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Introduction
Partial bladder outlet obstruction (PBOO) in human and
animal models results in numerous changes in the detrusor.
While in humans the degree, duration, and cause of
obstruction varies from individual to individual, all animal models
have the advantage of creating obstruction of the same
degree, which is beneficial for studying the bladder at any
given time. The rabbit bladder provides an excellent
opportunity to understand the physiological, histological, and
biochemical properties of a functioning
bladder[1,2].
In both men[3] and
rabbits[4] with PBOO, the detrusor smooth muscle (DSM) undergoes hypertrophy to
compensate for the increased force required to expel urine against
the obstruction. In some rabbits, as in men, DSM
hypertrophy following PBOO compensates for the increased muscle
contractility, which is required to overcome the increased
urethral resistance during micturition (compensation),
whereas other rabbits show bladder dysfunction (decompensation)
despite smooth muscle (SM)
hypertrophy[5,6]. With muscle strips from hypertrophied DSM from decompensated
bladders, physiological studies show a decrease of force in
response to carbachol (CCH) or field stimulation
(FS)[4]. The molecular mechanisms responsible for these changes in
contractility and emptying remain largely unknown.
The actin-associated protein caldesmon (CaD) is thought
to modulate the regulation of SM contraction by
myosin-mediated regulation via the phosphorylation of the myosin
light chain[7]. CaD is expressed as 2 dominant isoforms,
l-CaD and h-CaD with low and high molecular sizes (70 and
140 kDa), respectively. l-CaD is found in non-muscle cells of
various tissue types[8], whereas h-CaD is present in SM cells
from all sources. h-CaD is bound to thin filaments in SM,
where it inhibits the actin-myosin interaction and
actomyosin ATPase activity[9]. Both l-CaD and h-CaD have similar
regions to bind actin and myosin[10].
Zhang et al[11] found that the expression of l-CaD
increases significantly at the mRNA and protein levels in the
decompensated bladders compared to normal and
compensated bladders. The inability of decompensated bladders to
empty, despite detrusor hypertrophy, is associated with an
overexpression of l-CaD. The level of l-CaD overexpression
might be a useful marker in estimating the degree of detrusor
remodeling and contractile dysfunction in PBOO.
In the present study, we examined the expression of CaD
isoforms in rabbit DSM during the progression of PBOO,
and related them with time course of bladder obstruction.
Materials and methods
Partial bladder outlet obstruction The rabbit experiments,
including the surgical procedure, were approved by the
Institutional Animal Care and Use Committee of Xi'an Jiaotong
University (Xi'an, China). Thirty-two adult male New Zealand
white rabbits were divided into 4 groups, each consisting of
8 rabbits. Six rabbits in each group underwent partial outlet
obstruction. The other 2 rabbits in each group underwent
sham operations, in which the ligature was removed
immediately after placing it around the urethra; these rabbits served
as the control group. The rabbits in each group were
evaluated after 1, 2, 4, and 8 weeks of obstruction, respectively.
The data from all of the control animals showed no
significant differences and were combined into 1 group.
Under anesthesia, an 8Fr catheter was inserted into the
bladder via the urethra; the bladder neck was exposed
through a small vertical abdominal incision. Once the
ureters and vas deferens were identified, a 2-zero silk suture
was placed below the bladder neck with right angle clamps.
To maximize the standardization of the partial outlet
obstruction, a second 8Fr catheter was placed outside the
urethra, and the silk suture was tied around both catheters,
which were then removed. In the sham-operated group, the
silk suture was cut and removed after an identical dissection.
At 1, 2, 4, and 8 weeks' postoperatively, the animal were
euthanized and the bladder was opened via the same midline
incision; a 1×1.5 cm section of the ventral bladder wall was
excised with a scalpel. Three full-thickness strips were fixed
for the histological studies. The mucosal and serosal layers
were removed, and 3 longitutudinal bladder strips were
obtained from each rabbit bladder for the contractile studies.
The bladder muscle layer from the mid-body region was
isolated and frozen in liquid nitrogen for protein extraction.
Physiological studies The muscle strips were attached
by a 4-zero silk suture to a post in a 50 mL tissue organ bath
at one end and an isometric force transducer at the other
end. The transducer was calibrated with known weights,
and its output was directed to a polygraph. The tissue
organ baths were maintained at 37 °C and contained Tyrode's
solution (125 mmol/L sodium chloride, 2.7 mmol/L potassium
chloride, 1.8 mmol/L calcium chloride, 0.5 mmol/L
magnesium chloride, 23.8 mmol/L sodium bicarbonate, 0.4 mmol/L
sodium phosphoric acid, and 5.6 mmol/L glucose) perfused
by a bubbled mixture of 95% oxygen and 5% carbon dioxide.
