Human Trachealis and Main Bronchi Smooth Muscle Are Normo-Responsive in Asthma

Abstract

Rationale:
Airway smooth muscle (ASM) plays a key role in airway hyperresponsiveness (AHR) but it is unclear whether its contractility is intrinsically changed in asthma.

Objectives:
To investigate whether key parameters of ASM contractility are altered in subjects with asthma.

Methods:
Human trachea and main bronchi were dissected free of epithelium and connective tissues and suspended in a force-length measurement set-up. After equilibration each tissue underwent a series of protocols to assess its methacholine dose-response relationship, shortening velocity, and response to length oscillations equivalent to tidal breathing and deep inspirations.

Measurements and main results:
Main bronchi and tracheal ASM were significantly hyposensitive in subjects with asthma compared with control subjects. Trachea and main bronchi did not show significant differences in reactivity to methacholine and unloaded tissue shortening velocity (Vmax) compared with control subjects. There were no significant differences in responses to deep inspiration, with or without superimposed tidal breathing oscillations. No significant correlations were found between age, body mass index, or sex and sensitivity, reactivity, or Vmax.

Conclusions:
Our data show that, in contrast to some animal models of AHR, human tracheal and main bronchial smooth muscle contractility is not increased in asthma. Specifically, our results indicate that it is highly unlikely that ASM half-maximum effective concentration (EC50) or Vmax contribute to AHR in asthma, but, because of high variability, we cannot conclude whether or not asthmatic ASM is hyperreactive.

Full text

ORIGINAL ARTICLE
Human Trachealis and Main Bronchi Smooth Muscle Are
Normoresponsive in Asthma
Gijs Ijpma
1
, Linda Kachmar
1
, Oleg S. Matusovsky
1
, Jason H. T. Bates
2
, Andrea Benedetti
3,4,5
, James G. Martin
1
,
and Anne-Marie Lauzon
1
1
Meakins-Christie Laboratories,
3
Department of Epidemiology, Biostatistics and Occupational Health, and
4
Department of Medicine,
McGill University, Montreal, Quebec, Canada;
2
Department of Medicine, University of Vermont, Burlington, Vermont; and
5
Respiratory
Epidemiology and Clinical Research Unit, McGill University Health Centre, Montreal, Quebec, Canada
Abstract
Rationale: Airway smooth muscle (ASM) plays a key role in airway
hyperresponsiveness (AHR) but it is unclear whether its contractility
is intrinsically changed in asthma.
Objectives: To investigate whether key parameters of ASM
contractility are altered in subjects with asthma.
Methods: Human trachea and main bronchi were dissected
free of epithelium and connective tissues and suspended in
aforce–length measurement set-up. After equilibration each
tissue underwent a series of protocols to assess its methacholine
dose–response relationship, shortening velocity, and response
to length oscillations equivalent to tidal breathing and deep
inspirations.
Measurements and Main Results: Main bronchi and tracheal
ASM were significantly hyposensitive in subjects with asthma
compared with control subjects. Trachea and main bronchi did
not show significant differences in reactivity to methacholine and
unloaded tissue shortening velocity (Vmax) compared with
control subjects. There were no significant differences in
responses to deep inspiration, with or without superimposed tidal
breathing oscillations. No significant correlations were found
between age, body mass index, or sex and sensitivity, reactivity, or
Vmax.
Conclusions: Our data show that, in contrast to some animal
models of AHR, human tracheal and main bronchial smooth
muscle contractility is not increased in asthma. Specifically, our
results indicate that it is highly unlikely that ASM half-maximum
effective concentration (EC
50
) or Vmax contribute to AHR in
asthma, but, because of high variability, we cannot conclude
whether or not asthmatic ASM is hyperreactive.
Keywords: airway smooth muscle mechanics; airway
hyperresponsiveness; shortening velocity; asthma; smooth muscle
At a Glance Commentary
Scientific Knowledge on the Subject: Contraction of airway
smooth muscle is directly responsible for acute airway constriction
in asthmatic attacks. However, evidence on whether airway
smooth muscle contractility is altered in asthma is contradictory,
incomplete, and often derived from problematic tissue sources.
What This Study Adds to the Field: We have measured
a range of parameters of airway smooth muscle contractility
that have never been tested on reliable human airway smooth
muscle tissues. Our study found, at least in trachea and main
bronchi, no changes in contractility that could contribute to
airway hyperresponsiveness in subjects with asthma.
It is well established that airway smooth
muscle (ASM) contraction leads to the
airway constriction typical of airway
hyperresponsiveness (AHR) in asthma.
Nonetheless, it is unclear whether AHR is
the result of altered ASM contractility, or
even whether altered ASM function is
required at all for AHR. For example, the
inflammatory mediators typically present in
( Received in original form July 17, 2014; accepted in final form February 15, 2015 )
Supported by National Heart, Lung, and Blood Institute grant R01-HL 103405-02 and the Costello Fund. The Meakins-Christie Laboratories (McGill University
Health Centre Research Institute) are supported in part by a center grant from Le Fonds de la Recherche en Sant ´eduQu´ebec (FRSQ).
Author Contributions: G.I., acquisition of data, analysis and interpretation of data, drafting of manuscript. L.K. and O.S.M., acquisition of data and article
review. J.H.T.B. and J.G.M., analysis and interpretation of data and article review. A.B., statistical analysis and article review. A.-M.L., conception and design,
analysis and interpretation of data, and drafting and review of manuscript.
Correspondence and requests for reprints should be addressed to Anne-Marie Lauzon, Ph.D., Meakins-Christie Laboratories, McGill University, 3626 St-Urbain
Street, Montreal, PQ, H2X 2P2 Canada. E-mail: anne-marie.lauzon@mcgill.ca
Am J Respir Crit Care Med Vol 191, Iss 8, pp 884–893, Apr 15, 2015
Copyright ©2015 by the American Thoracic Society
Originally Published in Press as DOI: 10.1164/rccm.201407-1296OC on February 19, 2015
Internet address: www.atsjournals.org
884 American Journal of Respiratory and Critical Care Medicine Volume 191 Number 8 |April 15 2015

