Sensitivity of the airway smooth muscle in terms of force, shortening and stiffness

Full text

Respiratory Physiology & Neurobiology 325 (2024) 104264
Available online 9 April 2024
1569-9048/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Sensitivity of the airway smooth muscle in terms of force, shortening
and stiffness
Louis G´
elinas , Andr´
es Rojas-Ruiz , Magali Boucher , Cyndi Henry , Ynuk Boss´
e
*
Institut Universitaire de Cardiologie et de Pneumologie de Qu´
ebec (IUCPQ) – Universit´
e Laval, Qu´
ebec, QC, Canada
ARTICLE INFO
Edited by: Prof. Yu Ru Kou
Keywords:
Smooth muscle mechanics
Contractile properties
Elastance
Resistance
Lung physiology
ABSTRACT
Eight pig tracheal strips were stimulated to contract with log increments of methacholine from 10
-8
to 10
-5
M. For
each strip, the concentration-response was repeated four times in a randomized order to measure isometric force,
isotonic shortening against a load corresponding to either 5 or 10 % of a reference force, and average force,
stiffness, elastance and resistance over one cycle while the strip length was oscillating sinusoidally by 5 % at
0.2 Hz. For each readout, the logEC50 was calculated and compared. Isotonic shortening with a 5 % load had the
lowest logEC50 (-7.13), yielding a greater sensitivity than any other contractile readout (p<0.05). It was fol-
lowed by isotonic shortening with a 10 % load (-6.66), elastance (-6.46), stiffness (-6.46), resistance (-6.38),
isometric force (-6.32), and average force (-6.30). Some of these differences were signicant. For example, the
EC50 with the average force was 44 % greater than with the elastance (p=0.001). The methacholine sensitivity is
thus affected by the contractile readout being measured.
1. Introduction
While the physiological purpose of the airway smooth muscle is still
debated (Mitzner, 2004, 2007; Seow and Fredberg, 2001; Ameredes,
2007; Boss´
e et al., 2012; DuBois, 2007; Ford, 2007; Fredberg, 2007;
Gunst and Panettieri, 2012; Irvin, 2007; Mead, 2007c2007a, 2007b,
2008; Panettiere, 2007; Pare and Mitzner, 2012; Permutt, 2007; Seow
et al., 2007; Mead, 2007c2007c), its contractile activation with a
spasmogen triggers force, shortening and many other changes in me-
chanics, such as stiffening (Chin et al., 2010; Ijpma et al., 2015, 2020;
Noble et al., 2007; Gazzola et al., 2016; Fredberg et al., 1997). When
these changes occur in vivo, they translate into altered mechanics of the
airway wall and the lung (Mitzner et al., 1992; Boucher et al., 2022;
Chapman et al., 2014), with the potential to inuence airway caliber and
lung function. While force, shortening, and most other contractile
readouts progressively change in response to incremental concentra-
tions of a spasmogen, such as methacholine, their comparative sensi-
tivities are unknown. The extent by which sensitivity changes with the
contractile readout being measured may hint on the role played by the
airway smooth muscle in respiratory mechanics.
With in vitro preparations of airway smooth muscle, isometric force is
by far the most studied contractile readout. An entrenched conviction is
that sensitivity measured in isometric conditions applies to in vivo
physiology. Yet, isometric force does not typically exist in vivo. As
smooth muscle force develops, the muscle shortens and the airway
constricts, and in so doing, it rather adds to the passive recoil force of the
airway wall, which is continuously distended because the surrounding
attached parenchyma is stretched by the negative pressure of the pleural
cavity (Lambert et al., 1993; Oliver et al., 2007). The actual meaning of
isometric force on airway and lung mechanics is thus uncertain, and the
sensitivity to methacholine deduced from isometric force measurements
may well differ from the one affecting lung mechanics.
