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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Exp Physiol. 2019 Apr 22;104(7):1090–1099. doi: 10.1113/EP087558

Impact of Sarcopaenia on Diaphragm Muscle Fatigue

Matthew J Fogarty a, Carlos B Mantilla a,b, Gary C Sieck a,b,*
PMCID: PMC6602803  NIHMSID: NIHMS1020591  PMID: 30924589

Abstract

Type IIx and/or IIb DIAm fibres comprise more fatigable motor units that are more vulnerable to sarcopaenia - age-associated reductions of specific force and cross-sectional area. By contrast, type I and IIa DIAm fibres comprise fatigue-resistant motor units that are relatively unchanged with age. Fatigue resistance of the DIAm is assessed by normalising the residual force generated after a period of repeated supramaximal stimulation (e.g., 120 s) to the initial maximum force. Since sarcopaenia primarily affects more fatigable DIAm motor units, apparent fatigue resistance improves with ageing. However, the central question is whether there is an ageing-related difference in the residual force generated by the DIAm after repeated stimulation and whether this force is sufficient to sustain ventilatory behaviours of DIAm? In 6- and 24-month old Fischer 344 rats, we assessed the loss of ex vivo DIAm force across 120 s of repeated supramaximal stimulation at 10, 40 and 75 Hz. We found that relative fatigue resistance improved in older rats at 40 and 75 Hz stimulation. Across all stimulation frequencies, DIAm residual force was unchanged with age (~5 N/cm2). We conclude that ageing increases the relative contribution of type I and IIa fibres to DIAm force with decreased contributions of type IIx and/or IIb fibres. The residual force generated by the DIAm after repeated stimulation is sufficient to accomplish ventilatory behaviours, regardless of age.

Keywords: sarcopaenia, specific force, fibre type

INTRODUCTION:

Muscle fatigue is defined as a decline in force generation during repeated activation (Bigland-Ritchie 1984) The diaphragm muscle (DIAm) is comprised of multiple fibre types that vary in their susceptibility to fatigue, with type I and IIa fibres producing lesser forces though fatigue-resistant, and type IIx and/or IIb fibres producing higher forces though readily fatigueable (Fournier and Sieck 1988, Sieck and Fournier 1989, Lattari et al. 1997). In DIAm strip preparations, fatigue is influenced by the rate of stimulation, with less fatigue at low stimulation rates (10 Hz), and greater fatigue at higher stimulation rates (75 Hz). This phenomenon is due to activation of fatigueable type IIx and/or IIb fibres at higher frequencies of stimulation (Metzger and Fitts 1987, Ameredes et al. 2000). Fatigue is commonly assessed using the fatigue index, which relates the residual force after a period of repeated stimulation (e.g., 120 s) to the initial force. Accordingly, the fatigue index depends on the relative contribution and activation of type IIx and/or IIb fibres, producing the majority of the initial force. Conditions that reduce the relative contribution of type IIx and/or IIb fibres to the DIAm (e.g. denervation) increase the fatigue index and show an apparent improvement in fatigue resistance (Sieck et al. 1989, Lewis et al. 1996)

Sarcopaenia is the age-associated atrophy of muscle fibres and a decline in muscle specific force. In previous studies, we found that DIAm sarcopaenia is characterised by reduced relative contributions of type IIx and/or IIb fibres, underlying the age-related decrease in maximum specific force (Gosselin et al. 1994, Elliott et al. 2016, Khurram et al. 2018b), though we found no changes in the fatigue index (Gosselin et al. 1994). In old mice, the reduced relative contribution of type IIx and/or IIb DIAm fibres was associated with an increase in the fatigue index (Greising et al. 2015). However, past reports have only assessed fatigue at 40 Hz (Gosselin et al. 1994, Greising et al. 2015).