asthma could be responsible for triggering
abnormal airway narrowing even when the
ASM itself is entirely normal. However,
maximal concentrations of histamine in
healthy subjects do not reduce FEV
1
to
asthmatic levels (1), so an excess of other
contractile agonists is unlikely to cause
excessive ASM contraction either. It thus
remains a plausible hypothesis that ASM
contractility is intrinsically altered in
asthma. Nevertheless, the veracity of this
seemingly straightforward idea has so far
proved very difficult to establish or refute.
In the 1980s and early 1990s many
studies were conducted on human ASM
tissue (2–7), including some that compared
asthmatic with control ASM. These studies
were mainly focused on the isometric active
tension generated in response to a variety of
agonists (2, 3, 5, 7) with mixed results.
Some studies found asthmatic ASM to be
hyperresponsive (3), but most found either
no change or that the asthmatic ASM
was actually hyporesponsive (3, 5, 7).
Nevertheless, these studies all had a major
drawback in that they studied tissues
procured either from patients with lung
cancer (4, 6), many of whom were current
smokers, or from cadavers many hours
post-mortem (2, 3, 5, 7). It is likely that the
different tissue conditions used in these
studies, none of which were representative
of typical asthma, contributed at least in
part to the disagreement between results.
Recently, lungs donated for
transplantation that, for one reason or
another, do not meet transplantation criteria
have become available for use in medical
research. These organs are shipped
according to stringent transplantation
protocols that minimize decline in function,
so one would expect them to provide more
valid data about the properties of ASM in
either people with asthma or healthy
individuals compared with ASM from
surgically resected lungs that are usually
severely diseased. To date, however, the only
published study comparing asthmatic and
control ASM from this tissue source is
that of Chin and colleagues (8). Those
investigators found that human trachealis
exhibited no difference in either tension
or shortening velocity between control
subjects and subjects with asthma in
response to electrical field stimulation
(EFS). They did find small differences in
force recovery following 30 seconds of
large-amplitude oscillations and in length-
tension relationships, but the study did not
address ASM sensitivity to agonists or EFS.
Also, some of the results may have been
affected by the large difference in average
age of the subjects between the asthmatic
and control groups (15.0 65.9 vs. 31.7 6
17.5, respectively), because shortening
velocity has been shown in animal models to
decrease with age (9), and the shortening
velocity data themselves were highly variable.
There thus remains much that can be
learned about the nature of ASM in asthma
from further study of tissue from lungs
originally destined for transplantation. This
applies particularly to the question of
whether intrinsic ASM contractility is
altered in asthma. Accordingly, addressing
this question was the goal of the present
study. We expanded on the work of Chin
and colleagues (8) by assessing ASM
responsiveness to methacholine (MCh), the
response to a large stretch equivalent to
a single deep inspiration (DI) and unloaded
tissue shortening velocity, while using more
stringent subject selection criteria and
a higher-resolution force–length apparatus.
Most importantly, we made these
measurements not only in the trachealis but
also in ASM from the main bronchi to
establish whether our findings may be
generalized to more than a single
generation of the airway tree.
Some of the results of these studies have
been previously reported in the form of an
abstract (10).
Methods
Procurement and Dissection
Asthmatic and control transplant-grade
lungs were procured by the International
Institute for the Advancement of Medicine.
The demographics and clinical details of the
donors are shown in Table 1. The tissues
were stored in Custodial histidine-
tryptophan-ketoglutarate (HTK) or
University of Wisconsin (UW) solution
during shipment, with trachea and main
bronchi separately packed from the lungs,
which were used for a different study. On
arrival the trachea and main bronchi were
placed in oxygenated Hanks’balanced salt
solution (composition in mM: 5.3 KCl, 0.44
KH
2
PO
4
, 137.9 NaCl, 0.336 Na
2
PO
4
, 2.33
CaCl
2
, 0.79 MgSO
4
, 10 glucose, 10 HEPES
buffer, pH adjusted to 7.4 with NaOH) at
48C and used within 12 hours. Smooth
muscle (SM) bundles were dissected from
epithelium and connective tissue in
calcium-free Krebs solution (composition
in mM: 110 NaCl, 0.82 MgSO
4
, 1.2
KH
2
PO
4
, 3.4 KCl, 25.7 NaHCO
3
, 5.6 glucose,
pH at 7.4, bubbled with 95/5% O
2
/CO
2
gas
mixture) on ice and aluminum foil clips were
attached on either end of the tissue.
Tissue Mechanics
Equilibration. The tissue was attached
horizontally with foil clips to a length
controller (model 322C-I; Aurora Scientific,
Aurora, ON, Canada) and a force
transducer (model 400A; Aurora Scientific)
controlled by Aurora Scientific 600A
software at a reference length equal to the
in situ length in a relaxed state (intact
trachea or main bronchi ring in calcium-
free Krebs). The tissue was continuously
flushed with Krebs solution (as previously
with 2.4 mM CaCl
2
) at a rate of
approximately 1 ml/min in a 1-ml tissue
bath. The tissue was equilibrated for at least
30 minutes with EFS (10-s duration 25
V/cm, 50 Hz, 2-ms pulse width) every 5
minutes, followed by at least five contractions
with MCh 10
26
M. These EFS settings were
used in all protocols. Both equilibration
protocols were continued until a stable
baseline (without spontaneous contractions)
and contractile force were achieved.
Dose response. The tissue was exposed
to increasing concentrations of MCh every
minute, from a concentration of 10
27
M
up to 10
24
M (Figure 1A). The peak force
reached (relative to baseline force and
corrected for measurement noise) after
each administered dose was used as the
force representative of that dose. Maximum
stress was calculated from the maximum
force, extrapolated from the dose–response
curve fit divided by the SM cross-sectional
area. To determine the SM cross-sectional
area, the tissues were fixed in 10%
formalin for 12–24 hours, and embedded
in paraffin for histology. Five-micrometer-
thick slices were stained with Masson’s
trichrome, which provided the best contrast
between nonmuscle and SM tissue. The
average ratio of SM to total tissue area for
each tissue was calculated (average for all
tissues, 0.49 60.02) and multiplied by the
tissue cross-sectional area (average for all
tissues, 0.166 60.005 mm
2
).
EFS force–velocity. The tissue was
contracted using EFS for 10 seconds every
5 minutes immediately followed by
a measurement of the force (F
ref
) and
a rapid force clamp of 5, 10, 20, 40, or 80%
ORIGINAL ARTICLE
Ijpma, Kachmar, Matusovsky, et al.: Human Airway Smooth Muscle in Asthma 885