After equilibration for 30 min at slack length, the strips were
gradually stretched to the length where optimal force was
generated, and the maximal response to FS (32 Hz, 80 V, 1 ms
duration) was determined. After FS, the response to 200
µmol CCH was determined. Peak tension was recorded for
each of the different stimuli. Between the additions of
pharmacological agents, each strip was washed 3 times with fresh
buffer at 10 min intervals. After the completion of the
experiments, the muscle between the 2 silk sutures was
weighed. All of the results are expressed in gram
tension/100 mg tissue. The mean of the 3 strips from 1 animal
represented 1 individual preparation. The SEM for each
experiment was based on the 6 animals from each group.
The rabbits were divided into the control (sham operated)
and 1, 2, 4, and 8-week obstructed groups. Muscle strip
studies subcategorized the obstructed group of bladders
into compensated (force more than 10 g tension/100 mg
tissue at cholinergic stimulation) and decompensated (force
less than 10 g tension/100 mg
tissue)[5]. All of the results are presented with these definitions.
Histological studies Full-thickness strips from the
detrusor obtained from the bladder were fixed in 4% buffered
formalin. The strips were embedded in paraffin, and 6 mm
tissue sections were cut. The sections were stained with
conventional hematoxylin-eosin and Masson's trichrome
preparations. The muscle fraction was analyzed with a
microscope equipped with a digital camera and Image-Pro Plus 5.0
image analysis software. The percentage area was selected in
the program and generated automatically for each image.
Twenty random images per tissue were analyzed for each
tissue sample. The averages were then calculated for each group.
Protein extraction and Western blotting The total protein
was extracted in extraction buffer [20% glycerol, 50 mmol/L
Tris-HCl (pH 6.8) and 0.5% (v/v) Tween-20] and protease
inhibitor cocktail (Sigma, St Louis, MO, USA). After adding
10% SDS, the sample was mixed, boiled for 4 min, and
centrifuged at 11 000×g for 15 min at 4 °C to remove the
undissolved material. The protein concentration in the
supernatant was measured with the Bradford method. Aliquots of
the protein extract containing 30 µg of total protein were
electrophoresed in a 6% SDS-PAGE gel, and blotted
overnight at 4 °C to a P-membrane with Towbin buffer [25 mmol/L
Tris, 192 mmol/L glycine, and 20% (v/v) methanol]. The
membrane was blocked with 5% dry milk, and incubated with an
antibody against CaD (Santa Cruz Biotechnology, Santa Cruz,
CA, USA; catalog No sc-7574). After treatment with the
primary antibody, the membrane was washed in TBST buffer
(20 mmol/L Tris, 500 mmol/L NaCl, and 0.05% Tween-20), and
incubated with a secondary antibody (mouse antigoat
immunoglobulin G at 1:5000). The substrates were visualized
by ECL (Amersham Pharmacia Biotech) and by exposing the
membranes to autoradiographic films. The films were
scanned and analyzed with Image J Software (National
Institutes of Health). Standard curves were constructed to
establish the protein concentrations for the analysis fall within
the linear range.
Statistical analysis All data are expressed as the
mean±SEM with P<0.05 considered statistically significant.
ANOVA, followed by the Bonferonni test for individual
differences, were used for comparative purposes.
Results
Physiological studies The maximal contractile responses
to FS and CCH are presented in Figure 1. The responses to
all forms of stimulation decreased progressively in relation
to the duration of obstruction. The contractile responses to
FS were significantly sensitive to obstruction in comparison
to the contractile responses to CCH. The responses to CCH
decreased progressively, but significantly at 1 week
(P<0.05), and then decreased progressively, reaching a response less
than 10 g tension/100 mg tissue at 4 weeks of obstruction. It
should be noted that the 8-week obstructed group had the
lowest responses to FS and CCH.
Histological studies Figure 2 shows the representative
images of the control and 2 and 8-week obstructed rabbit
bladder body detrusor tissue sections showing SM and
collagen using Masson trichrome staining. The effect of
obstruction on the SM/collagen ratio is shown in Figure 3.
Obstructed bladders showed significantly hypertrophied
muscle bundles, and the collagen infiltration increased
progressively. The SM/collagen ratio increased
progressively before 2 weeks of obstruction, and decreased
gradually thereafter. After 1 week of obstruction, the ratio
increased significantly compared with the control
(P<0.05). After 4 and 8 weeks of obstruction, although significantly
lower than the earlier time points, the ratio was still higher
than that of the controls (Figure 3).
