Download Influence of intercellular tissue connections on airway muscle

Influence of intercellular tissue connections on airway
muscle mechanics
R. A. MEISS
Indiana University School of Medicine, Indianapolis, Indiana 46202
stiffness; smooth muscle contraction; tracheal muscle; shortening; connective tissue
IN A LARGE AND COMPLEX ORGAN such as the lung, smooth
muscle must operate within a complex mechanical
environment in which cartilaginous and parenchymal
attachments modify its contractile functions. Many
studies have examined the effect of forces and influences arising from various extramuscular components
on the mechanical function of pulmonary smooth muscle
(6, 7, 18, 21, 36). However, the mechanical performance
of smooth muscle tissue itself, even when freed from the
mechanical constraints of surrounding tissue, involves
complex interactions between the contractile cells and
the connective tissue of its own extracellular matrix.
Measurements of cellular mechanics in a multicellular
preparation, or the ability to predict the behavior of a
multicellular preparation from the properties of its
constituent cells, are limited by such interactions. In
those smooth muscles that undergo considerable shortening as part of their normal physiological function
(e.g., bronchial muscle, urinary bladder, or uterus), the
effects of tissue length on the contractile properties of
the whole tissue or organ are an important factor in
determining the overall function.
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Many mechanisms have been proposed to explain the
length dependence of smooth muscle mechanical behavior, including cellular processes such as length-dependent activation (i.e., calcium entry and myosin phosphorylation) (12, 14, 15, 22), length-dependent rearrangement of
the cellular constituents of the contractile mechanism
itself (5, 13, 37, 43), and the presence of length-dependent
internal mechanical loading (16). Much evidence supports these mechanisms, all of which are based on
processes that are primarily intracellular. The present
paper addresses an additional mechanism that is related to extracellular structures and has not yet been
studied extensively. In this regard, Gabella and Raeymaekers (11) describe the modified shortening behavior
of collagenase-treated taenia coli smooth muscle, and
Bramley et al. (2) have shown that collagenase treatment of isolated strips of bronchial muscle makes them
capable of additional shortening. This finding differs
from that of Ma and Stephens (20), who reported a
decrease in force development with no increase in
shortening. However, these results all imply that an
important component of the mechanical environment of
smooth muscle cells is provided by their extracellular
matrix and cell-to-cell interactions.
The experiments in this paper address the question
of extracellular mechanical constraints and are guided
by a set of assumptions and proposed interactions that
can be termed the ‘‘radial-constraint hypothesis’’ (26,
27, 32). They are designed both to provide further tests
of the validity of the hypothesis and to explain features
of the behavior of isolated smooth muscle in general.
The hypothesis holds that a strip of smooth muscle,
when very lightly loaded, will contract isotonically at
approximately constant tissue volume. As the extreme
of shortening is approached, the tissue must expand
significantly in a radial direction to accommodate constant volume. This expansion is counteracted by connective tissue acting in a radial direction. The strain
induced in this radial tissue acts as a load on the
contractile apparatus and causes shortening to be
limited, and it also causes the axial stiffness of the
preparation to rise as cross bridges are recruited to
bear the additional internal load. Thus the hypothesis
defines a way in which the transverse (or radial)
properties of the tissue can be investigated by making
measurements of shortening and stiffness that are
confined to the axial direction of the tissue strip.
Two approaches are reported here. In the one case,
additional radial constraints were added (reversibly) to
a contracting muscle strip, and the effects on the
contraction were characterized. In the second case,
radial constraints were removed (irreversibly) by mild
digestion of the intact tissue with collagenase, and a
similar mechanical characterization was done. In both
8750-7587/99 $5.00 Copyright r 1999 the American Physiological Society
5
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Meiss, R. A. Influence of intercellular tissue connections
on airway muscle mechanics. J. Appl. Physiol. 86(1): 5–15,
1999.—Contraction of smooth muscle in visceral organs is
modified by structures external to the muscle. Within muscle
tissue itself, connective tissue plays an important role in force
transference among the contractile cells. Connections arranged radially can affect contractile mechanics by limiting
tissue expansion at short lengths. Previous work suggests
that increased stiffness at extreme shortening is due to such
radial constraints. Two approaches to further study of these
effects are reported. To increase radial constraints, very thin
Silastic bands were placed loosely about strips of canine
trachealis muscle at rest length. The strips were allowed to
shorten under light afterloads, expanding until restrained by
the bands. Subsequent removal of the bands allowed increased shortening, with less increase in stiffness at short
lengths. Related isometric effects were observed. To reduce
constraints, muscle strips were partially digested with collagenase. Compared with control conditions, this treatment
permitted further shortening, with less increase in stiffness
at short lengths. These results emphasize the role of extracellular structures in determining mechanical function of smooth
muscle.
