/35 data-acquisition system and analyzed by use of LabChart (Millar Instruments). Pdi measurements were band-pass filtered (0.3–30 Hz) and sampled at 100 Hz by using LabChart. The data were then exported to MATLAB (The MathWorks, Natick, MA) for analysis with a custom-designed automated program that detected baseline and peak amplitude (26).

DIAm force.

Measurement of isometric force of the DIAm was conducted as previously described (23, 24, 36–38, 42, 52). Briefly, midcostal segments of the DIAm were dissected and positioned between a micrometer for length adjustment and a force transducer (model 6350, Cambridge Technology, Cambridge, MA). Muscle segments were incubated in a Reese-Simpson buffer circulated with 95% O₂ and 5% CO₂ and maintained at 26°C. The muscle segment was supramaximally stimulated by using platinum plate electrodes (0.5 ms pulse duration), and optimal length was established by maximal twitch (P_t) response. Maximal tetanic force (P_o) was obtained at a stimulation rate of 120 Hz in a 1-s duration train. DIAm specific force was determined as force normalized to physiological cross-sectional area, i.e., muscle mass/(optimal length × muscle density). Fatigability of the DIAm was determined during repetitive stimulation at 40 Hz in 330-ms-duration trains repeated each s for a 2-min period. A fatigue index was calculated as the ratio of the final force after 2 min to the initial force.

Statistical analysis.

Data were analyzed via JMP (versions 10.0; SAS Institute, Cary, NC). Data were analyzed by two-way ANOVA (age × sex) or by three-way ANOVA (behavior × age × sex). The Tukey-Kramer post hoc test was used when justified. Significance was accepted at P < 0.05 and all data are presented as means ± SE.

RESULTS

Animals.

Male and female (total n = 79) mice were used to examine Pdi and DIAm specific force at young (6 mo) and old (24 mo) age. The age-related increase in body mass of mice varied between sexes (interaction P = 0.014; Table 1). Specifically, body mass of male mice increased 15% at 24 mo compared with 6 mo, whereas body mass of female mice increased by 33%. It should be noted that young female mice at 6 mo were significantly smaller than young males; however, no sex differences were present in older mice (Table 1).

Table 1.

Group characteristics of all included mice

	Young		Old		Two-Way ANOVA
	Male (n = 17)	Female (n = 16)	Male (n = 26)	Female (n = 20)	P Value
Age, mo	6.1 ± 0.1	6.0 ± 0.1	24.1 ± 0.1	24.2 ± 0.1	—
Body Mass, g	31.6 ± 1.0	24.8 ± 1.0*	37.0 ± 0.9*†	36.8 ± 2.1*†	Interaction 0.014

Values are means±SE.

^*Significantly different from young male;

^†significantly different from young female.

Pdi.

There was a sex difference in the breathing frequency between male and female mice. The average breathing frequency of male mice (young and old) was higher at 156 ± 7 breaths/min compared with female mice (young and old) at 136 ± 7 breaths/min (main effect of sex P = 0.047; Table 2). Of note, there was merely a trend for an increase in breathing frequency between eupnea and hypoxia-hypercapnia and no effect of age (Table 2).

Table 2.

Respiratory frequency across behaviors and the lifespan in mice

	Young		Old
	Male*	Female	Male*	Female
Eupnea	144 ± 10	132 ± 10	155 ± 8	151 ± 9
Hypoxia/hypercapnia	168 ± 12	139 ± 9	166 ± 15	151 ± 9

Values are means±SE (n = 9/group). Data analyzed by 3-way ANOVA; main effect of age P = 0.094; main effect of sex P = 0.048; main effect of behavior P = 0.139; interaction P = 0.592.

^*Significantly different from female mice.

There was a significant effect of age on the ability of the DIAm to generate higher levels of force as reflected by Pdi (main effect of age P = 0.001; Figs. 1 and 2). There was no effect of age on Pdi generated during lower force ventilatory behaviors of the DIAm (eupnea and hypoxia/hypercapnia). The effect of age was apparent as motor behaviors became increasingly more forceful, such as with tracheal occlusion or bilateral phrenic nerve stimulation. Only small differences were seen across ages in the Pdi generated during sneezing (∼5%). However, with tracheal occlusion the Pdi generated in older mice was ∼41% lower, whereas the Pdi evoked by bilateral phrenic nerve stimulation was ∼27% lower in the older animals. In both age groups, the Pdi generated during tracheal occlusion was near maximal. In the present study, the maximal Pdi during tracheal occlusion (∼72 cmH₂O) in young mice (males and females combined) was very similar to that previously reported (26). In contrast, the maximal Pdi during tracheal occlusion in old mice was only ∼42 cmH₂O. Furthermore, the Pdi evoked by maximal phrenic nerve stimulation was ∼82 cmH₂O in young mice (both sexes), whereas the maximal evoked Pdi in old mice was ∼60 cmH₂O. These results clearly indicate a significant functional impact of aging on the ability of the DIAm to generate higher force, nonventilatory behaviors.