Western blot analysis of CaD isoforms in the bladder
Figure 4A shows the expression of h-CaD and l-CaD from
the control and obstructed rabbit bladders by Western
blotting. Data obtained from the Western blot analysis of
samples from the different groups are shown in Figure 4B.
The expression of l-CaD was very low in the control bladder.
Following obstruction-induced hypertrophy, the expression
of l-CaD increased significantly to approximately the same
extent as that of the 1_2-week obstructed groups and
increased further in the 4_8-week obstructed groups. However,
the increase was almost 8-fold higher in the 4-week obstructed
bladders and almost 25-fold higher in the 8-week obstructed
bladders. The expression of h-CaD increased in all of the
obstructed bladders, but at significantly higher levels in the
1_2-week obstructed bladders versus the control and
4_8-week obstructed bladders (P<0.05). The h-CaD/l-CaD ratio
decreased significantly in the 1_2-week obstructed groups
and decreased further in the 4_8-week obstructed groups.
Discussion
Previous studies on the rabbit model for bladder outlet
obstruction have identified several changes in whole
bladder function and contractile performance of the muscle
strip[11,13]. In the present study, we analyzed the molecular changes
in CaD isoform expression and correlated them with the time
course of obstruction. We found that bladder contractile
function decreased over the course of the obstruction, and
the 8-week obstructed group was the most severely compromised. In addition, we found that l-CaD and h-CaD
in DSM showed significant differences during the course of
PBOO. In particular, there was a progressive increase in
l-CaD and a significant overexpression in the 8-week
obstructed bladders compared to the normal and compensated
bladders, which indicated a close relationship with bladder
contractile function. The expression of h-CaD increased in
all obstructed bladders, but only at significantly higher
levels in the 1_2-week obstructed bladders.
Despite the acute onset, partial bladder outlet
obstruction in rabbits induces detrusor remodeling similar to that in
men with benign prostatic hyperplasia in terms of its impact
on structural and functional alterations in
SM[14]. In the animal model, the level of decompensation may be based on
more objective criteria, such as the contractile responses to
various forms of stimulation. In the present study, we
defined the muscle strips that force less than 10 g tension/100
mg tissue at cholinergic stimulation as
decompensated[5]. The DSM undergo compensatory hypertrophy required
to overcome the increased outlet resistance for the increased
force production. In some obstructed rabbits, as in humans,
detrusor hypertrophy and the associated remodeling are
sufficient to maintain a close to normal (compensated)
bladder function, whereas other rabbits show severe bladder
dysfunction (decompensated). Mannikarottu et
al[15,16] reported that bladder decompensation progresses over 28 d
based on bladder weight and response to various stimuli.
The responses of the rabbit bladders to partial outlet
obstruction may be divided into initial, compensated, and
decompensated phases[17,18]. The initial phase occurs
immediately after partial outlet obstruction. It is characterized by a
rapid increase in bladder mass until it becomes relatively
stable after surgery. The compensated state begins after
this initial growth period and is characterized by normal or
slightly lower than normal contractility. In some periods
after compensation, bladder function begins to deteriorate,
resulting in progressive decreases in contractile response to
all forms of stimulation, increased bladder mass, and
decreased compliance. In the final phase, decompensation is
characterized by connective tissue replacement of SM, which
finally results in a fibrous organ with little or no contractile
function[17,18]. Based on the morphological and
physiological studies, it can be concluded that the 1_2-week obstructed
bladders were in the compensated phase. At 4 weeks, the
obstructed bladders were in the decompensated phase, and
the 8-week obstructed bladders were severely
decompensated, but not in the final decompensated phase. Hence our
findings on changes in CaD isoforms can be correlated with these
well-accepted time points in bladder function after obstruction.
The SM is hypertrophied throughout the obstruction
period. Although hypertrophied, the response to FS and
CCH decreased, which indicates that the molecular
characteristics of SM have changed. An altered regulation of
contractile proteins is responsible for the decompensation of
detrusor muscles secondary to obstruction.