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CONNECTIVE TISSUE AND SMOOTH MUSCLE MECHANICS
cases, the results were consistent with the general
predictions of the hypothesis.
METHODS
Fig. 1. Placement and effects of constraint bands. Left:
with muscle at rest length, bands are evenly spaced and
offer no hindrance to shortening (top); Lo, optimal
length; as muscle shortens and increases in radius,
bands constrain radial expansion (middle); when bands
are removed, muscle is allowed to shorten further
(bottom). Right (A–C): force-stiffness plots corresponding to conditions at left, top to bottom, s, prevailing
length and stiffness conditions. Compare with data
traces in Fig. 3.
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Tissue preparation. All of the experiments described here
were performed on isolated canine trachealis strips. Mongrel
dogs were anesthetized with pentobarbital sodium and were
rapidly exsanguinated. A 10- to 15-cm-long segment of extrathoracic trachea was quickly removed and placed in a physiological saline solution of the following composition (in mM):
125 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 15.5 NaHCO3, 1.2
KH2PO4, and 11.5 glucose. The solution was bubbled throughout the experiment with 95% O2-5% CO2 to maintain a
physiological pH. The cartilaginous rings were cut at both
sides, and the trachea was pinned out in a dissecting dish.
The muscle area was cleaned of epithelial and adventitial
tissue. Under a dissecting microscope, small strips of muscle
tissue (,0.75 mm diameter and 8–12 mm long) were cut from
the muscle sheet, with care being taken to follow the natural
division of the tissue into discrete fiber bundles. To ensure a
low-compliance (and removable) attachment to the experimental apparatus, the ends of the strip were clamped in aluminum foil cylinders as previously described (24). Direct measurements of total system compliance, which included that of
the force transducer and all other mechanical components,
amounted to 0.93 µm/mN. This was equivalent to ,0.5% of
the muscle length at maximal force (e.g., 50 mN) at optimum
length (Lo ) (e.g., 10 mm).
After the tissue was mounted to the extension arms of the
apparatus, it was extended by adjusting the micromanipulator that bore the force transducer until a small force (,1–2%
of the anticipated maximum) was recorded. This length was
designated rest length (Lr ) and was ,10% less than Lo. This
procedure was used because the experimental protocols required that passive force be kept to a minimum. After the
tissue was mounted, the muscle bath, mounted on a rack-andpinion assembly, was elevated to immerse the muscle in
circulating, temperature-controlled, and oxygenated physiological saline solution. Muscles were stimulated by using
platinum electrodes along either side of the tissue, with
supramaximal voltage pulses of alternating polarity at a
frequency and voltage previously determined to produce a
maximal mechanical response.
Mechanical instrumentation. The protocols described below required close control of muscle force and/or length, along
with continuous measurement of the muscle stiffness under
both isotonic and isometric conditions. These requirements
were met by using a force-clamp system built around a
Cambridge 300H Ergometer with external force feedback.
The muscle was attached at one end to the lever of the driver
motor, while the other end was attached to the active element
of a photoelectric force transducer with a natural frequency of
670 Hz and a linear compliance of 0.28 µm/mN. A dedicated
digital computer system [suggested by the software design of
Smith and Barsotti (40)] was used to provide feedback for
isotonic force control. The output of the force transducer was
compared with an adjustable reference voltage to produce
isotonic conditions. Changes in experimental conditions were
controlled by signals from a second digital computer system
that also digitized and stored the experimental data.
Dynamic muscle stiffness was measured, as in previous
studies on other tissues (23, 27, 31), by applying a sinusoidal
length oscillation (32–100 Hz, amplitude ,0.5% Lr ) to the
muscle via the driver motor and analyzing the resulting force
perturbations, with stiffness defined as the ratio of the force
change to the length change (dF/dL). This system allowed
continuous measurement of muscle stiffness, a critical parameter in these studies, under both isotonic and isometric
conditions. At the perturbation amplitudes used, there was no
discernible effect of the oscillations on the muscle performance (24).
Experimental protocols. Before control and experimental
data were obtained, a preparation was equilibrated with a
series of electrically stimulated isometric contractions (usually 5–10, at 5-min intervals) until a constant peak isometric
force was obtained. During the subsequent control period,
isometric (at Lr ) and isotonic contractions were alternated.
An isotonic contraction (see Figs. 2 and 6, for example)
usually began with a brief period of isometric activity. As force
began to rise, the force-clamp system was switched to the
isotonic mode, with the afterload controlled at a level very
close to zero force (,5% of peak isometric force). The stimulus
duration was set to allow complete shortening before the
stimulus was turned off.