Many contractile readouts other than isometric force can be
measured in vitro (Chin et al., 2010; Ijpma et al., 2015, 2020; Noble
et al., 2007; Gazzola et al., 2016; Fredberg et al., 1997). They include,
inter alias, shortening, stiffness, elastance, resistance, and the ability to
relax in response to a bronchodilator. Each contractile readout is
potentially relevant to understanding the lung response to an inhaled
spasmogen (Boss´
e and Par´
e, 2013). The contractile readouts being
measured in a given experimental setting are often context-dependent
and are often restricted to one per study. Comparison between studies
are also difcult because different preparations (e.g., tracheal strips,
bronchial rings, precision-cut lung slices, isolated cells,…) from
different species are used in distinct experimental settings (Chin et al.,
2010; Ijpma et al., 2015, 2020; Noble et al., 2007; Gazzola et al., 2016;
Fredberg et al., 1997; Donovan et al., 2015; Ma et al., 2002; An et al.,
* Correspondence to: IUCPQ, Pavillon M, room 2687, 2725, chemin Sainte-Foy, Qu´
ebec, QC G1V 4G5, Canada.
E-mail address: ynuk.bosse@criucpq.ulaval.ca (Y. Boss´
e).
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology
journal homepage: www.elsevier.com/locate/resphysiol
https://doi.org/10.1016/j.resp.2024.104264
Received 5 February 2024; Received in revised form 2 April 2024; Accepted 6 April 2024

Respiratory Physiology & Neurobiology 325 (2024) 104264
2
Fig. 1. Concentration-response of porcine tracheal strips in the isometric condition. A representative force trace is shown in A. For each tracheal strip, the maximal
force at each methacholine concentration (purple asterisks) and the force near the end of each methacholine concentration (short green line) were collected.
Concentration-response curves were then constructed. The ones constructed with the maximal force are shown in B and the ones constructed with the end isometric
force are shown in C. The individual curves and their mean are shown on the left and right panels, respectively. n =8.
L. G´
elinas et al.

Respiratory Physiology & Neurobiology 325 (2024) 104264
3
2016; Harvey et al., 2013; Mailhot-Larouche and Boss´
e, 2019).
Herein, the changes in several contractile readouts in response to
incremental concentrations of methacholine were monitored on porcine
tracheal smooth muscle strips to directly compare sensitivity between
contractile readouts. More precisely, the concentration of methacholine
causing half of the maximal response (EC50) was measured and was
used as a surrogate for sensitivity. A lower EC50 means an increased
sensitivity because it typically implies that the whole concentration-
response curve is shifted to the left and that not only the response is
observed at lower concentrations but that any percentage of the
maximal response also typically occurs at lower concentrations. We
hypothesized that the sensitivity of the airway smooth muscle to
methacholine depends on by the contractile readout being measured.
2. Methods
2.1. Porcine tracheal smooth muscle strips
Tracheas from eight pigs were obtained from a local abattoir
(Boucherie Alphonse Cˆ
ot´
e, Sainte-Eulalie, Canada). The trachea was
excised after death and immersed into 4 ◦C Krebs solution (pH 7.4,
111.9 mM NaCl, 5.0 mM KCl, 1.0 mM KH
2
PO
4
, 2.1 mM MgSO
4
, 29.8 mM
NaHCO
3
, 11.5 mM glucose, 2.9 mM CaCl
2
), in which it was kept during
transportation and until further processing. One smooth muscle strip
from each trachea was isolated and mounted vertically in a 40-mL organ
bath at in situ length in 37 ◦C Krebs solution and underwent a period of
preconditioning as previously described (Gazzola et al., 2020). Indo-
methacin at 10
-6
M was also added during the entire experiment to avoid
spontaneous prostanoids-mediated contractions.
