Articles in PresS. Am J Physiol Renal Physiol (August 15, 2012). doi:10.1152/ajprenal.00261.2011
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TANCHUN WANG1#, DEREK M. KENDIG1#, SHAOHUA CHANG3, DANIELLE M.
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TRAPPANESE1, SAMUEL CHACKO3, AND ROBERT S. MORELAND1,2,3
Bladder Smooth Muscle Organ Culture Preparation
Maintains the Contractile Phenotype
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Department of Pharmacology and Physiology
Department of Pathology and Laboratory Medicine
Drexel University College of Medicine, Philadelphia, PA 19102,
3Division of Urology, Department of Surgery,
University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Running Title: Bladder smooth muscle organ culture
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# These authors contributed equally to this study
Address for correspondence:
Robert S. Moreland
Department of Pharmacology and Physiology
Drexel University College of Medicine
245 N 15th Street, MS #488
Philadelphia, PA 19102
215-762-5133
215-762-2299 (fax)
[email protected]
Copyright © 2012 by the American Physiological Society.
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Smooth muscle cells, when subjected to culture, modulate from a contractile to a
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secretory phenotype.
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techniques to study the regulation of smooth muscle biology. The goal of this study
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was to develop a new organ culture model of bladder smooth muscle (BSM) that would
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maintain the contractile phenotype and aid in the study of BSM biology. Our results
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showed that strips of BSM subjected to up to nine days of organ culture maintained
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their contractile phenotype, including the ability to achieve near control levels of force
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with a temporal profile similar to that of non-cultured tissues. The technical aspects of
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our organ culture preparation that were responsible, in part, for the maintenance of the
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contractile phenotype were a slight longitudinal stretch during culture and subjecting
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the strips to a daily contraction/relaxation.
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throughout the cross-section of the strips.
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collagenous matrix resulting in a leftward shift in the passive length-tension
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relationship.
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specific α-actin, calponin, h-caldesmon, total myosin heavy chain, protein kinase G,
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ROCK-I, or the ratio of the SM1/SM2 myosin isoforms. Moreover the organ cultured
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tissues maintained functional voltage-gated calcium channels and BKCa potassium
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channels. Therefore, we propose that this novel BSM organ culture model maintains
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the contractile phenotype and will be a valuable tool for the use of cellular/molecular
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biology studies of bladder myocytes.
This has hampered the use of cell culture for molecular
The tissues contained viable cells
There was an increase in extracellular
There were no significant changes in the content of smooth muscle
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Key words:
h-caldesmon, calponin, α-actin, myosin heavy chain, SM1/SM2, GFP
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encoding adenovirus
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In vitro cell culture is a valuable technique to manipulate individual cells in a controlled
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experimental environment to study cell signaling and the long-term effects of various
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exogenous stimuli. Methods to successfully culture smooth muscle cells have been
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available for decades (4, 10, 13, 42, 43, 56); however, the usefulness of these methods in
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studying the regulation of smooth muscle contraction has been questioned.
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primary concern is the well documented ability of smooth muscle cells to
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phenotypically modulate to non-contractile, migratory/secretory cells after a few sub-
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cultures (5, 33, 52, 57).
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expression of smooth muscle specific proteins and increased expression of non-muscle
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isoforms of those same proteins (37, 51, 57). These proteins include h-caldesmon (29,
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69), calponin (44), smooth muscle myosin isoforms (2), and α-actin (65). Moreover,
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culturing smooth muscle cells lowers the expression of calcium channels and receptors
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thus decreasing depolarization-induced contractions (26, 27, 67) and agonist-induced
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contractions (22, 68), respectively.
The
Smooth muscle cells grown in culture show decreased
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The contractile characteristics and smooth muscle specific protein
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expression in cultured bladder smooth muscle (BSM) cells may vary depending on
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culturing conditions and specific smooth muscle cell lines.