of F
ref
for 120 milliseconds (Figure 1B). The
shortening velocity was determined from
the rate of length change during the last 60
milliseconds of the force clamp, when the
force had stabilized. The data were rejected
if any of the force clamps had not stabilized
before this 60-millisecond period. To
compensate for force transducer drift, true
zero force was measured at the start of the
protocol by rapidly shortening the muscle
to 75% of L
0
followed by relengthening.
Unloaded tissue shortening velocity
(Vmax) was calculated by extrapolation
using a perpendicular least-squares fitting
method to a classic Hill curve of the
form V = b(F
0
2F)/(a 1F). For details,
see Reference 11.
Deep inspiration. Two protocols for
measuring DI effects were performed
(Figures 1C and 1D). For each protocol the
tissue was first exposed to three consecutive
EFS contractions 5 minutes apart to
establish a stable reference contractile force.
Subsequently, a length change equivalent
to a DI was applied to the tissue (half
sinusoidal wave, 0.2 Hz, 0.3 L
ref
amplitude)
followed by five EFS contractions, 5
minutes apart. The tissue was then
contracted with 10
26
M MCh and after
2 minutes the tissue was again exposed
to a DI equivalent length change (half
sinusoidal wave, 0.2 Hz, 0.2 L
ref
amplitude
to adjust for increased stiffness of the
Table 1. Subject Medical and Demographic Data
Subject Sex Age
Body Mass
Index Ethnicity Cause of Death Asthma History Other Medication(s) Medication in Hospital
Subjects with asthma
1 M 72 44.2 W CVA secondary
to ICH
Age of diagnosis
unknown
Smoking 16
pack-years,
quit 50 YA
Unknown Unknown
2 F 34 31.93 W Anoxia secondary to
drug intoxication
Diagnosed 20 YA,
hospitalized twice
with exacerbations
Inhaler–prednisone Solu-Medrol, Levophed
3 M 29 30.8 W Anoxia secondary to
cardiovascular
Diagnosed 7 YA Chewing tobacco
for 1 yr
Albuterol inhaler Esmolol, Levophed
4 F 60 25.59 W HT secondary to
blunt injury
Diagnosed 10 YA Complete
hysterectomy 40
YA, hypertension
Infrequent inhaler
use, hypertension
medication
Levophed, Solu-Medrol,
phentolamine
5 M 38 29.49 W CVA secondary
to ICH
Diagnosed at 4 mo Allergy medications Norepinephrine,
Levophed,
phenylephrine,
dobutamine
6 M 35 29.4 W Anoxia (asthma)
secondary to
cardiovascular
Asthma since
childhood
Albuterol, Pulmicort Levophed, Solu-Medrol,
albuterol
7 M 40 26.95 A Anoxia secondary to
cardiovascular
Asthma diagnosed
5YA
Dulera Levophed, Solu-Medrol
8 F 38 35.78 W Anoxia secondary to
cardiovascular
Asthma diagnosed
at 8
Singulair Levophed, epinephrine,
dopamine
Control subjects
9 M 22 23 W HT secondary to
SIGSW
Asthma as child,
not taken
medication in 7 yr
Smoked hookah
past year
Albuterol as child
and Pepcid
Levophed,
Neo-Synephrine,
Atrovent, albuterol,
Solu-Medrol
10 F 61 35.1 H CVA secondary
to ICH
Tobacco product
20 YA, diabetes,
hypertension
Levophed,
albuterol-ipratropium
11 M 47 26.2 W CVA secondary
to ICH
Neosynephrine
12 F 55 26 W CVA secondary
to ICH
Alcohol abuse,
marijuana and
cocaine use
Neosynephrine,
dopamine, Levophed,
Solu-Medrol,
dobutamine
13 F 35 21.87 W Anoxia secondary
to ICH
Teenage marijuana
use
Levophed
14 M 30 21.85 W Anoxia secondary to
asphyxiation
Marijuana, cocaine
use
Solu-Medrol
15 F 54 36.13 W CVA secondary
to ICH
Smoking 10
pack-years, quit
8YA
Epinephrine
16 F 62 30.86 W CVA secondary to
natural causes
Hypertension, basal
cell carcinoma in
nose 3 YA
Dopamine
17 M 55 26.79 W HT secondary to
blunt injury
Marijuana
occasionally,
alcohol abuse
Neosynephrine,
Solu-Medrol
18 F 58 33.87 W CVA secondary
to ICH
Neosynephrine,
Solu-Medrol
19 F 54 24.97 W HT secondary to
blunt injury
Smoking 30
pack-years, quit
16 YA,
hypertension
Levophed, albuterol
Definition of abbreviations: A = African American; CVA = cerebrovascular accident; H = Hispanic; HT = head trauma; ICH = intracerebral hemorrhage;
SIGSW = self-inflicted gunshot wound; W = white; YA = years ago.
ORIGINAL ARTICLE
886 American Journal of Respiratory and Critical Care Medicine Volume 191 Number 8 |April 15 2015