2.2. Protocol
For each strip, a concentration-response with methacholine was
repeated four times under four different contractile conditions in a
randomized order. Each time, four concentrations of methacholine were
added in log increments from 10
-8
to 10
-5
M. One contractile condition
was isometric, meaning that the length of the strip was held xed during
the entire concentration-response. During the isometric condition, the
concentrations of methacholine were added at 5-min intervals and both
the maximal force over the 5 min and the force near the end of the 5 min
were recorded at each concentration. The latter was more precisely
calculated 10 s before the administration of the subsequent concentra-
tion of methacholine using an average over 1 s (thus 100 data points of
isometric force, as the sampling frequency was 100 Hz). Two other
contractile conditions were isotonic, meaning that the load impeding
smooth muscle shortening was held xed. Two loads were tested, cor-
responding to 5 and 10 % of the reference force. The latter was obtained
at the end of the preconditioning period. It is more precisely the force
generated in response to an optimal electrical-eld stimulation (it nearly
represents the highest force that the muscle can generate). During
Fig. 2. Concentration-response of porcine tracheal strips in the isotonic condition against a 5 % load. A representative length trace is shown in A. The force trace is
also shown in the inset. For each tracheal strip, the maximal shortening at each methacholine concentration (purple asterisks) were collected. Concentration-response
curves were then constructed and are shown in B. The individual curves and their mean are shown on the left and right panels, respectively. n =8.
L. G´
elinas et al.

Respiratory Physiology & Neurobiology 325 (2024) 104264
4
isotonic conditions, the concentrations of methacholine were added at 8-
min intervals and the maximal shortening was recorded at each meth-
acholine concentration. The other contractile condition was during
cyclical motion, meaning that the length of the tracheal strips was
continuously uctuating before and during the concentration-response.
More precisely, the length of tracheal strips was sinusoidally oscillating
by 5 % (trough-to-peak) at 0.2 Hz, mimicking the strain the smooth
muscle undergoes in vivo due to tidal volume breathing. During the
oscillatory condition, the concentrations of methacholine were added at
5-min intervals and several contractile readouts were deduced at each
methacholine concentration, including peak force, average force, stiff-
ness, elastance and resistance. How they were calculated is described
below.
The order to which each strip was exposed to the four concentration-
responses was randomized using a 4×4 Latin square, which was
repeated twice for a sample size of eight. The Latin square was uniform,
meaning that each strip was subjected to all four concentration-
responses and each concentration-response occurred only once at a
particular order at every four experiments. The Latin square was also
balanced, meaning that the number of times a given concentration-
response occurred before or after all the other ones happened only
once every four experiments.
2.3. Data analyses
During the concentration-response in condition of cyclical motion,
ve contractile readouts were measured. The rst one is peak force,
representing the maximal force recorded at each methacholine con-
centration during one of the sinusoidal peaks in length. The other four
contractile readouts were calculated over 5 s, specically from trough to
trough in the sine wave with the peak force. The second readout is
average force, representing the average of all data points of force (500 of
them, as the sampling frequency was again at 100 Hz) of the sine wave
with the peak force. The third contractile readout is stiffness, repre-
senting the change in force over the change in length during the sine
wave with the peak force. The last two are elastance and resistance.
Since ASM tissue is innately non-linear (Ito et al., 2006; Bates and
Lauzon, 2005), they were calculated using analytical tools applicable to
both linear and non-linear systems (Fredberg and Stamenovic, 1989).
The method was described in detail by others (Fredberg et al., 1993).
Briey, it consists of measuring the area between the ascending and the
descending limbs of the force-length loops, which is then used to
sequentially deduce resistance, the phase angle, hysteresivity and ela-
stance. Put it simply, the phase angle quanties the extent by which the
resistive forces contribute to the total (elastic +resistive) forces. It has
been described in more details in two original papers (Fredberg and
Stamenovic, 1989; Fredberg et al., 1993), as well as in a recent review
where it is explained more simply (Boss´
e, 2022).
Fig. 3. Concentration-response of porcine tracheal strips in the isotonic condition against a 10 % load. A representative length trace is shown in A. The force trace is
also shown in the inset. For each tracheal strip, the maximal shortening at each methacholine concentration (purple asterisks) were collected. Concentration-response
curves were then constructed and are shown in B. The individual curves and their mean are shown on the left and right panels, respectively. n =8.
L. G´
elinas et al.