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however, found that cultured BSM cells lose their contractility to both agonist
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stimulation (carbachol) and membrane depolarization (KCl) and have significant
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changes in the expression of the smooth muscle specific α-actin isoform. These changes
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Cheng’s group, (30)
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that occur following culture of BSM cells complicate the interpretation of results
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obtained in vitro, and in particular, of those studies analyzing changes in response to a
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pathophysiological state or the long-term effects of pharmacological agents. DiSanto
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and Chacko’s group (72) established a homogeneous smooth muscle cell line from
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hypertrophied rabbit bladder that stably expressed smooth muscle myosin, myosin
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light chain (MLC) kinase, MLC phosphatase, and protein kinase G (PKG). The cells also
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exhibited a contractile response to agonist (bethanechol) stimulation. A human bladder
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smooth muscle cell line which express the smooth muscle phenotype, including the
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ability to contract in response to carbachol has been established (71).
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karyotypic analysis of both rabbit and human bladder cell lines showed that these cells
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are tetraploid. Thus, published reports show that, unless smooth muscle cells become a stable
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cell line with chromosomal alterations, they lose their phenotype upon continued culturing.
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Three-dimensional BSM cell culture techniques with sustained uni-directional tension
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exhibited improved contractility and relatively less phenotypic modulation in culture
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(42). However, a completely representative tissue model of the physiological conditions
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of BSM in vivo has still not been developed. Atala’s group (1, 12) has successfully
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utilized stem cells in the bioengineering and reconstruction of whole bladder organ
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which restores and maintains normal function in diseased and injured tissues.
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However, there are no reports to date that describe an in vitro model of normal BSM
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that is capable of maintaining contractile phenotypic properties.
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However,
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In contrast to classical cell culturing techniques, organ culture of smooth muscle
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tissues has the potential to maintain the contractile phenotype and may therefore be a
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useful alternative method. Organ culture of smooth muscle tissues has already been
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applied to several smooth muscle types, such as carotid and pulmonary arteries, in
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conjunction with genetic manipulation of specific smooth muscle regulatory proteins
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(23, 30, 40, 48, 49). Early studies by Hellstrand's and Karaki's laboratories (35, 66)
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demonstrated that force could be maintained in an organ cultured vascular preparation
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for up to four days if cultured in a serum-free media. Myosin content however in the
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Hellstrand study (35) was decreased by 50%. This is consistent, in terms of force
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development, with our previous work showing that a vascular preparation could be
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subjected to organ culture for up to 9 days in a serum-free media (21, 48, 49). More
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recently, Corteling et al. (16) again using a vascular preparation, showed no loss of
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contractility in response to UTP stimulation, no decrease in caldesmon content, but a
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50% loss of calponin. Murata et al. and Lopes et al. subjected vascular rings to 7 or 14
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days of organ culture, respectively, but did not compare their cultured results to the
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non-cultured control tissues (36, 39). Therefore, although several studies have reported
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the use of vascular smooth muscle in organ culture, with mixed results, to the best of
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our knowledge there is no literature systematically evaluating the viability of organ
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culture as a useful model for BSM research.
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The goal of this study was to develop a novel organ culture model of BSM tissue
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containing smooth muscle cells close to native conditions and evaluate if this model
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retains the contractile phenotype and therefore may be useful in studies of contractile
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regulation as well as other aspects of smooth muscle biology. Specifically, in this study,
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we determined the phenotype of the organ cultured tissue in terms of smooth muscle
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specific-isoforms of contractile proteins, contractility, viability of cells within the tissue,
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and functional membrane ion channels. Based on our previous studies using vascular
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smooth muscle subjected to organ culture (48, 49), we hypothesized that BSM strips
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would maintain their contractile phenotype including tissue contractility, maintained
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expression of smooth muscle specific protein isoforms, and typical morphology for up
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to 9 days in organ culture if cultured under stretch in a serum-free media and subjected
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to a daily contraction/relaxation. We have provided a novel and what we believe will
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be a very valuable tool for the application of molecular biology techniques to study not
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only the basic regulation of BSM contraction, but also the changes that occur in
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pathophysiological states such as overactive bladder or voiding dysfunction that occurs
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in response to benign prostatic hyperplasia or diabetic uropathy, using a simple in vitro
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system which would allow modification of the external milieu.