tissue), followed by flushing with Krebs
solution for 5 minutes and five EFS
contractions, each 5 minutes apart. In
both protocols a continuous sinusoidal
length oscillation was superimposed, with
theonlydifferencebeingtheamplitude
and frequency of this oscillation. One
protocol had a 30-Hz 0.0125 L
ref
oscillation applied to measure stiffness
throughout the protocol; the other had
a 0.2-Hz 0.04 L
ref
oscillation applied
to simulate the effect of continuous
breathing oscillations. The order of the
two protocols was randomized for each
tissue.
0
5
10
A
C
B
D
E
Force (mN)
00 100 200 300
Time (s)
400 500 600
0.5
1
Length (fraction L ref)
10–4 M MCh
10–7 M MCh
10–6 M MCh
10–5 M MCh
EFS
.25% Lref oscillation
DI
10-6 M MCh
EFS
4% Lref oscillation
DI
10-6 M MCh
0
4
2
6
Force (mN)
0.8
0 100 200 300 400
Time (s)
500 600 700 800
1
0.9
1.1
Length (fraction L ref)
Zero force calibration
Force clamp
Measurement Fref
EFS
0
10
20
Force (mN)
30
0 1000 2000
Time (s)
3000 4000
1
1.2
Length (fraction L ref)
1.4
0
10
20
Force (mN)
30
0 1000 2000
Time (s)
3000 4000
1
1.2
Length (fraction L ref)
1.4
Figure 1. Traces from all airway smooth muscle tissue mechanics experiments, and a histology sample. Traces are the average of all tissues from
subjects with asthma. (A) Trace of methacholine (MCh) dose–response protocol. (B) Trace of force–velocity protocol. (Cand D) Traces of deep inspiration
(DI) protocols. (E) Sample of a histology image of smooth muscle cross-section with Masson’s trichrome staining. EFS = electrical field stimulation.
ORIGINAL ARTICLE
Ijpma, Kachmar, Matusovsky, et al.: Human Airway Smooth Muscle in Asthma 887