Respiratory Physiology & Neurobiology 325 (2024) 104264
5
Fig. 4. Concentration-response of porcine tracheal strips in cyclical motion. A representative force trace at 10
-6
M of methacholine is shown in A, together with a
zoomed trace (inset) showing three cycles that include the peak cycle (green). For each tracheal strip, the peak force at each methacholine concentration (purple
asterisks) were collected. From the cycle with the peak force (green traces), several contractile readouts were calculated, including average force, stiffness, elastance
and resistance. Concentration-response curves were then constructed for each of these readouts and are shown in B to F. The individual curves and their mean are
shown on the left and right panels, respectively. n =8.
L. G´
elinas et al.

Respiratory Physiology & Neurobiology 325 (2024) 104264
6
•R =4 A/
πω
(
ε
2
), where R is resistance, A is area of the loop,
ω
is
angular frequency and
ε
is strain.
•ϕ =arcsin
ωε
R/ΔF, where ϕ is the phase angle and F is force.
•tan ϕ =
η
, where
η
is hysteresivity.
•E =
ω
R/
η
, where E is elastance.
For each contractile readout, the log EC50 was calculated. More
precisely, a log(methacholine) vs. response with three parameters (top,
bottom and EC50) was tted by the least square method to each
concentration-response of each tracheal strip for each contractile
readout. Individual results, together with means ±standard deviations
are presented. A repeated measures ANOVA was then used to compare
the logEC50 between contractile readouts, which was followed by a
Tukey multiple comparisons test to compare each contractile readout
with each other. Statistical analyses were performed using Prism 10
(Version 10.1.1, GraphPad Software, San Diego, CA) and p≤0.05 was
deemed signicant.
3. Results
The results in the isometric condition are depicted in Fig. 1. As ex-
pected, the force rose with incremental concentrations of methacholine
(Fig. 1A). Interestingly, the maximal force at each concentration was not
maintained over time (Fig. 1A). Instead, the isometric force typically
reached its highest value at a rate related to the methacholine concen-
tration, and then receded (Fig. 1A). When the logEC50 was calculated
using the maximal force at each methacholine concentration, it ranged
from -6.87 to -5.95 M with a mean of -6.32 ±0.28 M (Fig. 1B). When the
logEC50 was calculated using the isometric force near the end of the
5 min of contraction at each methacholine concentration, it ranged from
-6.89 to -6.08 M with a mean of -6.38 ±0.28 M (Fig. 1C). The difference
between the logEC50 with the maximal force and the end isometric force
was not signicant (p=0.96).
The results in isotonic conditions with a load corresponding to either
5 or 10 % of the reference force are depicted in Figs. 2 and 3, respec-
tively. As expected, the shortening increased with incremental concen-
trations of methacholine (Fig. 2A & 3A). Interestingly, the maximal
shortening at each concentration was mostly maintained over time
(Fig. 2A & 3A). When the logEC50 was calculated using the shortening
against a 5 % load, it ranged from -7.68 to -6.36 M with a mean of -7.13
±0.40 M (Fig. 2B). When the logEC50 was calculated using the short-
ening against a 10 % load, it ranged from -7.11 to -6.37 M with a mean
of -6.66 ±0.42 M (Fig. 3B). The logEC50 with the shortening against a
5 % load was lower than the one measured with the shortening against a
10 % load (p=0.03).
The results in the condition of cyclical motion are depicted in Fig. 4.
As expected, all contractile readouts increased with incremental con-
centrations of methacholine. Similar to the isometric condition, the
Fig. 4. (continued).
Table 1
LogEC50 for contractile readouts measured during the condition of cyclical
motion.
Readouts Range (M) Mean ± SD (M)
Peak force -6.93 to -5.70 -6.33 ±0.41
Average force over the peak cycle -6.89 to -5.65 -6.30 ±0.41
Stiffness during the peak cycle -7.14 to -5.86 -6.46 ±0.43*
†
Elastance during the peak cycle -7.11 to -5.84 -6.46 ±0.42*
†
Resistance during the peak cycle -7.17 to -5.64 -6.38 ±0.49
*Signicantly different from the peak force (p<0.001)
†Signicantly different from the average force (p<0.001)
L. G´
elinas et al.