Fig. 1.

Representative transdiaphragmatic pressure (Pdi) tracings from young and old mice (male) across motor behaviors: eupnea, hypoxia (10% O₂)-hypercapnia (5% CO₂), spontaneously occurring deep breaths, sneezing, tracheal occlusion, and bilateral maximal phrenic nerve stimulation. Bar represents 10 cmH₂O.

Fig. 2.

Pdi generated across motor behaviors: eupnea, hypoxia (10% O₂)-hypercapnia (5% CO₂), spontaneously occurring deep breaths, sneezing, tracheal occlusion, and bilateral maximal phrenic nerve stimulation for both male (A) and female (B) mice at 6 and 24 mo of age, young (open bars) and old (solid bars), respectively (n = 9/group). Data analyzed by 3-way ANOVA: interaction P = 0.988; main effect of age P = 0.001; main effect of sex P = 0.153; main effect of behavior P < 0.001. Significant main effect of behavior: eupnea and hypoxia-hypercapnia less than deep breaths and sneezing; and eupnea, hypoxia-hypercapnia, deep breaths, and sneezing less than tracheal occlusion and nerve stimulation. *Significant main effect of age different from young at same behavior.

There were no differences in Pdi generated across all behaviors between male and female mice (main effect of sex P = 0.153; Fig. 2). In both young and old mice, there were significant differences in Pdi across motor behaviors, with increasingly greater Pdi generated during nonventilatory behaviors compared with ventilatory behaviors (main effect of behavior P < 0.001; Fig. 1). Specifically, eupnea and hypoxia-hypercapnia generated the lowest Pdi, followed by deep breaths, sneezing, and tracheal occlusion, which was associated with the highest Pdi, approximating that elicited by maximal bilateral phrenic nerve stimulation.

DIAm force.

Sarcopenia was evidenced by a decrease in both P_t and P_o of the DIAm in older mice of both sexes (∼30% less P_o in older male and female mice; main effect of age P < 0.001; Fig. 3; Table 3). There were no significant differences in P_t and P_o across sexes or interactions between age and sex (P ≥ 0.144). Sarcopenia significantly reduces maximum specific force of the DIAm in old age in both sexes. There was no difference in the ratio of specific P_t to P_o across age or sex (Table 3).

Fig. 3.

Diaphragm muscle (DIAm) force is decreased at 24 mo of age (n = 8/group). Specific force of the DIAm is normalized for the physiological cross-sectional area of the muscle. Data analyzed by 2-way ANOVA: interaction P = 0.145; main effect of age P < 0.001; main effect of sex P = 0.363. *Significantly different from young.

Table 3.

Diaphragm muscle properties across the lifespan in mice

			Two-Way ANOVA P Value
	Male	Female	Age effect	Sex effect	Interaction
Pt, N/cm2			<0.001	0.166	0.358
Young	6.2 ± 0.3	6.4 ± 0.3
Old*	4.3 ± 0.5	5.1 ± 0.3
Pt/Po			0.474	0.354	0.881
Young	0.31 ± 0.01	0.32 ± 0.01
Old	0.33 ± 0.03	0.34 ± 0.02

Values are means±SE (n = 8/group). Maximal twitch force (P_t) and isometric tetanic force (P_o) are normalized to physiological cross-sectional area.

^*Significantly different from young group.

Fatigue resistance of the DIAm increased with age in both sexes (P = 0.036; Fig. 4). There was no difference in DIAm fatigue resistance across sexes or interaction between age and sex (P ≥ 0.063; Fig. 4).

Fig. 4.

DIAm fatigue resistance following 2 min of repetitive stimulation calculated as the ratio of force after 2 min of repetitive stimulation to that of the initial force (2-way ANOVA: main effect of age P = 0.036; main effect of sex P = 0.063; interaction P = 0.882). *Significantly different from young.