In humans, DIAm sarcopaenia specific to type IIx and/or IIb fibres may underlie age-associated impairments in maximum transdiaphragmadic pressure (PdiMAX) generation (Enright et al. 1994, Tolep et al. 1995, Polkey et al. 1997). The inability to generate PdiMAX necessary to accomplish expulsive manoeuvres (coughing and sneezing) may underlie the increased morbidity and susceptibility to respiratory disease observed in older humans (Polkey et al. 1997). In older humans, increased fatigue resistance following maximum efforts (Kent-Braun 2009, Hunter et al. 2016) is entirely consistent with DIAm sarcopenia predominantly affecting type IIx and/or IIb fibres. Thus in both humans and rodents it appears likely that DIAm sarcopenia is selective for IIx and/or IIb fibres. It remains unclear as to whether ventilatory behaviours (eupnoea and breathing against an occluded airway) or expulsive manoeuvres (PdiMAX) can be accomplished following fatigue of DIAm.

The objective of the current study is to assess if the effects of fatigue and sarcopaenia on DIAm specific force are additive. We hypothesize that in sarcopaenic rats (with lower DIAm initial forces), the fatigue index following 40 and 75 Hz stimulations will be higher than in young rats. Regardless of changes in relative fatigue, we hypothesize that the residual specific forces will be unchanged following 120 s of stimulation, due to the contributions of type I and IIa DIAm fibres, that are unaffected by sarcopaenia. We predict that these residual forces will be sufficient to accomplish ventilatory behaviours (eupnoea and occlusion) in young and old rats.

METHODS:

Ethical approval:

Protocols were approved by the Mayo Clinic IACUC (00003068–17), complied with State and Federal laws and NIH guidelines. All animal experiments herein complied with the ethical principles outlines by the Journal and follow the publication standards of The Physiological Society (Drummond 2009, Grundy 2015). Animals were deeply anaesthetized (indicated by absence of both deep pain and palpebral reflexes) with intramuscular ketamine (10 mg/kg) and xylazine (60 mg/kg) and euthanized by exsanguination.

Animals and diaphragm muscle strip preparation:

We used young (6-months old, n=12) and old (24-months old, n=12) female and male Fischer 344 rats obtained from the aged colony at the National Institute of Aging. Food and water were available ad libitum and animals were housed on a 12 hour light/dark cycle. These ages were selected based on survival information (100% and 50%, respectively) (Cameron et al. 1985, Turturro et al. 1999). The DIAm was excised and placed in a tissue bath containing Rees-Simpson’s buffer (pH 7.4) at 26°C and gassed with carbogen (95% O2/5% CO2). A ~2 mm wide strip was dissected from the mid-costal portion of the left DIAm and suspended within a tissue bath, with the costal margin clamped and the central tendon tied with silk and attached to a force transducer (6350, Cambridge Technology, MA). Optimal DIAm length (Lo) and supramaximal stimulus settings were established in a manner identical to past rat studies (Gosselin et al. 1994, Zhan et al. 1998, Ameredes et al. 2000, Elliott et al. 2016, Khurram et al. 2018a, Khurram et al. 2018b). Electrical field stimulation was achieved via platinum plate electrodes placed on either side of the muscle, with stimulation current provided using a stimulator (701C, Aurora Scientific, ON, Canada). The maximum tetanic specific force was assessed at 5, 10, 20, 40, 50, 75 and 100 Hz in a manner identical to past studies (Gosselin et al. 1994, Zhan et al. 1998, Ameredes et al. 2000, Elliott et al. 2016, Khurram et al. 2018a, Khurram et al. 2018b).

Assessment of DIAm fatigue:

Fatigue of the DIAm was assessed using a pattern of direct muscle stimulation as previously described (Sieck et al. 1989, Sieck et al. 1991, Lewis et al. 1992a, Lewis et al. 1992b, Watchko and Sieck 1993, Lewis et al. 1994, Lewis et al. 1996, Lattari et al. 1997, Zhan et al. 1998, Ameredes et al. 2000, Ermilov et al. 2010). Briefly, supramaximal (~150 mA) stimulus pulses (0.05 ms duration) were delivered at 10, 40 or 75 Hz in 333 ms trains repeated each s (33 % duty cycle, approximating the duty cycle of ventilation (Sieck et al. 1984, Richards et al. 2018)) for 120 s. Output of the force transducer data was digitized (1 kHz sampling rate) and recorded in LabChart software (ADInstuments, Dunedin, New Zealand). After each 120 s fatigue trail, there was a 4 minute recovery period, with the entire experimental protocol taking ~35 minutes to perform. An index of fatigue resistance was calculated as a ratio of the residual force after 120 s to the initial force. Specific force of the DIAm was calculated by normalizing force to the estimated cross-sectional area of the DIAm strip (muscle cross-sectional area = muscle strip weight (g)/(Lo (cm) × 1.056 g/cm3). Figure 1 shows examples of force fatigue induced by direct muscle stimulation in a 6-month old and a 24-month old rat at 10 (Figure 1 A), 40 (Figure 1 B) and 75 Hz (Figure 1 C). Fatigue was characterized by a decrease in maximum specific force as well as the relative force decline across time.