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MATERIALS AND METHODS
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Bladder smooth muscle tissue preparation: Male New Zealand White rabbits
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weighing 2 - 2.5 kg were used in this study. All animal studies and procedures were
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approved by the Drexel University College of Medicine's Institutional Animal Care and
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Use Committee. Rabbits were euthanized by a pentobarbital overdose administered via
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the ear vein followed by exsanguination. The bladders were then quickly removed and
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placed in ice cold physiological salt solution (PSS). PSS contained (in mM) 140 NaCl,
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4.7 KCl, 1.2 MgSO4, 1.6 CaCl2, 1.2 Na2HPO4, 2 MOPS (pH 7.4), 5 D-glucose, and 0.02
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EDTA. The bladder neck, trigone, and base region were removed, leaving only the
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mid-portion of the bladder body for experimentation. The bladder body was dissected
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in ice-cold PSS under a dissecting microscope. The mucosal and serosal layers were
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carefully removed and detrusor smooth muscle strips (~1.5 × 6 mm) were cut along the
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central axis of the bladder in the longitudinal orientation as previously described (55)
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and stored in PSS at 4 °C until used. Storage was never longer than 24 hours.
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Bladder smooth muscle organ culture:
Muscle strips (~1.5 × 6 mm) were
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mounted onto polymerized silicon elastomer (Dow Corning Corp, Midland, MI) in six-
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well culture dishes, using 0.2-mm stainless steel pins. The pins were positioned so that
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the BSM strips were slightly stretched longitudinally while raised 2 to 3 mm above the
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surface of the elastomer. Each well held up to two muscle strips in a volume of 5 ml of
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serum-free medium composed of DMEM/F-12 (1:1 ratio, Cellgro, Mediatech, Inc.,
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Manassas, VA), 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B,
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35 µg/ml L-ascorbic acid, 5 µg/ml transferrin, 3.25 ng/ml selenium, 2.85 µg/ml insulin,
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and 200 µg/ml L-glutamine. The muscle strips were maintained at 37 °C, 5% CO2, and
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100% relative humidity in an incubator. The medium was changed daily for up to 9
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days. The medium changing process included a 10 min wash with sterile PSS, then
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stimulation with 110 mM sterile KCl-PSS (equimolar substitution for NaCl) for 5 min,
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and a second wash in sterile PSS for 10 min, before the strips were incubated in 5 ml of
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fresh serum-free medium overnight. The 5 min stimulation in 110 mM sterile KCl-PSS
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followed by a wash in sterile PSS constitutes the daily contraction/relaxation cycle.
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Selected organ cultured tissues were either: not stretched but were subjected to daily
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contraction/relaxation,
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contraction/relaxation,
or
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contraction/relaxation.
Tissues not subjected to daily contraction/relaxation were
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simply washed in sterile PSS while the other tissues were subjected to
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contraction/relaxation before being returned into fresh serum-free medium overnight.
slightly
neither
stretched
slightly
but
not
stretched
subjected
nor
subjected
to
to
daily
daily
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Measurement of isometric force: The bladder tissue strips were removed from
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culture and allowed to equilibrate in 37 °C PSS for 10 minutes. Tissue strips were then
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mounted between a Grass FT.03 force transducer and a stationary clip in water-jacketed
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muscle organ baths containing 37 °C PSS and aerated with 100% O2. The strips were
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stretched to a force of ~2 grams and allowed to stress-relax until a passive force of ~1
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gram was achieved. This passive force approximates the optimal length for maximal
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active stress development (Lo) (55, 61). The strips were then allowed to equilibrate for
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at least 40 min until a stable basal force recording was obtained.
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After the equilibration period, the strips were stimulated with 110 mM KCl-PSS
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then relaxed with PSS; this contraction/relaxation cycle was repeated four times. The
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strips were also subjected to 30 µM carbachol stimulation for 5 minutes and then
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relaxed by rinsing with PSS for 5 minutes. Peak force during carbachol and KCl-PSS
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contractions were recorded.
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expressed as grams force or active stress (stress = force/cross-sectional area) and cross-
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sectional area was determined using tissue length and calculated wet weight as
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previously described (38).