To produce an artificial radial constraint of shortening
tissue, five to six very thin (,0.2 mm) restraint bands cut
from Silastic tubing were placed around the muscle strip
before applying the aluminum foil attachment fixtures (see
Fig. 1.) Care was taken to ensure that the bands fit loosely
around the strip at the Lr and that they were evenly spaced
(there was very little lateral migration of the bands during
the experiment). After the control contractions on the banded
CONNECTIVE TISSUE AND SMOOTH MUSCLE MECHANICS
RESULTS
Isotonic behavior of constrained muscle. Figures 2
and 3 show the isotonic contraction of the same tra-
Fig. 3. Effects of constraint bands on isotonic length-stiffness relation (cf. Fig. 2). Stiffness is plotted as function of muscle length. Note
restricted shortening and increased stiffness as result of presence of 7
constraint bands with inside diameter of 0.75 mm. In this example,
there was no apparent change in stiffness during early portion of
shortening.
cheal muscle strip with and without the presence of
applied constraints (5 bands, 1.2-mm inside diameter,
0.2-mm thick, evenly spaced). Compared with the control condition (Fig. 2B), the constrained tissue (Fig. 2A)
shortened less (by ,15% of Lr ), and the shortening was
accompanied by almost twice the increase in peak
isotonic stiffness. During the isometric and early isotonic portions of the contraction, there was no apparent
effect of the constraint bands on muscle behavior,
although there were some changes in the initial isotonic stiffness and shortening velocity. For the data set
summarized in Fig. 4 and Table 1, the mean stiffness at
the onset of isotonic shortening was 0.061 6 0.033
mN/µm with the bands applied. This fell to 0.051 6
0.020 mN/µm on their removal. Similarly, initial shortening velocity for the banded muscles was 2.650 6
Fig. 2. Effects of constraining bands on isotonic
mechanical activity. A: length (L), force (F), and
stiffness traces of a muscle contracting with 5 constraining bands (1.2-mm diameter) applied. Contraction started isometrically; switch to isotonic conditions allowed the muscle to shorten as fully as
possible with prevailing afterload and constraint
bands. dF/dL, change in F/change in L. Dashed
lines, peak shortening and stiffness. B: same muscle,
at same afterload, with constraint bands removed.
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strip were finished, the muscle was removed from the apparatus, keeping the foil attachment fixtures intact. The bands
were carefully dissected away, and the strip was remounted
to the apparatus by using the same mounting holes as in the
control situation. After the remounting, a set of measurements complementary to those done in the banded condition
were carried out. The physical manipulations involved in
these procedures did not appear to have a detrimental effect
on the performance or viability of the preparations, although
small changes in the shortening velocity and the isotonic
stiffness at the onset of shortening were noted (see RESULTS ).
Twelve muscles, with a mean Lr of 9.75 6 0.62 (SD) mm and
weight of 4.8 6 1.6 mg, were studied in this manner.
To reduce the (hypothetical) degree of radial constraint, 17
muscles (mean Lr, 9.57 6 0.54 mm; mean weight, 5.9 6 1.5
mg) were subjected to mild collagenase digestion, by using
collagenase type III (Worthington Biochemical, Freehold, NJ)
at a mean concentration of 539 6 9.1 U/ml. The muscle was
left attached to the experimental apparatus, and an auxiliary
bath (volume 3.0 ml) with thermal connections to the experimental tissue bath was used to maintain the muscle at ,37°C
during the digestion. During the digestion period, which
averaged 21.4 6 0.44 min, the solution was gently stirred, but
care was taken to prevent any mechanical perturbation of the
muscle preparation. At the end of the digestion period, the
auxiliary bath was removed and was replaced with the main
experimental muscle bath. Washing of the preparation was
accomplished by the continuous circulation of physiological
saline in the larger volume (35 ml) of the muscle bath. Sham
digestions, using the above protocol but without collagenase,
were performed as controls. There was no significant change
in muscle properties due to the experimental manipulations.
Statistical comparisons. All experimental protocols were
arranged so that each muscle served as its own control. This
procedure minimized animal-to-animal variation and allowed the detection of small but significant variations with
the use of a paired t-test procedure (SigmaStat; Jandel
Scientific).
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CONNECTIVE TISSUE AND SMOOTH MUSCLE MECHANICS
0.746 mm/s, whereas that for the same muscles unbanded was 2.370 6 0.706 mm/s. This represented a
9.0% decline in velocity. A paired t-test showed that this
decline was statistically significant (P 5 0.006). However, since the paired before and after measurements
spanned a significant amount of time (up to 2 h), this
slight fall could be attributed to the expected rundown
of the preparation rather than to a consistent effect of
the constraint bands. In those instances in which the
entire curves were determined, the force-velocity relationship (not shown) was unchanged by the removal of
the constraint bands, because all velocities were measured at lengths close to Lr, where the constraint bands
were still slack. The shortening length at which the
effect of the bands did become evident depended significantly (for a constant size of band) on the size (mass) of
the muscle (P , 0.001).