Figure 1:

Figure 1:

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Traces of force production during 120 s fatigue tests during 10 Hz stimulation (A), 40 Hz stimulation (B) and 75 Hz stimulation (C) of young (left column) and old (right column) rat DIAm.

Fibre type proportions, cross-sectional areas and relative contributions:

Muscle strips were stretched to 150 % of their resting length (approximating Lo) and flash-frozen in melting isopentane. Serial transverse sections of DIAm strips were cut at 10 μm using a Reichert Jung Frigocut 2800 Cryostate (Reichert Microscope Services, Depew, NY, USA) and immunoreacted with the following antibodies specific to different MyHC isoforms: MyHCSlow (BA-F8, 1:3 dilution; Developmental Studies Hybridoma Bank, Iowa City, IA), MyHC2A (SC-71, 1:3 dilution; Developmental Studies Hybridoma Bank) and Laminin (Sigma L9393, 1:200 dilution; Sigma-Aldrich, St. Louis, MO). In adjacent sections, MyHC2X (6H1, 1:1 dilution; Developmental Studies Hybridoma Bank) were incubated together, with the pattern of staining allowing the distinguishing of DIAm fibre types in a manner extensively used and previously validated (Schiaffino et al. 1989, Prakash and Sieck 1998, Elliott et al. 2016). Muscle cross-sections were imaged using a 20x oil-immersion objective (NA 1.0) on an Olympus FV2000 laser confocal microscope (Olympus America, Melville, NY) in a manner identical to past rat reports (Elliott et al. 2016, Khurram et al. 2018a, Khurram et al. 2018b).

Statistical methods:

The number of rats in each age-group (n=12), was determined by power analysis based on pilot data (n=6 young rats) of DIAm specific force following the 120 s 40 Hz fatigue test (4.7 ± 0.6 N/Cm2). The effect size (Cohen’s d) was calculated with an a priori biologically-relevant expected difference of 15% and equal variance, with sample size required estimated using d=0.8, α=0.05 and β=0.8. A second cohort (young: n=7; old: n=10) were assessed across 10, 40 and 75 Hz stimulations. Fibre type percentage, cross-sectional area and relative contributions of different fibre types to the DIAm were assessed in a subset of rats (n=9 for young and old). Statistical analysis was done using Prism 7 (Graphpad, CA) with Two-way ANOVA and Bonferroni post hoc tests used to compare groups and factor. A three-way ANOVA and Bonferroni post hoc tests used to compare groups, and two factors. All data was assessed for normality with Shapiro-Wilk tests. Significance was set as P<0.05, all data are presented as mean ± 95% confidence intervals (CI).

RESULTS

DIAm sarcopaenia and reduced contribution of type IIx and/or IIb fibres in 24-month old rats:

Sarcopaenia of the DIAm was observed in 24-month old rats, as reflected by a 32% reduction in maximum DIAm specific force (evoked by 75 Hz stimulation; young: 24.4 ± 4.1 N/cm2, n=8; old: 16.4 ± 2.4 N/cm2, n=10; P=0.0006) and a 29 % reduction in the cross-sectional area of type IIx and/or IIb DIAm fibres (young: 3,144 ± 340 μm2, n=9; old: 2,234 ± 90 μm2, n=9; P=0.0003; Figure 2).

Figure 2:

Figure 2:

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Example DIAm cross-sections identifying type I (green), type IIa (red) and type IIx and/or IIb (black) fibres in young (A) and old (B) rats. production during 120 s fatigue tests (40 Hz stimulation) of young (A) and old (B) rat DIAm. Plots of (mean ± 95% CI) DIAm fibre type proportions (C), DIAm cross-sectional areas (μm2; D) and relative contributions (E) show an increased percentage of type I fibres, decreased cross-sectional areas of type IIx and/or IIb fibres, increased relative contributions of type I fibres and reduced relative contribution of type IIx and/or IIb fibres in old (squares) compared to young (circles) rats. Two-Way ANOVA with Bonferroni post hoc test, n=9 rats per age; *P<0.05.