DISCUSSION

We previously determined that DIAm sarcopenia disproportionally affects type IIx and/or IIb fibers that comprise more fatigable fast-twitch motor units capable of generating higher forces. Accordingly, the results of the present study support our hypothesis that sarcopenia impacts the performance of expulsive, higher force, nonventilatory behaviors. In contrast, lower force ventilatory behaviors are maintained. Indeed, as a result of aging, the presence of sarcopenia diminishes functional capacity of the DIAm in male and female mice as evidenced by decreases in maximum force and Pdi. There were no differences between sexes in DIAm force or Pdi. The presence of respiratory muscle sarcopenia and reduction in Pdi generation during higher force, nonventilatory motor behaviors in both males and females is likely a central factor in respiratory complications associated with old age.

Activation of ventilatory muscles such as the DIAm is necessary for breathing throughout life, with the DIAm being the primary muscle responsible for inspiration (21). Additionally, the DIAm contributes in coordinated fashion with other muscle groups to accomplish near-maximal expulsive behaviors such as coughing or sneezing, which are necessary for airway clearance (39, 41, 53–55). Pdi measurements are used clinically to evaluate respiratory muscle weakness as outlined in the American Thoracic Society and the European Respiratory Society Statement on Respiratory Muscle Testing (1). The present study found that the forces required for breathing room air (eupnea) are not impacted in old age. Even when breathing was stimulated by hypoxia-hypercapnia, the generation of Pdi was not affected by age. The limited impact of sarcopenia on Pdi generated during ventilatory behaviors was not unexpected, since we recently demonstrated that even after unilateral DIAm paralysis (following unilateral phrenic denervation) there is no change in Pdi during eupnea and hypoxia-hypercapnia (20).

The selective impact of sarcopenia on higher force, nonventilatory behaviors of the DIAm highlights the importance of the reserve capacity of the DIAm. Higher force nonventilatory behaviors of the DIAm require recruitment of type FInt and FF motor units, whereas ventilatory behaviors require only type S and FR motor units (15, 39, 41, 53, 54, 56). Previously we reported that DIAm sarcopenia in males is fiber type specific, with significant atrophy of only type IIx and/or IIb fibers (24); those comprising type FInt and FF motor units (2). Thus it appears that ventilatory behaviors of the DIAm are preserved into old age while higher force, nonventilatory behaviors are compromised by fiber type-specific sarcopenia.

The decrease in higher force, nonventilatory Pdi in old age was evident in both male and female mice. In both sexes, maximal bilateral phrenic nerve stimulation results in 27% less force in old mice. The Pdi generated during tracheal occlusion is near maximal in younger animals [∼70% of maximal Pdi (26)], but, with the age-related decrease in maximum Pdi, the Pdi generated during tracheal occlusion was 41% less in old mice. The functional and clinical implications of higher force nonventilatory behaviors are related to a decrease in the ability of the DIAm to generate forces in old age needed for airway clearance or to withstand apneic events common in obstructive sleep apnea.

Force loss of the DIAm due to sarcopenia was evident in both male and female mice at 24 mo of age (∼30% decrease in P_o in older mice). In male mice, we previously reported DIAm sarcopenia at 24 mo of age (23, 24). The results of the present study now expand our findings to include female mice.

In the present study, DIAm sarcopenia was related to an increase in fatigue resistance. Specifically, both male and female mice had ∼15% greater DIAm fatigue resistance in old age. This increase in DIAm fatigue resistance may be related the fiber type-specific atrophy of type IIx and/or IIb fibers associated with age (24). Previously, we reported an aging-related decrease in the proportion of IIx and/or IIb DIAm fibers in mice (24, 25). Importantly, type IIx and/or IIb fibers comprise type FInt and FF motor units, and it is likely that the reduced contribution of these more fatigable fibers would result in an overall increase in DIAm fatigue resistance. Collectively, the impact of sarcopenia on nonventilatory Pdi may represent a decrease in the ability to sustain high-force behaviors over time. Sarcopenia may also ultimately impact ventilatory behaviors as a result of the reduced force generation and increased demands imposed by concomitant changes in the lung parenchyma and or chest wall mechanical properties in old age as well as coexisting disease. Results of the present study indicated a trend for sex differences in DIAm fatigue resistance at both young and old ages, which is consistent with previous reports that muscles in females exhibit more fatigue resistance than males (31).