A closer view of the effect of applied constraints is
shown in Fig. 3, in which the muscle stiffness is plotted
as a continuous function of length during isotonic
shortening. Early in the contraction the curves do not
differ greatly, but after a few millimeters of shortening,
the stiffness of the banded muscle (k) begins to increase rapidly and reaches its highest value at the end
of the (somewhat restricted) shortening. The same
preparation, with the bands now removed (s), is free to
shorten further, and less stiffness is developed as
shortening continues. These results are summarized in
Fig. 4 and Table 1, in which 47 paired contractions of 12
muscle preparations are compared. The differences
between both parameters, as determined with a paired
t-test, are highly significant (P , 0.0001). Expressed in
other terms (and with the same degree of statistical
significance), the banded muscles shortened to 44.1% of
Lr, whereas the same muscles, after unbanding, shortened further to 31.5% of Lr. Concomitantly, the banded
muscles showed a stiffness increase during shortening
of 32.2%, whereas the same muscles unbanded showed
Table 1.
Shortening, DL/L
Velocity, mm/s
Initial dF/dL, mN/µ
Peak dF/dL, mN/µ
Muscle constrained with Silastic bands (n 5 47)
Condition
Mean 6 SD
95% C.I.
Ratio (U/B)
P value
Banded
Unbanded
0.560 6 0.096 0.685 6 0.103
0.1070 to 0.1440
1.234
,0.001
Condition
Mean 6 SD
95% C.I.
Ratio (D/I)
P value
Intact
Digested
0.788 6 0.064 0.802 6 0.059
0.0362 to 0.0645
1.019
,0.001
Banded
Unbanded
2.650 6 0.746 2.370 6 0.706
0.0858 to 0.4760
0.918
0.006
Banded
Unbanded
0.061 6 0.033 0.051 6 0.020
0.0050 to 0.0146
0.896
,0.001
Banded
Unbanded
0.218 6 0.137 0.113 6 0.069
0.0803 to 0.1290
0.541
,0.001
Muscle subjected to collagenase digestion (n 5 17)
Intact
Digested
3.071 6 0.490 3.360 6 0.462
0.0215 to 0.0078
1.103
,0.001
Intact
Digested
0.046 6 0.023 0.054 6 0.017
0.0011 to 0.0146
1.288
0.029
Intact
Digested
0.271 6 0.104 0.220 6 0.106
0.1590 to 0.4190
0.793
,0.001
All statistical comparisons were made on a paired basis, with each muscle serving as its own control. U/B, unbanded to banded; D/I, digested
to intact. 95% C.I., 95% confidence interval is for difference of means (paired t-test).
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Fig. 4. Statistical analysis of effects of radial
constraints and their removal. Each muscle
served as its own control, with each constrained contraction being compared with contraction with bands removed. A–D: center
plot shows before-and-after values of parameter in question for each individual muscle;
box plots (right and left) indicate characteristics of whole population. In box plots, horizontal band represents median; top and bottom
edges of box are 75th and 25th percentiles,
respectively; extensions are 90th and 10th
percentiles, respectively; s, 95th and 5th
percentiles, respectively. Increase in peak
shortening (A) and decrease in peak isotonic
stiffness (B) are quite marked, whereas effects on initial velocity (C) and initial isotonic
stiffness (D), although statistically significant, are less clear cut. Data are from 47
paired contractions of 12 muscles. For clarity
in plotting individual comparisons (center
plots), data were pooled and averaged for each
muscle, leaving only 12 sets of points to plot.
See Table 1 for numerical summary of complete data set.
CONNECTIVE TISSUE AND SMOOTH MUSCLE MECHANICS
sured on paired contractions (intact vs. digested) of 17
muscle preparations. Because of the potentially destructive effects of the enzyme treatment on muscle viability,
comparisons were made only between the last predigestion contraction and the first postdigestion contraction.
Thus there was only one contribution to the data set
from each muscle (in contrast to the case for the banded
muscle experiments). Data were analyzed for significant differences with a paired t-test (see Table 1 and
Fig. 8). Although the effect of digestion on peak shortening was not very large, it was highly significant (P 5
0.0003). The effect on peak isotonic stiffness was considerably greater and was also highly significant (P 5
0.0001), as was the effect on the initial isotonic stiffness, although there was greater variation in the initial
stiffness data. Postdigestion shortening velocities (at
the beginning of the isotonic phase of the contraction)
were consistently and significantly higher that the
predigestion control values, an effect that may reflect a
reduced internal load on the contractile cells (see
Fig. 5. Relationship between isometric and isotonic effects of constraint bands. A: 2 isometric length-tension curves from same
muscle, before and after removing constraint bands. Data have been
fitted with arbitrary 2nd degree polynomial functions, with 95%
confidence intervals shown (dotted lines). Approximate point at
which divergence begins is marked by vertical dashed line. B: 2
length-stiffness curves from same muscle, before and after removing
bands (compare with Fig. 3). Again, approximate point of divergence
is marked by vertical dashed lines. C: for each of 5 muscles so treated,
correspondence between isotonic (ordinate) and isometric (abscissa)
estimates of divergence is shown. There is good correlation between
the 2 measurements. Dashed lines, 95% confidence intervals.