For DIAm fibre proportions, there was an effect of fibre type (F(2,48)=47.2; P<0.0001) and age*fibre type interaction (F(2,48)=4.8; P=0.01), but no effect of age alone (F(1,48)=0.1; P>0.99). Post hoc analysis showed that there was a 15% increase in the proportion of type I fibres in older rats (P=0.047), with no change in the proportion of type IIa (P>0.99) and IIx and/or IIb fibres (P=0.34, Figure 2C). For DIAm fibre cross-sectional area, there was an effect of age (F(1,48)=16.5; P<0.0001), fibre type (F(2,48)=283.7; P<0.0001) and age*fibre type interaction (F(2,48)=19.3; P<0.0001). Post hoc analysis showed that there was a 29% reduction in the cross-sectional areas of type IIx and/or IIb fibres in older rats (P=0.0001), with type I (P>0.99) and IIa fibre cross-sectional areas unchanged (P>0.99; Figure 3D). The relative contribution of different fibre types to DIAm mass was defined as the product of the proportion and cross-sectional areas of each fibre type, divided by the total of all fibre types (Elliott et al. 2016, Khurram et al. 2018b). There was a statistically significant effect of fibre type (F(2,48)=297.2; P<0.0001) and age*fibre type interaction (F(2,48)=37.7; P<0.0001) to the relative contribution to DIAm mass, but no effect of age alone (F(1,48)=0.1; P>0.99). Post hoc analysis showed that there was a 39% increase in the relative contribution of type I fibres (P<0.0001), a 21% decrease in the contribution of type IIx and/or IIb fibres (P<0.0001) in older rats, with no changes in type IIa fibres (P=0.25; Figure 2E).

Figure 3:

Figure 3:

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The first row of plots shows DIAm specific force decline (N/Cm2; mean ± 95% CI) during 120 s of repeated stimulation at 10 (A), 40 (C) and 75 Hz (E) in young (circles) and old (squares) rats. The second row of plots shows relative DIAm force declines (%; mean ± 95% CI) during the 120 s fatigue tests at 10 (B), 40 (D) and 75 Hz (E). Two-Way ANOVA with Bonferroni post hoc test; young: n=8 (13 for 40 Hz), old: n=10 (17 for 40 Hz); *P<0.05.

Fatigue of the DIAm during 120 s of repeated stimulation:

Fatigue of the DIAm was induced by repeated direct muscle stimulation at 10, 40 and 75 Hz over 120 s in both young and old rats. At all ages, DIAm force generation decreased progressively during repeated stimulation (P<0.0001) reaching a comparable residual force at all frequencies after 120 s (~5 N/cm2; Figure 3).

At 10 Hz stimulation, DIAm specific force was reduced from ~6.5 N/cm2 to ~5 N/cm2 after 120 s in both young and old animals (Figure 3A; P<0.0001). There was no significant effect of age (F(1,16)=1.1; P=0.32) or age*time interaction (F(4,64)=0.5; P=0.73) on DIAm specific force induced by 10 Hz (Figure 3A). There was no significant effect of age (F(1,16)=0.2; P=0.67) or age*time interaction (F(4,64)=0.4; P=0.81) on the relative decline in DIAm force (% relative to initial force; Figure 3B).

At 40 Hz stimulation, DIAm specific force was reduced from initial values of ~22.5 N/cm2 in younger and ~13 N/cm2 in older rats to ~4.5 N/cm2 at both ages (P<0.0001; Figure 3C). There was a significant effect of age (F(1,28)=10.2; P=0.004), time and age*time interaction (F(4,112)=20.5; P<0.0001) on DIAm specific force (N/cm2), with post hoc analyses showing significantly lower initial specific forces in older compared to younger rats (sarcopaenia - 42% lower in older rats; P<0.0001) and after 30 s of repeated stimulation (37% lower in older rats; P=0.0005; Figure 3C). Thereafter as repeated stimulation continued the specific forces generated by younger and older DIAm converged to a similar residual value (Figure 3C). When fatigue was characterized as the relative force decline (% initial), there was also a significant effect of age (F(1,28)=8.4; P=0.007) and age*time interaction (F(4,112)=5.5; P=0.0004; Figure 3D). Post hoc analyses showed that the differences in relative DIAm force emerged after 60 s of repeated stimulation with older rats generating higher relative forces compared to younger rats (P=0.001; Figure 1D).