Rejection Criteria
Tissues were rejected if control EFS and
MCh 10
26
M contractions did not achieve
a maximal force level within 30% of the
reference contractions at the end of the
equilibration phase. Tissues were also rejected
if they failed to relax fully after a contraction
with either EFS or MCh 10
26
Morif
spontaneous contractions failed to subside
before the end of the equilibration phase.
Rejection rates were similar between subjects
with asthma (10%) and control subjects (15%).
Data Analysis and Statistics
Linear mixed models were used to estimate
the expected difference in maximum
contractile force (Fmax), the half-maximum
effective concentration (EC
50
), and Vmax
between subjects with asthma and control
subjects, and site, adjusted for one another.
We included a random intercept to account
for correlation between measures on the
same subject. Two-way repeated measures
analysis of variance was used for the DI
protocols. Error bars are standard errors.
All protocols have been applied to tissues
from all the lungs described in Table 1. For
each trachea and main bronchi (unless they
were not provided, damaged, or rejected)
two tissues were tested and the results
averaged (n = number of subjects). The 95%
confidence intervals of the difference of the
mean of the control subjects compared with
the subjects with asthma was calculated by
averaging all available tissues for each
subject. The confidence intervals were
expressed as a percentage of the mean
of the control subjects.
Results
Dose–Response Curves
Dose–response curves are shown in
Figure 2 as absolute stress (Figure 2A) and
stress normalized to maximum contractile
stress (Figure 2B). Absolute stress was
not significantly different with location
(P= 0.60) or disease (P= 0.66) (Figures
2A and 2C). EC
50
, the dose at which 50%
of the maximum stress is generated, was
0
10–8 10–7 10–6 10–5
MCh Dose(M)
Stress (kPa)
10–4 10–3
40
80
0
100
200
300
Stress (kPa)
MB-Control
T-Control
MB-Asthma
T-Asthma
400
–7.5 –100 EC50 σmax
–50
0
50
–7.0
–6.5
–6.0
–5.5
EC50 (10x M MCh)
% of Control average
MB-Control
T-Control
MB-Asthma
T-Asthma
–5.0 *
120
160
200
A
CD E
B
MB-Control n=5
T-Control n=6
MB-Asthma n=6
T-Asthma n=6
10–8 10–7 10–6 10–5
MCh Dose(M)
Stress normalized to σmax
10–4 10–3
0.2
0.4
0.6
0.8
1MB-Control n=7
T-Control n=8
MB-Asthma n=6
T-Asthma n=6
Figure 2. Methacholine (MCh) dose–response curves. Triangles represent trachea and circles main bronchi tissues; solid symbols are control subjects
and open symbols are subjects with asthma. (A) Absolute stress dose–response of main bronchi (MB) and trachea (T) in subjects with asthma and control
subjects. (B) Dose–response curves normalized to maximum stress (s
max
). (C)s
max
derived from curve fits of the dose response. No significant differences
were found. (D)EC
50
derived from dose–response curves. EC
50
showed significant differences with disease state (*P= 0.05) but not location. (E)
Confidence interval of the difference of the means of pooled trachea and main bronchi data for EC
50
and s
max
in control subjects versus subjects with
asthma. EC
50
= half-maximum effective concentration.
ORIGINAL ARTICLE
888 American Journal of Respiratory and Critical Care Medicine Volume 191 Number 8 |April 15 2015