The peak force in each equilibrated muscle strip was
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Concentration-response curves to carbachol were generated by the cumulative addition of
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carbachol from 0.01 - 100 µM. Concentration-response curves to KCl were generated by the
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cumulative addition of KCl from 4.7 - 110 mM. Similar osmolarity was maintained in the higher
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[KCl] solutions by the equimolar substitution of KCl for NaCl. All responses were normalized
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to the maximal response of each individual bladder strip. EC50 values were calculated for each
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bladder strip using GraphPad Prism software. The average EC50 ± SE were determined using the
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EC50 values from each individual concentration-response curve.
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Inhibition of membrane BKCa channels with paxilline: After the equilibration
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period, the strips were incubated with the BKCa channel inhibitor paxilline (10 µM) for 5
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min. The paxilline concentration was chosen based on data from a previous study (17).
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After the inhibitor was added to the muscle bath, the frequency and magnitude of
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spontaneous contractions were recorded using a Grass FT.03 force transducer and AD
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Laboratory Chart 5 software for Windows.
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Measurement of passive length-tension curve:
To measure the passive length-
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tension relationship, the strips were stretched in ~1 - 2 mm length increments and then
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allowed to stress-relax until a stable recording of passive force was reached. This was
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repeated several times until high levels of passive force were achieved.
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experimental points for each strip were plotted as passive stress produced as a function
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of length.
The
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Histology: BSM strips were cultured as described above and then removed,
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placed in Histochoice, and sent to AML labs (Rosedale, MD) for paraffin embedding
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and sectioning.
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embedded BSM strips and stained with Masson’s Trichrome stain.
Cross-sections of 5 µm thickness were taken from the paraffin
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Immunohistochemistry: Paraffin slides of thin sections of BSM strips (organ
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cultured for 1 or 9 days) were placed in xylene and subjected to descending
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concentrations of alcohol (100%, 95%, 70%, 30%, and phosphate-buffered saline, PBS) to
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remove the paraffin and rehydrate the samples. Citrate buffer (10 mM citric acid,
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0.05%, Tween 20, pH 6.0) was used for antigen retrieval treatment (10 minutes at 90 °C).
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The tissue sections were blocked in a 3% bovine serum albumin solution for 30 minutes
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then incubated with anti-collagen I antibody (Abcam, Cambridge, MA) at 1:300
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overnight at 4 °C. After three washing with PBS, the tissue sections were incubated in a
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secondary anti-mouse FITC antibody (1:400; Sigma-Aldrich, St. Louis, MO) for 1 hour at
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room temperature.
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Ontario, Canada) was added to each slide after another three washings in PBS. The
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slides were viewed under a Nikon Eclipse E800 Fluorescence Microscope and images
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were captured using Image-Pro software. A negative control to confirm the specificity
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of the primary antibody was performed using non-immune mouse serum in place of the
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primary antibody.
A drop of mounting medium (Vector Laboratory, Burlington,
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Infection with adenovirus encoding green fluorescent protein (GFP) to determine
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viability of organ cultured bladder smooth muscle: Recombinant adenoviral vector
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encoding GFP was purchased from Stratagene (La Jolla, CA). The viral vector was
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introduced into BSM strips subjected to organ culture for 7 days.
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adenovirus encoding GFP was 2 x 107/ml. After co-culture in the culture medium
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described above for an additional 2 days, resulting in 9 days of total organ culture,
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muscle strips were embedded in optimal cutting temperature (OCT) compound (Sakura
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Finetek USA, Inc, Torrance, CA) for cryostat sectioning. The frozen sections (5 µm
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thick) were mounted onto slides with a drop of mounting medium containing DAPI to
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stain nuclei (Vector Laboratory, Burlington, Ontario, Canada). The fluorescence of the
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frozen sections was detected using a Nikon eclipse E800 microscope. Images were
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captured using Image-Pro Plus software (Media Cybernetics, Bethesda, MD).