At 75 Hz stimulation, DIAm specific force was reduced from initial values of ~24.5 N/cm2 in younger and ~16 N/cm2 in older rats to ~5 N/cm2 at both ages after 120 s (P<0.0001; Figure 3E). There was a significant effect of age (F(1,16)=4.8; P=0.04), time and age*time interaction (F(4,64)=28.6; P<0.0001) on DIAm specific force (N/cm2), with post hoc analyses showing significantly lower initial specific forces in older compared to younger rats (sarcopaenia; 33% lower in older rats; P<0.0001) and after 30 s of repeated stimulation (24% lower in older rats; P=0.006; Figure 3E). When the fatigue was characterized as the relative force decline (% initial), there was also a significant effect of age (F(1,16)=37.2; P<0.0001) and age*time interaction (F(4,64)=18.2; P<0.0001) on relative DIAm force (Figure 3F). Post hoc analyses showed that the differences in relative DIAm force emerged after 30 s of repeated stimulation with older rats generating higher relative forces compared to younger rats (P=0.003; Figure 3F).

Residual DIAm force following 120 s of repeated stimulation:

Following 120 s of repeated stimulation at 10, 40 ad 75 Hz, the specific force generated by the DIAm was reduced to ~5 N/cm2 in both young and old rats. Across frequencies, neither age (F(1,16)=0.4; P=0.51), frequency (F(2,32)=0.5; P=0.59) or age*frequency interaction (F(2,32)=2.4; P=0.26) had any influence on the final residual specific force generated by the DIAm after repeated stimulation (Figure 4A). By contrast, there was a frequency-dependence of relative DIAm fatigue (% of initial) at 10, 40 and 75 Hz stimulations (Figure 4B), with a significant effect of age (F(1,16)=18.1; P=0.0006), frequency (F(2,32)=190.9; P<0.0001) and age*frequency interaction (F(2,32)=5.0; P=0.01). Post hoc analyses showed that there was less relative DIAm fatigue in older compared to younger rats at 40 Hz (P=0.0002) and 75 Hz (P=0.01) stimulation, with no effect at 10 Hz (P>0.99; Figure 4B).

Figure 4:

Figure 4:

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Plots of (mean ± 95% CI) DIAm specific force (N/Cm2; A) and DIAm relative force (B) and following the 120 s fatigue test at 10, 40 and 75 Hz stimulations in old (squares) and young (circles) rats. Two-Way ANOVA with Bonferroni post hoc test, n=7 young, n=10 old; *P<0.05.

Modelling DIAm pressure generation in initial and fatigued conditions:

We modeled the production of maximum initial force and residual force following 120 s of repeated stimulation (Figure 5), based estimates of DIAm single fibre specific force as previously reported (Geiger et al. 2000, Geiger et al. 2001, Geiger et al. 2002) (Table 1). The model also included the relative contribution of each fibre type to total DIAm mass (Figure 2E). The model also assumed a relative force loss for each DIAm motor unit/muscle fibre type was based on past reports (Sieck and Fournier 1987, Fournier and Sieck 1988) (Table 1).

Figure 5:

Figure 5:

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The left y-axis shows specific force (N/cm2) generated by DIAm type I (white), type IIa (light grey) and type IIx and/or IIb fibres (dark grey) in young and old rats before (initial) and after 120 s fatigue tests (residual). In this model, the initial DIAm specific force of type IIx and/or IIb fibres is 43% lower in old compared to young rats. The residual (fatigued) DIAm specific force of type IIx and/or IIb fibres is 44% lower in old compared to young rats. The specific force generated by type 1 and type IIa fibres in unchanged, regardless of age or fatigue. The residual DIAm specific force of type IIx and/or IIb fibres compared to initial is reduced by ~75% in young and old rats. The right y-axis shows these forces normalised to Pdi in Fischer 344 rats, with Pdimax 100% of maximum DIAm specific force. Note that the model indicates that regardless of age or fatigue, ventilatory behaviours such as eupnoea and occlusion are able to be accomplished by recruitment of type I and IIa fibres. The model shows that activation of fibres within the DIAm is insufficient to achieve the pressures required for expulsive manoeuvres such as sneezing or coughing. Three-Way ANOVA with Bonferroni post hoc tests, n=9 rats per age.