significantly reduced (hyposensitive) in
subjects with asthma (P= 0.050), with no
significant difference with location (P=
0.1718) (Figures 2B and 2D). The
confidence interval of the difference of the
means of subjects with asthma versus
control subjects (Figure 2E) shows the large
variability in the absolute stress and a less
than 2.5% chance that asthmatic ASM (n =
8) is hypersensitive to MCh compared with
control subjects (n = 11).
Shortening Velocity
To assess whether Vmax is changed in
subjects with asthma, it was calculated from
force–velocity curves of five force clamps
during five separate, consecutive EFS
contractions. Figure 3 shows the average
data for the individual force clamps as well
as the Hill-curve–extrapolated Vmax.
No significant difference was found with
disease state (P= 0.38) or location(P=
0.42). The 95% confidence interval of the
difference of the means (Figure 3C) showed
a less than 2.5% chance that the Vmax is
more than 9.8% increased in subjects with
asthma (n = 8) compared with control
subjects (n = 11).
Deep Inspiration
The effect of DI on the contractile force in
successive contractions is shown in Figure 4.
Because no significant differences between
main bronchi and trachea were found, only
the pooled data for all tissues per subject
are shown. No significant differences were
found between subjects with asthma and
control subjects. The force of the first
contraction after a DI in relaxed muscle was
less than the force in all subsequent
contractions for control subjects and
subjectswithasthmabothwithand
without superimposed breathing
oscillations, but not when the DI
was applied to contracted muscle.
Furthermore, a significant difference was
found between the force prior to DI and
the second and third contraction in
subjects with asthma and the second
contraction only in control subjects and
subjects with asthma after a DI in both
relaxed and contracted ASM when
breathing oscillations were superimposed.
Although subjects with asthma showed
a trend toward less contractile force after
a DI, particularly in the first contraction
after the DI in relaxed muscle, none of the
differences between subjects with asthma
and control subjects were statistically
significant. The superimposed breathing
oscillation did not have a significant effect
ontheresponsetoDIs.
Body Mass Index, Age, and Sex
Effects
The asthmatic and control groups were not
significantly different in age (43.3 62.2 vs.
48.5 64.0) and body mass index (BMI)
(31.8 61.0 vs. 27.9 61.6), with a small
difference in sex distribution (62% male vs.
36% male). Three main parameters from
the protocols (EC
50
and maximal stress
from the MCh dose–response and Vmax)
were tested for correlation with age, sex,
and BMI (Figure 5). Only the pooled data
for all tissues per subject are shown. None
of the parameters showed any significant
correlation.
Discussion
We examined several key indicators of
human ASM contractility in subjects with
asthma and control subjects at two sites in
the bronchial tree. We found strong
evidence that contractility as expressed by
EC
50
andVmaxisunlikelytobealteredto
favor AHR in asthma. Furthermore, no
differences in reactivity or DI response
were found. Although peripheral ASM
may show increased contractility in
asthma, our results indicate that such
differences are not intrinsic to ASM. In
addition, our study did not find any
evidence of age, sex, or BMI effects on
tracheal and main bronchial ASM
contractility.
0
0
0–40
–30
Vmax
–20
–10
% of Control average
0
10
MB-Control
T-Control
MB-Asthma
T-Asthma
0.2
0.4
0.05
0.1
0.15
Velocity (L0 s–1)
Vmax (L0 s–1)
0.2
0.25
0.3
0.35
A
BC
T-Asthma n=5
T-Control n=8
MB-Asthma n=6
MB-Control n=7
0.2 0.4
Load (fraction of isometric force)
0.6 0.8 1
Figure 3. (A) Electrical field stimulation force–velocity curves. Shortening velocity was measured
at five force clamps, and Vmax was calculated using extrapolation of a Hill-curve curve fit. (B) Vmax
for main bronchi (MB) and trachea (T) in asthma and control. Triangles represent trachea and circles
main bronchi tissues; solid symbols are control subjects and open symbols are subjects with
asthma. No significant differences were found. (C) Confidence interval of the difference of the
means of pooled trachea and main bronchi data of control subjects versus subjects with asthma
ORIGINAL ARTICLE
Ijpma, Kachmar, Matusovsky, et al.: Human Airway Smooth Muscle in Asthma 889