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The titer of
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Immunoblots and quantitation of smooth muscle contractile phenotype marker
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proteins: BSM strips were removed from culture at days 3, 5, 7, or 9 and incubated in 37
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°C PSS for 10 minutes, rinsed in acetone, and then completely air dried. The strips were
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homogenized in a 1% SDS, 10% glycerol, and 1 mM DTT, and Protease Inhibitor
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Cocktail solution (1:100; Sigma-Aldrich Inc., St. Louis, MO) using glass/glass
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homogenization. Samples were centrifuged at 12,000 rpm for 5 minutes at 4 °C and
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assayed for protein concentration using the Bio-Rad DC assay (Hercules, CA). The
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samples were subjected to SDS-PAGE (4% SDS stacking gel; 7.5% separating gel for h-
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caldesmon and myosin, 14% separating gel for calponin, or 12% separating gel for PKG
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and ROCK-I) and transferred to a nitrocellulose membrane at 0.8 A for 2 hours at 4 °C.
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Following transfer, the membranes were blocked with 3% non-fat dry milk-phosphate
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buffered solution (PBS) for 60 minutes at room temperature and then incubated in 1%
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milk-PBS containing primary antibody against h-caldesmon (1:1,000 Upstate Cell
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Signaling, Billerica, MA), calponin (1:1,000,000, Sigma-Aldrich Inc., St. Louis, MO),
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smooth muscle myosin (1:10,000 Sigma-Aldrich Inc, St. Louis, MO), PKG (1:2000, Assay
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Designs Inc., Ann Arbor, MI) or ROCK-I (1:2000, Chemicon International Inc.,
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Temecula, CA). α-Actin (1:100,000, Sigma-Aldrich Inc., St. Louis, MO) was also probed,
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as a loading control.
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incubated with a goat anti-mouse IRDye 800cw conjugated secondary antibody or goat
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anti-rabbit IRDye 680cw conjugated polyclonal secondary antibody for 45 min at room
Following washes in 0.1% Tween-PBS, the membranes were
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temperature (1:10,000 Li-COR Biosciences, Lincoln, Nebraska) and then were washed
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three times in 0.1% Tween-PBS for 10 minutes each.
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visualized with the Li-Cor Odyssey Infrared Imaging System.
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Electrophoresis of myosin isoforms:
The membranes were then
Aliquots (15 µg) of total protein from
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bladder tissues at various days of culture were subjected to SDS-PAGE using a highly
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porous slab gel (1 mm thick, 3% SDS stacking gel, and 5% separating gel). Proteins on
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the gels were stained with Coomassie-blue to visualize SM1 and SM2 isoforms of
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myosin. Densitometric analysis of the Coomassie-blue stained gels using a Bio-Rad GS-
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800 Quantitative Densitometer (Hercules, CA) was used to quantify the SM1/SM2 ratio.
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Chemicals and statistics: All reagents for solutions described above, unless
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otherwise specified below, were purchased from Fisher Scientific (Pittsburgh, PA) and
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were of analytical grade or better.
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obtained from Bio-Rad Laboratories (Hercules, CA). Carbachol and paxilline were
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purchased from Sigma-Aldrich (St. Louis, MO). Statistical significance between means
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was determined using one-way ANOVA followed by the Tukey post hoc test or a
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Student’s t-test when appropriate. A P value < 0.05 was taken as significant. All "n"
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values refer to the number of bladder muscle strips; each strip was taken from a
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different animal.
All electrophoretic and blotting reagents were
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RESULTS
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Effect of organ culture on bladder smooth muscle isometric contraction: BSM
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strips were dissected and subjected to organ culture as described in the METHODS
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section. At 3, 5, 7, or 9 days of organ culture the BSM strips were removed from culture,
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mounted for isometric force recording and stimulated by either 110 mM KCl-PSS or 30
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µM carbachol. Isometric force was recorded at the peak of contraction. 110 mM KCl
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and 30 µM carbachol were used as the stimuli to reduce diffusional delays and produce
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uniform activation of the bladder strip. Lower concentrations of stimuli can result in
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activation of the outer layers of a multi-cellular preparation with an unstimulated inner
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core. This will then be followed with activation of the inner core while the outer cells
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are in a state of decreasing activation.