Table 1:

Single Fibre Characteristics for DIAm Force Model

Fibre Type Initial Specific Force - Young (N/Cm2) Residual Specific Force – Young (N/Cm2) Initial Specific Force - Old (N/Cm2) Residual Specific Force - Old (N/Cm2)
Type I 20a 18c 20 18c
Type IIa 24a 22c 24 22c
Type IIx/b 36a 9c 25b 6c
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a

Specific force data from DIAm single fibres was published previously (Geiger et al. 2000, Geiger et al. 2001, Geiger et al. 2002).

b

Derived from observed reductions in maximum specific force generation of ~30 % in DIAm of old Fischer 344 rats (Khurram et al. 2018b).

c

Residual force following 120 s of repeated stimulation based on past reports for DIAm motor units (Sieck and Fournier 1987, Fournier and Sieck 1988, Sieck 1991).

Based on our model, there are differences in the relative contribution of different DIAm fibre types to the initial maximum specific force in both younger and older rats that changed following 120 s of repeated stimulation (P<0.0001; Figure 5). The relative contributions of different fibre types varied with age (F(1,96)=421.0; P<0.0001), fibre type (F(2,96)=320.4; P<0.0001), fatigue (F(1,96)=32.5; P<0.0001) and interactions with age*fibre type (F(2,96)=386.2; P<0.0001), age*fatigue (F(2,96)=20.7; P<0.0001), fibre type*fatigue (F(2,96)=96.9; P<0.0001) and age*fibre type*fatigue (F(2,96)=22.9; P<0.0001). Post hoc analyses showed no difference in the specific force generated by type I or type IIa fibres across age, fatigue or age*fatigue (P>0.99 in all cases). By contrast, initial specific force contributed by type IIx and/or IIb DIAm fibres was reduced by 43% in older compared to younger rats (P<0.0001). Residual specific forces contributed by type IIx and/or IIb fibres decreased following 120 s of repeated stimulation, with a 75% reduction in both younger and older rats (P<0.0001). Following 120 s of repeated stimulation, Residual specific forces contributed by type IIx and/or IIb fibres decreased by 44% in older compared to younger rats (P<0.0001; Figure 5)

These fibre type specific estimates of specific force were plotted against the transdiaphragmatic pressures (Pdi) required to accomplish different motor behaviours of the DIAm, as previously reported in Fischer 344 rats (Khurram et al. 2018b). In this previous study we found that the maximum Pdi (Pdimax) induced by bilateral phrenic nerve stimulation was reduced in older rats compared to younger rats (~89 vs. 112 cm2 H2O, respectively). In both younger and older rats, we found that the Pdi generated during eupnea is ~10 cm2 H2O (~9–11% Pdimax). During airway occlusion, Pdi increases to ~28 cm2 H2O (~25–30% Pdimax). The Pdi generated during expulsive behaviours (sneezing or coughing) is 90% Pdimax. Notably, in both the initial and fatigued conditions, the model predicts that younger and older rats are equally capable of producing occlusion pressures with the recruitment of type S and FR motor units (type I and IIa fibres). By contrast, our model suggests that the ability of older rats to generate sufficient Pdi during expulsive behaviours is compromised even without fatigue. With repetitive activation at higher frequencies, the decrease in DIAm force generation would compromise the ability to generate Pdi for expulsive behaviours in both younger and older rats (Figure 5).