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an increase of 17.8%. The relatively small changes in
shortening velocity and initial stiffness, as mentioned
earlier, may reflect temporal changes in the muscle
unrelated to the presence of the constraint bands. Note
that 12 pairs of values are shown for the individual
muscles in Fig. 4. For clarity in plotting, multiple
determinations on individual muscles were averaged to
provide one set of data per muscle. This provisional
data pooling did not result in any significant changes in
the statistical parameters. The statistical calculations
in Table 1 and the box plots in Fig. 4 were based on all
47 individual comparison pairs. The unbanded-tobanded ratio [Ratio (U/B)] in Table 1 is arranged to
show the effects of removing the bands; it is thus
analogous to the results of enzymatic digestion to be
reported below.
Isometric behavior of constrained muscle. The relationship between the length and diameter of a muscle
(with constant volume) should be relatively independent of whether the muscle is contracting under isotonic or isometric conditions. A test of this assumption
is shown in Fig. 5. Conventional isometric lengthtension curves were constructed for a muscle strip
before and after removal of the constraint bands (Fig.
5A). The data points have been fitted with arbitrary
second-degree polynomial functions, and the 95% confidence intervals are shown. It is apparent that the
presence of the constraint bands reduced the muscle
force at the shorter lengths while having no detectable
effect at lengths nearer to Lr. Lightly loaded isotonic
contractions were also made under the same conditions, and a plot of their length vs. stiffness relationship
is shown in Fig. 5B. Although it was difficult to define
precisely the length at which the two length-tension
curves diverged, this parameter could be estimated by
choosing the point at which the 95% confidence intervals no longer overlapped. When this point was compared with the divergence shown by the lengthstiffness plots in Fig. 5B, a close correspondence was
found. For five muscles of differing weights that underwent this isotonic-isometric comparison protocol, the
lengths at which the isometric length-force and isotonic
length-stiffness curves diverged showed a significant
correlation (Fig. 5C).
Effects of partial digestion on shortening and stiffness. Figure 6 shows the results of a 17-min digestion
(with 270 U/ml of collagenase type III) on the isotonic
behavior of a muscle strip. Compared with the control
record (Fig. 6A), the digested strip shortened further
but became considerably less stiff at the peak of shortening (Fig. 6B). Note also that the stiffness at the
beginning of isotonic shortening was greater for the
digested muscle. The initial isotonic shortening velocity
was also increased by a small, but statistically significant, amount.
The contraction data from Fig. 6 are further illustrated in Fig. 7 (compare with Fig. 3 for banded
muscle). For use in analyzing multiple contractions,
data values for peak shortening (1), peak isotonic
stiffness (2), initial isotonic stiffness (3), and the initial
shortening velocity (not shown in Fig. 7) were mea-
9
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CONNECTIVE TISSUE AND SMOOTH MUSCLE MECHANICS
Fig. 6. A: length, force, and stiffness traces of muscle
contracting under control conditions. As in Fig. 2,
contraction began under isometric conditions; then
muscle was allowed to shorten as fully as possible at
low afterload. Dashed lines, peak shortening, peak
stiffness, and initial stiffness (top, middle, bottom,
respectively). B: same muscle, after 17 min of collagenase digestion. Nos. 1–3 identify regions that correspond to similar regions in length-stiffness plot in
Fig. 7.
The decline in isometric muscle function was also
followed after the period of digestion (unpublished
observations). In a typical strip, in which contractions
were made every 6 min, the peak isometric force fell
steadily in an approximately exponential manner. Increasing the interval between contractions decreased
the rate of decline, but when the data were analyzed on
the basis of each contraction, the rate of decline became
constant. This indicates that the decline was due
primarily to mechanical damage to weakened connective tissue caused by the force generated by the cells.
Peak isometric force and stiffness fell proportionately
(i.e., the ratio of stiffness to force was preserved, as
noted in the previous section), indicating a common
locus for the elements bearing the developed force and
those providing the axial resistance to elongation.
DISCUSSION
Fig. 7. Data replotted from experiment shown in Fig. 6. Stiffness is
plotted as function of muscle length. Note additional shortening (1)
and reduced peak stiffness (2) as result of enzyme treatment. Initial
isotonic stiffness (3) was increased after digestion.