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stimulation with either 110 mM KCl or 30 µM carbachol has no deleterious effect on our
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preparation of BSM (55, 61). The responses to 110 mM KCl-PSS and 30 µM carbachol
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were calculated as stress (force/cross-sectional area) to normalize for differences in
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tissue size, and are shown in Figure 1A. There was a significant decrease in stress
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development as early as 3 days after the start of organ culture and this decrease
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continued over the 9 days of organ culture. Because a decrease in stress could be due to
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either a decrease in contractile force or an increase in tissue mass, which would result in
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an increase in cross-sectional area and decreased calculated stress, cross-sectional area
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of all tissues were calculated and the results are shown in Figure 1B.
We have previously shown that repeated
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Cross-sectional
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area increased after 3 days in culture to a maximum after 5 - 7 days in culture, a time
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course similar to the decrease in calculated stress. We then analyzed the amount of
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force developed in terms of grams force in response to carbachol and KCl-PSS, as
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shown in Figure 1C. This analysis was independent of any potential increase in tissue
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mass. Grams force in response to KCl-PSS stimulation decreased by approximately 20%
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after 5 days of organ culture with a maximal decrease of 30% after 9 days of organ
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culture. Grams force in response to carbachol was slightly but significantly decreased
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at 7 - 9 days of organ culture. These results suggest that organ culture increased tissue
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weight but did not have a large effect on force development.
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The suggestion that the decrease in stress in the organ cultured tissues was due
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to a significant increase of tissue mass while still maintaining contractile viability is
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supported by the results shown in Figure 1B. An increase in cross-sectional area would
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have a significant effect on stress possibly accounting for the decreases shown in Figure
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1A. Therefore, in terms of contractile viability, grams-force as shown in Figure 1C is
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more representative of the state of the tissues.
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The maintenance of stress at high, albeit lower than non-cultured values, and the
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maintenance of force to near non-cultured values was due in part to the stretch imposed
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on the tissues and daily contraction/relaxation during culture. The far right bars in
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Figure 1A and 1C show the stress and force values for tissues not subjected to stretch,
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the daily contraction/relaxation, or either stretch or contraction. Removal of the tissue
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stretch or removal of both stretch and contraction resulted in significantly lower levels
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of stress or force development suggesting that stretch and a daily contraction/relaxation
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are critical for the maintenance of contractility over the course of organ culture.
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Effect of organ culture on bladder smooth muscle contractile profile: BSM is
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categorized as a typical phasic-like smooth muscle based on its contractile pattern.
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Upon membrane depolarization or agonist stimulation the contractile response is
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characterized by a rapid increase to peak force followed by a slow decrease to a lower
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level of quasi-maintained force. To investigate whether organ culture changes the time
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course of a contractile event in BSM, we recorded force development at several time
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points during KCl-PSS or carbachol stimulation. Figure 2A shows typical force tracings
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in response to carbachol and KCl from bladder tissues not cultured (day 0) and those
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subjected to 9 days of organ culture. The results of several experiments such as those
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shown in Figure 2A are provided in Figure 2B (carbachol) and 2C (KCl) expressed as a
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percentage of the maximal force attained. Tissue strips subjected to organ culture
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stimulated by carbachol exhibited significantly slower decreases from peak force during
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the first 90 sec; later time points were not significantly different between non-cultured
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and cultured tissues (Figure 2B). Tissue strips subjected to 3, 5, 7, or 9 days of organ
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culture showed no significant differences when compared to non-cultured tissues in the
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time course of a contraction in response to KCl-PSS (Figure 2C).
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Effect of organ culture on bladder smooth muscle sensitivity: Organ culture had
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minimal effects on maximal force generation by BSM strips subjected to stretch and
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daily contraction/relaxation to KCl. In order to determine the effect of 9 days of organ
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culture on the sensitivity of the BSM strips, we subjected control, non-cultured BSM and
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BSM organ cultured for 9 days to the cumulative addition of either carbachol or KCl.
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Moreover, to determine the influence of the slight stretch and daily KCl
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contraction/relaxation, we also subjected BSM strips that were organ cultured for 9 days
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with stretch but without daily contraction/relaxation, without stretch but with daily
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contraction/relaxation, or without stretch and daily contraction/relaxation to the
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cumulative addition of carbachol or KCl. The results of these studies are shown in
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Figure 3. The calculated EC50 values for carbachol and KCl concentration-response
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curves for all five conditions are shown in Table 1.