DISCUSSION:

There were four main findings of the present study: i) Sarcopaenia of the DIAm is present in our cohort of older Fischer 344 rats; ii) Increased fatigue index (apparent fatigue resistance) in the DIAm of old rats compared to young following repeated stimulation at 40 and 75 Hz; iii) The residual specific force generated by the DIAm following 120 s of repeated stimulation is equivalent across stimulation frequencies (~5 N/cm2 at 10, 40 and 75 Hz) and is unaffected by sarcopaenia; and iv) The residual force generated by the DIAm following 120 s of repeated stimulation is sufficient to sustain ventilatory function ranging from eupnoea to airway occlusion in both younger and older rats. Taken together, these results suggest that there is no additive effect of DIAm sarcopaenia and fatigue. Our data supports the notion sarcopaenic changes in DIAm occur primarily in type IIx and/or IIb fibres comprising FInt and FF motor units. Importantly, the resilience of type I and IIa DIAm fibres ensures adequate Pdi can be generated to sustain ventilation.

DIAm Sarcopaenia:

The results of the present study confirmed the presence of sarcopaenia in the DIAm of Fischer 344 rats. The magnitude of maximum DIAm force loss (~30), the reduction in the cross-sectional areas (~30%) and relative contribution (~25%) of type IIx and/or IIb DIAm fibres to total mass is consistent with previous reports in Fischer 344 rats (Gosselin et al. 1994, Elliott et al. 2016, Khurram et al. 2018b). In a recent study we reported that in old Fischer 344 rats, there is marked (~25 %) phrenic motor neuron (MN) loss compared to young rats (Fogarty et al. 2018b). This loss disproportionately affects the larger phrenic MNs (Fogarty et al. 2018b), likely to innervate IIx and/or IIb fibres (Sieck and Fournier 1989, Sieck 1991, Fogarty and Sieck 2019) most affected by sarcopaenia. Thus, it is likely that there is a denervation of DIAm fibres in sarcopaenic rats. In concordance, previous studies show unilateral DIAm denervation leads to a reduction in maximum specific force and selective atrophy of type IIx and/or IIb fibres in DIAm strips (Miyata et al. 1995, Lewis et al. 1996) and permeablilised single fibres (Geiger et al 2001).

DIAm Fatigue:

In the present study, in both young and old Fischer 344 rats, there was only a ~20% relative decline in DIAm force generation following 120 s of repeated stimulation at 10 Hz. This minimal DIAm force fatigue at 10 Hz is consistent with results from our previous study in adult Sprague-Dawley rats (Johnson and Sieck 1993). By contrast, repeated stimulation at 40 and 75 Hz for 120 s induced a substantial decline in relative DIAm force (~80% fatigue in young and 70% fatigue in old rats), which is equivalent to data previously reported for Sprague-Dawley rats (Sieck et al. 1989, Watchko and Sieck 1993, Lattari et al. 1997, Zhan et al. 1998, Prakash et al. 1999, Ameredes et al. 2000, Ermilov et al. 2010). Age-related differences in DIAm relative force fatigue was entirely attributed to the lower initial specific forces at 40 or 75 Hz stimulation in old Fischer 344 rats. Regardless of the stimulation frequency or age, residual specific forces following fatigue were ~5 N/cm2. Together, these results indicate that the relative force decline with repetitive stimulation is dependent on the relative contribution of type IIx and/or IIb DIAm fibres. With sarcopaenia, the relative contribution of type IIx and/or IIb fibres decreases and the relative fatigue of the DIAm is reduced. However, it is striking that the residual force generated by the DIAm after repeated stimulation at 10, 40 and 75 Hz was similar in young and old rats, indicating that the relative contribution of fatigue resistant type I and IIa fibres is preserved. These results are entirely consistent with the sparing of smaller phrenic MNs (Fogarty et al. 2018b) and the preservation of ventilatory Pdi (Khurram et al. 2018b) in old Fischer 344 rats.

Preservation of DIAm Force Generation to Accomplish Ventilatory Behaviours:

Obviously, in old age, resilience of the force generating capacity of the DIAm to accomplish ventilatory behaviours ranging from eupnoea to airway occlusion without fatigue is essential to sustain life (Belman and Sieck 1982, Sieck and Fournier 1989, Sieck 1991, Mantilla and Sieck 2011, Fogarty and Sieck 2019). Previously, we reported a model for DIAm motor unit recruitment to accomplish Pdi pressure generation across a range of motor behaviours (Sieck and Fournier 1989, Sieck 1991, Mantilla and Sieck 2011, Fogarty et al. 2018a). In relation to PdiMAX, the lower Pdis during eupnoea were achieved in both young and old rats by recruitment of only fatigue resistant DIAm motor units, comprising type I and type IIa fibres. Following fatigue, activation of type I and IIa fibres was sufficient to generate the Pdi necessary to accomplish eupnoea and breathing against an occluded airway - even when DIAm sarcopenia was apparent (old rats). These modelling results are also consistent with our previous observation that in younger humans, the generation of Pdi less than 40% of PdiMAX could be sustained without appreciable fatigue (Belman and Sieck 1982).