Respiratory Physiology & Neurobiology 325 (2024) 104264
7
maximal value for each contractile readout at each concentration was
not maintained over time (Fig. 4A). Instead, they reached their highest
value at a rate related to the methacholine concentration, and then
receded (Fig. 4A). The range, mean and standard deviation of the
logEC50 for the peak force, as well as for the average force, stiffness,
elastance and resistance over the peak force cycle are shown in Table 1.
Signicant differences in logEC50 were observed with stiffness vs. peak
force (p=0.003), stiffness vs. average force (p=0.003), elastance vs.
peak force (p=0.002), and elastance vs. average force (p=0.001).
The logEC50s with all contractile readouts are depicted in Fig. 5. The
repeated measures ANOVA was highly signicant (p<0.0001). Apart
from the signicant differences stated above, the logEC50 with isotonic
shortening against a 5 % load was signicantly lower than the ones
measured with all other contractile readouts (p<0.002), and the one
with isotonic shortening against a 10 % load was signicantly lower
than the one measured with maximal isometric force (p=0.03).
4. Discussion
This study was specically designed to compare methacholine
sensitivity between different contractile readouts within the same
tissues. The results point to many statistically signicant differences.
This nding has not only implications in many experimental settings,
but may also provide insights regarding the elusive role played by the
airway smooth muscle on respiratory mechanics. The more sensitive
contractile readouts are indeed more likely to be affected by a physio-
logical range of contractile agonist concentrations and, thereby, more
likely to inuence airway wall and lung mechanics.
Unsurprisingly, the range of sensitivity between contractile readouts
was modest. It was suspected that, upon activation, a contractile level
sufcient to alter stiffness would be near to the one causing force
development and shortening. This narrow range of logEC50, uctuating
between -7.13 and -6.30, was thus anticipated. Yet, it is still important to
emphasize that the two extremes represent a 6.8-fold difference (7.4
×10
-8
vs. 5.0 ×10
-7
M). It is thus possible that the lower spectrum of this
range is physiologically relevant, while the higher spectrum may not.
The logical order of sensitivity, according to our opinion, should
have been stiffness, together with its related contractile properties such
as elastance and resistance, followed by force and shortening. Short-
ening is presumably the less desirable behavior of the airway smooth
muscle in vivo as it would translate to airway constriction. It thus came as
a surprise that shortening yielded a signicantly greater sensitivity than
any other contractile readout, at least under a low counteracting load.
However, two important confounders need to be considered in our
experimental settings. First, the shortening was done at low loads. This
implies a substantial amount of shortening, and shortening can obvi-
ously not proceed innitely. It is rather limited by physical constraints.
Since the isotonic load was low, maximal (or near maximal) shortening
could have been attained at low methacholine concentrations, therefore
blocking (or limiting) further shortening by subsequent concentrations.
This effect would shift the concentration-response curve to the left,
thereby decreasing the EC50. Second, in our settings, the shortening can
only be initiated once the load is attained. This can be seen in the inset of
Figs. 2 and 3. Therefore, greater is the isotonic load, greater is the
concentration required to initiate shortening. This effect shifts the
concentration-response curve to the right, thereby increasing the EC50.
The fact that sensitivity decreased with increasing load in our experi-
ments supports this notion. Based on this trend, we surmised that by
further increasing the load, the EC50 would have kept increasing. It is
therefore possible that under physiological conditions with an auxotonic
load (Oliver et al., 2010), the EC50 may surpass the ones observed for
stiffness and force. More studies are clearly needed.