Previous studies have reported sex differences in DIAm properties in young adult rats (34, 48). Specifically, Lawler and colleagues (34) found that, in female rats at 12 mo of age, the DIAm had greater total protein content and oxidative enzymatic activity. Unfortunately, this study did not examine the functional impact of the biochemical differences. Future studies could examine possible sex- and age-related differences in myosin heavy chain content per half sarcomere and myosin heavy chain coexpression in single type-identified DIAm fibers, both of which would affect muscle fiber specific force (19). In another study exploring the effects of undernutrition, Prezant and colleagues (48) reported that, in control female rats at 4.5 mo of age, type I and II DIAm fibers were smaller and DIAm specific force was ∼23% greater compared with males. Undernutrition significantly impacted DIAm fiber cross-sectional area and specific force, but these effects were similar across sexes. Unfortunately, this study did not distinguish type IIa fibers from type IIx and/or IIb fibers. It is possible that sex differences in DIAm properties vary across species, e.g., rats vs. mice.

In the present study, Pdi was analyzed in anesthetized spontaneously breathing young and old male and female mice of a hybrid background, in accordance with experimental considerations for the use of animals in gerontological research that recommend using less inbred mouse strains (43). In support, we previously reported in young male mice that there was no difference in Pdi generated across a range of behaviors between the C57Bl/6 × 129 mouse strain used in the present study and the C57Bl/6J strain (26). The C57Bl/6 × 129 strain is very commonly used to backcross and maintain genetically manipulated mice. We selected 24 mo of age based on reported survival statistics [70–75% survival (14, 59)] that were validated in our own mouse colony. This age represents a time when female mice are ovarian deficient, which occurs at ∼20 mo of age (22). As such, the aging effects in the present study are not likely confounded by varying sex hormone signaling between males and females. The absence of sex differences in DIAm at 6 and 24 mo of age does not exclude the possibility that sex differences are present at intermittent ages between 6 and 24 mo of age. In this respect, varying rates of sarcopenia have been noted between men and women around the time of menopause (46). It is possible that evaluating DIAm activity in anesthetized mice may be impacted by differences in the age-related response to ketamine and/or xylazine; however, this possible limitation cannot be specifically accounted for in the present study.

Respiratory muscle sarcopenia (both force and Pdi loss) is likely a central factor contributing to respiratory complications in the aging population. Indeed, old age is associated with greater risk for intensive care unit admission for respiratory failure, frequency of prolonged mechanical ventilation, and the resulting intensive care unit-acquired weakness (9–11, 29, 49, 58). Although the specific link between DIAm sarcopenia and respiratory complications has not been determined, it is probable that the 20–40% impairment in maximum DIAm force due to sarcopenia could be a confounding factor in old age. Furthermore, other confounding age-related respiratory complications (i.e., pneumonia or respiratory distress syndrome) may subsequently impact the DIAm due to higher work of breathing requiring activation of higher force, more fatigable motor units during ventilatory behaviors. The combined impact of these comorbidities and sarcopenia needs to be determined.

Mechanisms underlying DIAm sarcopenia may be related to motor neuron loss in old age (5). Some studies have reported a concurrent loss of motor neuron with sarcopenia (5, 12, 16, 33), whereas others report no loss of motor neurons (8). It is possible that any motor neuron loss may be specific to type FInt and FF motor units, which would explain both the fiber type-specific atrophy and decrease in specific force. Previously, we have demonstrated that denervation of the DIAm results in selective atrophy of type IIx and IIb fibers and a decrease in specific force (18, 44).

This project sought to examine functional components (both force and Pdi) of the respiratory muscles in male and female mice during old age. By elucidating age-related effects on the respiratory system, it may be possible to develop therapeutic strategies to mitigate sarcopenia and age-related respiratory complications. Collectively, the results of the present study indicate that there is an age-related decline in DIAm function in both male and female mice that is fiber type (motor unit type) specific. Importantly, the loss of DIAm function in old age is associated with a loss in the capacity to generate higher force nonventilatory behaviors, which are required for airway clearance.

GRANTS

This research was supported by grants from the National Institute of Health AG-044615 (C. B. Mantilla and G. C. Sieck) and T32-HL-105355 (S. M. Greising), and the Mayo Clinic.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

S.M.G., C.B.M., and G.C.S. conception and design of research; S.M.G., J.S.M.-M., and J.M.S. performed experiments; S.M.G., C.B.M., J.S.M.-M., and J.M.S. analyzed data; S.M.G., C.B.M., J.S.M.-M., and G.C.S. interpreted results of experiments; S.M.G. prepared figures; S.M.G. and G.C.S. drafted manuscript; S.M.G., C.B.M., and G.C.S. edited and revised manuscript; S.M.G., C.B.M., J.S.M.-M., J.M.S., and G.C.S. approved final version of manuscript.