Since PdiMAX is reduced, there is likely to be marked impairment of expulsive manoeuvres of the DIAm (e.g., sneezing and coughing) in sarcopaenia. Furthermore, since these expulsive manoeuvres require the recruitment of fatigable DIAm motor units, these motor behaviours are also susceptible to fatigue if activation is recurring.

Clinical Implications:

The current work and our past reports on DIAm sarcopenia in Fischer 344 rats (Gosselin et al. 1994, Elliott et al. 2016, Fogarty et al. 2018b, Khurram et al. 2018b) are consistent with human studies showing reduced maximum voluntary contractions of DIAm in older individuals (Polkey et al. 1997) and impaired expulsive airway clearing behaviours (Chong and Street 2008). The interaction of age and various comorbidities may cause additional reductions in DIAm function. These comorbidities can be categorized into conditions selectively affecting type IIx and/or IIb fibres, and those that affect all DIAm fibres.

Comorbidities with ageing, such as mechanical ventilation (Sassoon et al. 2002, Sassoon et al. 2014), undernutrition (Lewis et al. 1986), chronic obstructive pulmonary disease (Ottenheijm et al. 2005, Mantilla and Sieck 2013) and corticosteroid treatment (van Balkom et al. 1997) are conditions that cause selective DIAm weakness and atrophy in type IIx and/or IIb fibres. Importantly, in studies where the residual specific forces following fatigue were assessed, these residual forces were sufficient to accomplish ventilation requirements (Sassoon et al. 2014). Of course, the atrophy of type IIx and/or IIb DIAm fibres and functional deficits in airway clearance manoeuvres in these conditions is likely to predispose patients to respiratory infection and sepsis – leading to a more generalised atrophy of DIAm fibres and deleterious consequences for the endurance of ventilation.

In conditions where DIAm fibre atrophy occurs in all fibre types, such as congestive heart failure (Supinski et al. 1994, Howell et al. 1995), sepsis (Hussain et al. 1985, Boczkowski et al. 1988, Leon et al. 1992) and cancer cachexia (Roberts et al. 2013a, Roberts et al. 2013b), there is a marked reduction in the endurance of the DIAm to perform ventilatory manoeuvres. In cases where both generalised atrophy and sarcopaenia are present, severe ventilatory disability and impaired airway clearance would be disastrous for patient outcomes. In patients, investigations into these factors are confounded by a plethora of interventions (e.g. corticosterioid treatment) and/or chronic illness (e.g. diabetes, obesity) (Mantilla and Sieck 2013). In rodent models of generalised DIAm atrophy, use of young animals (mostly less than 3-months old) precludes the assessment of age-associated factors (Starr and Saito 2014). Future studies concentrating on the nexus between conditions of generalised DIAm atrophy and sarcopaenia will be of immense value to gerontologists and critical care specialists alike.

New Findings:

Central questions of this study?

1) Is the residual force generated by the DIAm after repeated activation reduced with sarcopaenia? 2) Is the residual force generated after fatiguing activation sufficient to sustain ventilatory behaviours of DIAm in young and old rats?

Main findings:

Following DIAm fatigue, the residual specific force after 120 s of repeated stimulation was unaffected by ageing and is sufficient to accomplish ventilatory behaviours, but not expulsive manoeuvres (e.g. coughing). The inability to perform expulsive behaviours may underlie the increased susceptibility of older individuals to respiratory tract infections.

Acknowledgements:

We would like to thank Rebecca Macken and Yun-Hua Fang for their assistance in the completion of this project.

Funding: Supported by a National Institutes of Health grants R01-AG044615 and R01-AG057052 (GCS and CBM).

Footnotes

Conflicts of Interest: None of the authors has any conflict of interest to disclose.

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