In the present study, we focused on the relationship
between CaD and the progression of PBOO. The major
mechanism that regulates SM contraction is mediated via myosin
light chain phosphorylation. However, physiological data
indicate that there is also a secondary, actin-linked system
of SM regulation involving CaD, tropomyosin, and
calmodulin[19,20]. The thin filament-associated proteins
h-CaD have been known to modulate actomyosin ATPase and
contraction in SM[21]. h-CaD inhibits the
in vitro motility of actin filaments over myosin heads, but the role of l-CaD in
contractility is less clear. In the present study, we proved
that the expression of l-CaD increased significantly to
approximately the same extent at that of the 1_2-week obstructed
groups and increased further in the 4_8-week obstructed
groups. The expression of h-CaD increased in all of
obstructed bladders, but at significantly higher levels in the
1_2-week obstructed bladders compared to the control and
4_8-week obstructed bladders. It is possible that excess
h-CaD prevents its replacement with l-CaD in the thin filaments
and the alteration of contractile characteristics in the
1_2-week obstructed groups. Yet the role of l-CaD in
contractility or cell motility is less clear; the overexpressed l-CaD may
be associated with remodeling of the cytoskeletal structure,
which enables the SM cells to resist high intravesical
pressure during micturition. The overexpressed l-CaD displaces
h-CaD from the thin filaments, since the binding sites for
both l-CaD and h-CaD are the same, and the thin filaments
containing l-CaD may interfere with the generation and/or
maintenance of the additional force required for
compensating the obstruct-induced contractile dysfunction. Therefore,
the overexpression of l-CaD is associated with bladder
decompensation and could be a marker for the status of
detrusor muscle remodeling and dysfunction.
Control bladders show very little l-CaD, and l-CaD may
be from the interstitial cells. Compensated bladders of the
1_2-week obstructed groups showed an increased
expression of both l-CaD and h-CaD, whereas the decompensated
bladders of the 4_8-week obstructed groups showed an
overexpression of l-CaD, but very little change in h-CaD
compared to the controls. Therefore, the h-CaD/l-CaD ratio in
the control groups was approximately 9:1, in the
compensated bladders of the 1_2-week obstructed groups, the ratio
decreased significantly to 3:1. In the decompensated
bladders of the 4_8-week obstructed groups, the ratio decreased
to less than 1:1. Therefore, the h-CaD/l-CaD ratio has a close
relationship with bladder function. h-CaD and l-CaD have
the same structure, so they can be analyzed by 1 polyclonal
antibody simultaneously, which makes it possible to
measure the h-CaD/l-CaD ratio more objectively. Although the
implication of the h-CaD/l-CaD ratio is unclear, the
h-CaD/l-CaD ratio could be a relatively precise marker for bladder
function after PBOO.
We clearly show the incremental changes in CaD with
the progression from compensated to decompensated function. In the only comparable study, Zhang
et al[11] reported that h-CaD was unchanged and l-CaD was
overexpressed in the SM of the decompensated bladders in response to
partial bladder outlet obstruction for 2 weeks. Our study is
different. The time course of obstruction in our study was
much longer, and we clearly documented deterioration into
decompensation over time.
An additional characteristic response of the rabbit
bladder to obstruction is the increased collagen deposition
between and within SM bundles[22] (Figure 2). In the present
study, we noticed that the relative area of collagen increased
significantly after 2 weeks' obstruction, and the SM/collagen
ratio was downregulated from then on. We can conclude
that before the 2-week obstruction, the increase in bladder
mass was mainly due to SM hypertrophy, and that the
increase in bladder mass after 2 weeks' obstruction was mainly
due to the increased deposition of collagen. The fibroblast
was hyperplastic in the obstructed groups and produced
more collagens, which induced the bladder and resulted in
decompensation. One would suspect that the
overexpression of l-CaD may be from the hyperplastic fibroblast; however,
Zhang et al[11] proved that the CaD and SM myosins were
colocalized in the bladder myocytes from the decompensated
bladders, and SM cells outnumbered fibroblasts, which
indicated that a large portion of the overexpressed l-CaD in the
hypertrophied detrusor were derived from SM cells.
The present study demonstrates an association between
alterations in the contraction of the rabbit bladder after
obstruction with l-CaD and h-CaD in the bladder muscle. The
expression of l-CaD increased significantly to approximately
the same extent as that of the 1_2-week obstructed groups
and increased further in the 4_8-week obstructed groups.
The expression of h-CaD increased in all of the obstructed
bladders, but at significantly higher levels in the 1_2-week
obstructed bladders compared to the control and 8-week
obstructed bladders. The h-CaD/l-CaD ratio decreased
significantly in the 1_2-week obstructed groups and decreased
further in the 4_8-week obstructed groups. This suggests
that the overexpression of l-CaD and the h-CaD/l-CaD ratio
could be markers for the status of detrusor muscle
remodeling and dysfunction.
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