Asthmatic ASM Is neither Intrinsically
Hyperreactive nor Hypersensitive
We studied tissues from both main bronchi
and trachea, because previous animal
studies have shown that ASM contractility is
not uniform throughout the lung (12),
which may be more so in subjects with
asthma. Our MCh dose–response data
indicate that tracheal and bronchial ASM
are not intrinsically hyperresponsive in
asthma. In fact, our data show that
ASM EC
50
is slightly, but significantly,
hyposensitive. Large variability in our
maximal stress data leaves some
uncertainty to the contribution of ASM
reactivity to AHR. Furthermore, we cannot
exclude the possibility that ASM is
hyperresponsive in the asthmatic
intrapulmonary airways. However, studies
on human intrapulmonary bronchial SM
from cadavers with fatal asthma mostly
showed hyposensitivity to a range of
agonists (2, 5, 7). One of those studies
showed hyperresponsiveness to histamine,
but hyporesponsiveness to acetylcholine
and in the EFS frequency response (2).
However, in these studies the connective
tissue and epithelium were not removed
(usual practice for the spiral strip dissection
technique) and the tissues were dissected
up to 14 hours post-mortem. Also, forces
were normalized by tissue weight.
Because the remodeling in the airway
wall may have changed the quantity of ASM
relative to that of the connective tissues and
epithelium, the results on reactivity may
have been misinterpreted (13). Whole-
airway comparisons in MCh dose–response
between control subjects and subjects with
asthma have also been done recently, and
showed a clear decrease in airway diameter
at every dose in subjects with asthma, but
no changes in sensitivity of the airways to
MCh (14). However, it is unknown to what
extent those dose–responses were affected
by the epithelium and other airway wall
tissues. Consequently, our data should be
more representative of the actual intrinsic
ASM contractility.
The hyposensitivity observed in our
data may be the result of desensitization
from prolonged agonist exposure, which
would be expected to occur in asthma. This
has previously been shown in cultured
vascular SM cells (15) and more recently in
rabbit tracheal SM cells (16). Furthermore,
the increase in ASM mass found in asthma
is caused by hypertrophy and hyperplasia,
and both have been shown to reduce the
contractility of ASM. Our laboratory has
shown that rat tracheal ASM responds
to repeated allergen challenge with
a reduced contractility of SM cells and
a commensurate increase in SM cell
number (17). Hypertrophy of ASM has
also been shown to result in reduced
contractility in some studies (18), although
not consistently (19). Although detailed
medication intake of the donors in the
last weeks of life is difficult to get, it is
possible that end-of-life drugs or asthma
medication, particularly long-acting
b-agonists, may have reduced contractility.
However, it is unlikely that pharmaceutical
agents remain in effective concentrations
after dissection and equilibration protocols,
and prolonged exposure to b-agonists has
been shown to lead to aggravated AHR
(20, 21).
Shortening Velocity
The force–velocity data shown in Figure 3
directly contradict a range of findings in
both human and animal studies. Several
animal models of AHR have shown an
increase in Vmax in MCh contracted
trachea (22–24). In humans, asthmatic
bronchial ASM cells harvested by
endobronchial biopsies were shown to have
an increased Vmax and total shortening
when exposed to contractile agonists
compared with control subjects (25).
Our laboratory has also shown in
a mathematical model that an increase in
shortening velocity could, in principle,
0.6
0.7
0.8
Normalized Stress
0.9
Passive
Control
Asthma
**
**
**
A
1
Contracted
+
B
0.6
0.7
51015
Minutes since DI
20 250
0.8
Normalized Stress
0.9
**
**
*
*
C
1
51015
Minutes since DI
20 250
*
++ *
*
D
Figure 4. Deep inspiration (DI) response. All stresses are normalized to the average contractile stress
over three electrical field stimulation (EFS) contractions prior to the first DI. (Aand C) EFS contractile
stress after a DI in passive, relaxed airway smooth muscle. (Band D) EFS contractile stress after
a DI in methacholine 10
26
M contracted airway smooth muscle. Cand Dfollow the same protocol
as Aand Bbut with a continuous superimposed length oscillation equivalent in amplitude and
frequency to tidal breathing. Black squares are control subjects (n = 6) and gray squares subjects with
asthma (n = 6). Two-way repeated measures analysis of variance showed statistically significant
differences within the same group of subjects, but not between subjects with asthma and control
subjects. Markers indicate significant differences: *different from force prior to DI; **same as * but also
different from all subsequent EFS contractions;
1
different from force at 20 and 25 minutes;
11
different from force at 25 minutes.
ORIGINAL ARTICLE
890 American Journal of Respiratory and Critical Care Medicine Volume 191 Number 8 |April 15 2015