Summary of major findings. Mechanically constraining the increase in diameter of a shortening muscle
resulted in a decrease in the amount of shortening,
coupled with an increase in the stiffness at the shorter
lengths. Partial digestion of a muscle strip produced
the opposite effects, suggesting that the enzyme treatment has reduced internal constraints on tissue expansion. Smaller but significant effects were found on the
initial isotonic shortening velocity and on the initial
isotonic stiffness. These results will be discussed in the
context of the radial-constraint hypothesis, with its
inherent implications for the function of smooth muscle
in organs where muscle is subject to large amounts of
shortening.
The role of muscle in any organ whose function
requires an active dimensional change is obviously
critical. As is the case in many other organs, the muscle
components of the lung shorten on their own, but they
also are stretched and released by external forces
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DISCUSSION ). Statistical analysis of the stiffness during
the rise of isometric force from the data set reported in
Fig. 8 (before the sudden step to the afterload; see Fig.
6, bottom set of traces) showed that it was not significantly affected by the digestion (paired t-test).
Longer term effects of mild digestion. Figure 9 shows
data from a muscle strip digested for 22 min with 552
U/ml of collagenase type III. Compared with the control
record (left), the peak shortening and the initial velocity of shortening were maintained at a nearly constant
level for .30 min after the washout of the enzyme
preparation, although some of the later contractions
did not show the enhanced shortening. However, the
peak stiffness associated with the maximal shortening
showed a steady and marked decline over the same
period of time.
CONNECTIVE TISSUE AND SMOOTH MUSCLE MECHANICS
11
Fig. 8. Statistical analysis of effects of collagenase digestion. Each of 17 muscles served as
its own control, with intact condition compared with first isotonic contraction after
digestion. DL, change in length; Lr, resting
length. Data are displayed as in Fig. 4: increase in peak shortening (A); decrease in
peak isotonic stiffness (B); initial velocity (C);
initial isotonic stiffness (D). Data are summarized in Table 1.
mechanical activity, and mechanical connections are
made at numerous locations on the lateral surfaces of
the cell via membrane-associated dense bodies and
their intra- and extracellular connections (1, 4, 42)
rather than just at the ends of the cell. A cell in isolation
can be connected by its ends to experimental apparatus, but such a connection presents an arrangement
much different mechanically from the natural tissuebased connections. For these reasons, it would appear
that there are significant advantages to developing
ways of making detailed mechanical studies of smooth
muscle tissues in a relatively intact state, with the goal
of drawing inferences about cellular mechanical function. The validity of such inferences must rest on an
understanding of both tissue structure and the mechanical
relationships that the structural arrangements would de-
Fig. 9. Left: control contractions before digestion. Right:
each parameter was followed after digestion. Peak
shortening and velocity did not decline seriously, but
there was a steady and rapid decline of peak isotonic
stiffness.
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during the respiratory cycle (7). Even when airway
muscle is studied in isolation, the contractile activity of
its cells is subject to significant modification by connections and interactions within the muscle tissue itself. It
might, therefore, appear that study of isolated muscle
cells would be a fruitful and relatively uncomplicated
source of basic information about the mechanical function of smooth muscles. In fact, this approach has
yielded much of value in our understanding of muscle
function and continues to do so, despite the considerable technical difficulties involved. However, as a number of structural studies have emphasized, the mechanical conditions facing the contractile apparatus of isolated
cells are significantly different from those experienced
in an intact tissue (8–10, 19). In particular, a cell
within a tissue is both a generator and a transmitter of
12
CONNECTIVE TISSUE AND SMOOTH MUSCLE MECHANICS
even at moderate lengths (Figs. 2 and 6) suggests that
intracellular loads do influence the speed of muscle
shortening.
Results from banded muscle strips. The effects of
externally applied constraints in limiting the expansion of shortening muscle strips are, to a first approximation, qualitatively consistent with the hypothesis
under investigation, but some caution must be applied
to interpretation of the results. The constraint bands do
not contain the tissue uniformly, as would be the case if
the muscle were contained in a continuous rigid cylinder. To allow longitudinal freedom of movement, the
bands were made very thin (,0.2 mm), thus minimizing mechanical interaction between adjacent bands.
However, unconstrained tissue would be allowed to
expand between adjacent bands, and the expected
effects would be reduced. In addition, for smaller
muscles, their own internal radial connective tissue
elements would begin to come into play before the
constraint bands produced much of an effect. Despite
these quantitative shortcomings of the constraint-band
experiments, the results do support the basic qualitative predictions of the hypothesis.
The agreement between the isotonic and isometric
effects of constraining bands (Fig. 5) makes it apparent
that, for full isometric force to be developed, the muscle
strip must be free to assume the appropriate diameter
when active. These results imply strongly that one of
the determinants of the isometric length-tension relationship in intact smooth muscle is lateral connections
within the tissue that prevent full external (axial)
expression of internally generated force. This is further
supported by measurements which show that isometric
muscle stiffness undergoes less of a relative decline in
force at short lengths than does the isometric force (23).