Perhaps the most relevant comparisons between contractile readouts
are the ones measured in the same contractile condition. During cyclical
motion, ve different contractile readouts were measured, namely peak
force, average force, stiffness, elastance and resistance. Both stiffness
and elastance were more sensitive than both the peak force and the
average force. This, at least, ts with the logical order of sensitivity
stated above. As outlined in the Introduction, force development in vivo
is very likely to cause shortening and airway narrowing. However, if the
stiffening of the smooth muscle precedes force development (i.e., if it
truly takes place at lower concentration), it would allow airways to resist
dilation during breathing maneuvers without causing constriction. In
turn, this would minimize dead space excursion during breathing,
optimizing ow into the alveoli and maximizing gas exchange at every
breath. Notably, it is worth mentioning that we and others have previ-
ously demonstrated that stiffness is more sensitive than force to meth-
acholine (Gazzola et al., 2016; Ansell et al., 2013). In both cases, the
difference was greater than the one reported herein, amounting to log
differences. Since these studies were done with human bronchial rings
and intact sheep airways (and not with strips that are mostly made of
muscle), it is possible that this greater sensitivity to stiffness vs. force is
amplied when the smooth muscle is surrounded by the other structural
components of the airway wall.
Another interesting observation was that maximal isometric force at
each methacholine concentration was not maintained while maximal
shortening mostly was. It thus seems that even when the activation drive
Fig. 5. A scatter plot showing the methacholine logEC50 calculated using
different contractile readouts. For each readout, points are from individual
tracheal strips and the grey horizontal bar is the mean. They are shown in order
of sensitivity from left to right to facilitate comparison. A repeated measures
ANOVA was conducted followed by a Tukey’s multiple comparisons test to
compare the logEC50 between contractile readouts. Asterisks indicate pairwise
signicant differences (*,** and *** are p<0.05, 0.01 and 0.001, respectively).
n =8.
L. G´
elinas et al.

Respiratory Physiology & Neurobiology 325 (2024) 104264
8
decreases, the shortening can be maintained. This is consistent with a
mounting literature on the molecular mechanisms governing airway
smooth muscle contraction, showing that many evanescent structures
can be erected to physically link the contractile machinery inside the cell
with its plasmalemma, the extracellular matrix and the surrounding
cells, which, at the tissue scale, efciently works to maintain shortening
passively (Zhang et al., 2015; Tang, 2018; Zhang and Gunst, 2019; Seow,
2013). It is also consistent with previous work, showing that the increase
in respiratory system resistance caused by inhaled methacholine in
humans is lasting longer than the time required for methacholine
clearance (Chapman et al., 2014; Cartier et al., 1983).
5. Conclusion
This study demonstrates that, although all within the same log range,
the sensitivity to methacholine is signicantly affected by the contractile
readout being measured. It was shown that direct comparisons between
certain contractile readouts can be misleading and should thus be
interpreted with caution. Yet, when measured within the same con-
tractile condition, some readouts, such as stiffness, change in response to
lower concentrations than others, such as force. This can be key ele-
ments to take into account when contemplating the role played by the
airway smooth muscle in respiratory mechanics.
Funding
Natural Sciences and Engineering Research Council of Canada
(NSERC) (RGPIN-2020-06355), the Canadian Institutes of Health
Research (CIHR) (508356-202209PJT); and the Fondation de l’IUCPQ
(Institut Universitaire de Cardiologie et de Pneumologie de Qu´
ebec)
CRediT authorship contribution statement
Magali Boucher: Writing – review & editing, Supervision, Meth-
odology, Investigation, Data curation, Conceptualization. Cyndi Henry:
Writing – review & editing, Supervision, Project administration. Ynuk
Boss´
e: Writing – original draft, Visualization, Validation, Supervision,
Software, Resources, Project administration, Funding acquisition,
Formal analysis, Conceptualization. Louis G´
elinas: Writing – review &
editing, Validation, Methodology, Investigation, Formal analysis, Data
curation. Andr´
es Rojas-Ruiz: Writing – review & editing, Methodology,
Investigation, Data curation.
Acknowledgements
The authors are indebted to owners and employees of the Boucherie
Alphonse Cˆ
ot´
e for donating pig tracheas for research purposes.
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