account for the differential response to DI
in subjects with asthma because increased
shortening velocity may lead to a faster
return to a prestretch length (26).
Furthermore, Jackson and colleagues (27)
showed that immediately after a DI the rate
of increase of airway resistance is much
higher in subjects with asthma compared
with control subjects, which may indeed be
caused by an increase in shortening
velocity.
Nonetheless our current study showed
no change in Vmax, with high confidence
that Vmax in subjects with asthma is not
considerably increased compared with
control subjects. One possible explanation
may be that, because Vmax is not uniform
throughout the lung (12), ASM shortening
velocity may also not be changed uniformly
throughout the lung in asthma. Trachea
and main bronchi are likely exposed to
a different inflammatory and mechanical
environment than peripheral bronchi and
consequently the trend of decreased Vmax
in asthmatic main bronchi may not extend
into the periphery. The only other study
of EFS shortening velocity in human
trachealis SM also found no differences
between subjects with asthma and control
subjects (8), in agreement with our data.
DI Response
Adefining feature of asthma is the lack of
response to DIs, whereas in healthy subjects
DI bronchodilating and bronchoprotective
abilities surpass any currently available
medication (28, 29). Several studies have
shown that DIs can reduce subsequent
contractile force generation in animal ASM
and this effect has been hypothesized to be
reduced in asthma (30, 31). Our results do
indicate that the contractile force is reduced
0
100
Stress (kPa)
200
300
Control
Asthma
–7
–6.5
Dose (10X M)
–6
–5.5
0.2
30 40
BMI
50
0.25
0.3
40 60
A
g
e (Years)
80 Female Male
Gender
0.35
Vmax (L0 s–1)
0.4
0.45
Figure 5. Body mass index (BMI), age, and sex correlations for three contractility parameters. None of the parameters showed a significant correlation
with BMI, age, or sex.
ORIGINAL ARTICLE
Ijpma, Kachmar, Matusovsky, et al.: Human Airway Smooth Muscle in Asthma 891

after a DI, but subjects with asthma only
show a nonsignificantly greater force
reduction following a DI in relaxed muscle
compared with control subjects. Even
length oscillations equivalent to continuous
breathing did not have much effect on the
contractile force in subjects with asthma
or control subjects. The subject with
severe asthma showed contractile force
potentiation after a DI in contracted
muscle, but this was not seen in any of the
other subjects with asthma, including the
subject with fatal asthma.
The DI response in relaxed muscle in
subjects with asthma versus control subjects
has previously been assessed by Chin and
colleagues (8), showing a reduced effect on
subsequent EFS contractions in subjects
with asthma. However, their protocol used
10-minute 30% L
ref
length oscillations,
whereas we simulated a physiologic single
DI with a single half-sinusoidal stretch of
30% of L
ref
in relaxed muscle and a similar
20% L
ref
stretch in 10
26
M MCh contracted
muscle. Although there are some
differences in the type of subjects (age and
asthma severity), the difference likely lies in
the applied protocols. Perhaps stretch-
activated mechanisms in ASM respond
differently to a single stretch than to a long
duration of repeated stretches. Another
study, on whole airway segments taken
from lung resections of control subjects
andmildtomoderatesubjectswith
asthma, showed an immediate effect of DI
similar to ours on the dose–response to
MCh in control subjects and subjects with
asthma, but over time, they observed
a greater narrowing in the subjects with
asthma (31). While this narrowing did not
surpass the narrowing prior to the DI,
these results were different from ours,
which showed no significant difference
between the equilibrium contractile force
after the DI and the contractile force prior
to the DI.
Age, Sex, and BMI Effects
Age effects on ASM response have been
found in animal studies (9) and both weight
(32) and sex (33) have been implicated in
human asthma. Although not enough
subjects could be tested for conclusive
answers regarding correlations, no
obvious trends were apparent for any of
the three tested parameters. The lack of
sex effects may be attributed to age,
because sex effects are more obvious in
teenagers (33). Alternatively, any short-
term hormonal effects may not be present
because the hormones would be washed
out during equilibration or degraded
during transport. Maturational studies in
sheep (34) and guinea pigs (9) show
changes in both contractile force
(increase with age) and shortening
velocity (strong decrease with age).
However, these studies focused on very
young animals and little is known about
changes in adulthood.
Conclusions
Our data suggest that, in contrast to many
animal models of AHR, human tracheal and
main bronchi SM does not contribute to
AHR through persistent changes in
contractile properties. Our results indicate
that it is highly unlikely that ASM EC
50
or
Vmax contribute to AHR in asthma, but,
because of high variability, we cannot
conclude whether asthmatic ASM is
hyperreactive. We conclude that ASM
is probably not intrinsically and
homogenously altered in asthma. Further
research will have to address whether
transient or more peripheral ASM changes
do occur. n
Author disclosures are available with the text
of this article at www.atsjournals.org.
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