Both of these observations emphasize that, in addition
to cellular mechanisms such as myofilament overlap
and varying activation, the overall tissue geometry
must be considered as a significant contributor to the
isometric length-tension relationship.
Mechanics of partially digested tissues. This portion
of the experimental protocol was designed to address
the question of radial constraints by attempting to
remove them, rather than by adding them as in the first
set of experiments. Efforts were made to keep the
protocols as similar as possible, although the effects of
the digestion made it impossible to complete a reliable
length-tension curve on the digested muscle. In several
instances, both the banding and digestion protocols
were carried out on the same muscle strip. This practice was not generally used, however, because of the
possibility of the deterioration of the preparation, especially after the period of digestion.
The continued decline in isometric force after digestion (see RESULTS ) and the decline in peak isotonic
stiffness (Fig. 9) might be viewed as such a decline in
viability. However, other measures of contractility, such
as the peak velocity of shortening or the maximal
degree of shortening, do not show a steady and continued decline. These findings may have two explanations.
If the activity of the collagenase had continued even
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termine. The present paper is an attempt to further such
an experimental and analytical framework.
Assumptions of the radial-constraint hypothesis. Important elements of this hypothesis have been addressed in experiments on other tissues (26, 27, 29, 30),
and a brief synopsis of its salient features has been
given in the introduction to this paper. Because the
essence of the proposal is that the length-dependent
increase in stiffness is due to geometrical, not biochemical, factors, it should be applicable to a wide range of
smooth muscle tissues. One element of the hypothesis,
however, does concern activities occurring within the
cells. Although there have been recent cautions expressed in the case of skeletal muscle, it is generally
accepted that the stiffness of contracting muscle is
related to the size of the active cross-bridge population.
The close relationship between developed force and
stiffness in isometrically contracting smooth muscle
has been demonstrated for a number of smooth muscles
(13, 17, 23, 34, 44), and deviations from this relationship have been used to provide evidence for particular
conditions of the contractile system (13, 25, 31). In the
present case, because the force-clamp system keeps the
external force low and constant, it is assumed that the
measured increases in axial stiffness represent increased cellular cross-bridge activity, which would vary
in response to a mechanical load developed within the
tissue. An attempt has been made to calculate the size
of such an internal load, for instance in ovarian ligament muscle (30), on the basis of the measured relationship between force and stiffness under isometric conditions. At short lengths and low afterloads, it was shown
to be an appreciable fraction of the externally manifested force. These arguments assume that the size of
the active cross-bridge population in smooth muscle is
capable of rapid adjustment in response to a changing
load, whatever its source. Unpublished experiments
from this laboratory show that this is the case for
trachealis muscle; a change in applied load causes a
rapid adjustment to the force-velocity-length conditions appropriate to the new load. These properties also
hold for step increases in load. The rapid adjustment in
force-dependent muscle stiffness that occurs when external loading conditions change (for example, in the
transition from isometric to isotonic contraction; see
Fig. 2) further suggests that rapid changes in the size of
the cross-bridge population are possible and may play a
role in the adaptation to changing internal loads at
constant external force.
The internal load revealed by the radial-constraint
measurements is probably not to be identified with the
intracellular internal loads revealed by measurements
of single cells (16), because, in that case, the load was
proportional to cell length over a wide range and was
not associated with a significant rise in stiffness. The
radial-constraint internal load is highly nonlinear with
muscle length (as predicted by the hypothesis), and
thus it is likely to be tissue based. The presence of other
(cellular) internal loads in tracheal muscle (see Ref. 39)
is not ruled out by these experiments. In fact, the
continuous fall in velocity during isotonic shortening
CONNECTIVE TISSUE AND SMOOTH MUSCLE MECHANICS
more sensitive to degradation of radial (circumferential) elastic elements than to longitudinal elements.
The observed increase in initial isotonic stiffness
shown by the digested muscle appears anomalous at
first. However, it may be explained by a net shift,
caused by the cutting of critical internal connections,
from a more series-connected to a more parallelconnected orientation of the connective tissue throughout the muscle strip. Such behavior has been demonstrated for an artificial network of elastic elements and
their interconnections (3) and appears to be a phenomenon that is a general property of some types of
networks.
Implications of the findings. The present study has
attempted to view tracheal smooth muscle as an integrated tissue, intact but removed from the mechanical
constraints of its usual attachments in the trachea. The
aim of the isolation was to permit a focus on mechanical
constraints inherent within the structure of the tissue
itself. The degree of shortening of tracheal muscle in
situ is limited by its connections to the cartilaginous
rings of the trachea, and it is possible that this muscle
in vivo never experiences the extreme shortenings that
are associated with the sharp increase in axial stiffness. However, it is possible that muscle at more distal
regions of the respiratory tree does experience shortening sufficient to produce an increase in internal load (2,
21). In any event, the presence of the phenomenon in
isolated muscle does reveal features of tissue assembly
that are not mechanically evident under conditions of
less extreme shortening but which nonetheless are
features of its functional arrangement and may play a
role in airway mechanics.
Most studies of the mechanical properties have been
confined to the analysis of forces and connections
arranged in the axial direction of the tissue. Reports of
the measurement of the transverse elastic properties of
isolated muscle tissue, especially smooth muscle, are
not common in the literature. Tsuchiya and co-workers
(41) have used an ultrasound technique to measure
transverse stiffness in skeletal muscle, and there have
been attempts to use ultrasound to make dimensional
and mechanical measurements on vascular muscle in
situ (38). However, the tissue structure of smooth
muscle is quite unlike that of skeletal muscle, and in
the case of the vascular muscle, geometric factors of the
vessel wall obscure the properties of the muscle itself.
This present study offers an indirect but mechanically
straightforward means of investigating multidimensional mechanical properties of muscle tissue from a
number of organs and with a variety of experimental
goals. In addition to revealing the presence of radial
forces in intact tissue, these experiments suggest the
possibility of physically mapping the mechanical interconnections of the tissue by selective disruption of the
connective tissue with a variety of histolytic agents
(33). Work in other laboratories has also demonstrated
the continued viability of smooth muscle tissues subjected to partial digestion, and enzymatic and structural methods have been used to investigate the effects
of interactions between cells and tissue (2, 8, 10, 11).
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during the period of washout after digestion, then the
decline could represent further proteolysis of supporting structures. However, it is also likely that continued
mechanical activity, which would produce internal longitudinal (isometric conditions) or radial (isotonic conditions) mechanical stresses, resulted in physical disruption of internal connections weakened by the digestion.
This is borne out by the observed exponential decline in
peak isometric force (see RESULTS ), which suggests that
the amount of internal disruption experienced with
each contraction is proportional to the force produced in
the preceding one. Thus the decline becomes less as the
self-generated disrupting force becomes less and the
weaker connections have been progressively broken.
Viewing the digestion results in light of the findings
from the artificial constraint experiments, it is apparent that different degrees of constraint were present in
the two cases. Removal of the natural constraints by
digestion did allow the muscle strips to shorten further.
Although the amount of additional shortening was not
large compared with that seen in the banding experiments, the effect was consistent, with a very high
statistical significance. This smaller increase in shortening could be explained in two ways. In the first place,
the natural radial constraints were likely to be less
robust than those provided by the Silastic bands;
secondly, the degree of proteolysis was probably not
uniform across the diameter of the strip. With digestion
conditions kept mild enough to preserve the mechanical integrity of the preparation, a considerable portion
of the radially disposed connective tissue would have
escaped degradation. The small increase in shortening
velocity after digestion (see Fig. 8) may have been due
to reduced internal load associated with loss of connective tissue, but it is also possible that the period of
mechanical quiescence during the digestion period
allowed a more complete recovery of contractility than
did the usual 6 min between successive contractions.
However, there was no significant correlation between
the increased velocity and the degree of enhanced
shortening in individual muscles.
The peak stiffness data also showed different degrees
of sensitivity to the experimental protocols. Whereas
the artificial constraint bands almost doubled (1.85
times) the peak stiffness at the shortest lengths, the
stiffness of the digested muscle fell to about threequarters (0.793 times) of its control value. These differences are again probably due to the factors discussed in
regard to maximal shortening. It could also be argued
that the digestion process, in addition to its effects on
the radial connective tissue elements, also affected
connective tissue elements in series with the muscle
cells, and this might account for the reduced peak
isotonic stiffness. However, because the ratio between
force and stiffness was maintained during the degradation of the tissue and because the axial force was kept
very low, this factor should have minimal effect. A
quantitative analysis performed on a mathematical
model of the radial-constraint hypothesis (29) revealed
that the decrease in axial stiffness was considerably
13
14
CONNECTIVE TISSUE AND SMOOTH MUSCLE MECHANICS
The author thanks Dr. Susan Gunst for generous provision of
tracheal muscle tissue and for many helpful discussions.
The author also acknowledges the financial support of the Dept. of
Obstetrics and Gynecology, Indiana University School of Medicine.
A portion of this work has appeared previously in abstract form (32).
Address for reprint requests: R. A. Meiss, Depts. of Physiology and
Biophysics, and Obstetrics and Gynecology, Indiana Univ. School of
Medicine, Rm. 111, Med. Research Facility, 1001 Walnut St., Indianapolis, IN 46202 (E-mail: [email protected]).
Received 5 June 1998; accepted in final form 9 September 1998.
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