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Figure 3A shows the averaged cumulative concentration-response curves to KCl.
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Neither organ culture nor any of the altered organ culture conditions resulted in a
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significant change in sensitivity to KCl as compared to control, non-cultured BSM
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(Table 1). In fact the KCl concentration-response curves of BSM not organ cultured,
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tissues organ cultured for 9 days with stretch and daily contraction/relaxation, and
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tissues organ cultured for 9 days with stretch but not contracted daily were
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superimposable.
Although the lack of stretch on the tissues did not produce a
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significant change in the EC50 for KCl stimulation, the lack of stretch appears to decrease
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sensitivity at lower [KCl].
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Figure 3B shows the averaged cumulative concentration-response curves to
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carbachol. Subjecting the BSM strips to 9 days of organ culture with stretch daily
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contraction/relaxation or with just stretch alone had no significant effect on the
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sensitivity of the tissues to carbachol as compared to control, non-cultured tissue strips
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(Table 1). BSM organ cultured for 9 days in the absence of stretch were significantly less
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sensitive to carbachol than control non-cultured tissues (Table 1).
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however, it appears that organ culture alone shifted the carbachol concentration-
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response curve to the right, but the shift was not statistically significant.
Qualitatively
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Effect of organ culture on the morphology and viability of bladder smooth
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muscle: In order to further investigate the potential effects of organ culture on BSM
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morphology and extracellular matrix content, we subjected tissues to Masson’s
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Trichrome stain. Representative BSM tissues subjected to organ culture for 0, 5, or 9
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days were sectioned, and then stained with Masson’s Trichrome as shown in Figure 4.
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Non-cultured BSM tissues (Day 0) showed red staining of the smooth muscle cells with
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a dense extracellular matrix in blue. BSM tissues subjected to 5 or 9 days of organ
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culture showed an apparent increase in extracellular matrix surrounding the smooth
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muscle layers and between the bundles of smooth muscle within the longitudinal and
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circular muscle layers, defined by orientation of the tissue sectioning. These findings
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are consistent with our hypothesis that organ culture increased the weight of the tissues
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resulting in an increase in cross-sectional area and a decrease in calculated stress
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development, with little effect on grams-force.
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Due to the fact that organ cultured alterations in the Masson’s Trichrome stained
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sections were noted, we were interested in determining more precisely if this was due
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to an increase in collagen. Therefore, we performed immunohistochemisty on the organ
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cultured BSM strips to examine collagen content as described in METHODS.
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Representative images are shown in Figure 5. Figure 5A shows the collagen content in
381
tissues organ cultured for 1 day and Figure 5B shows the collagen content in tissues
382
organ cultured for 9 days. Collagen is shown as green. Tissues organ cultured for 9
383
days show a qualitative increase in collagen content as compared to those organ
384
cultured for only 1 day.
385
increases matrix content, and specifically collagen.
386
control to confirm the specificity of the primary antibody using non-immune mouse
387
serum in place of the primary antibody in tissues cultured for 9 days.
These results support our conclusion that organ culture
Figure 5C shows the negative
388
We investigated the potential effects of organ culture on BSM tissue viability
389
using adenoviral infection of GFP as an indicator of live cells. GFP will be expressed
390
only in the smooth muscle cells which take up the adenovirus and therefore retain
391
viability (29). The results of these studies are shown in Figure 6B. Sections of BSM
392
organ cultured for 9 days (7 days of organ culture followed by 2 additional days of
20
393
organ culture in the presence of GFP-adenovirus) expressed green fluorescent signals
394
which appear to be evenly distributed across the tissue. In addition we used DAPI
395
staining to determine if the cultured bladder strips contained intact nuclei. Figure 6A
396
shows a representative DAPI stained cultured BSM strip. The DAPI staining shows
397
even distribution of the nuclei in BSM cells within the cultured tissue.
398
Effect of organ culture on the passive length-tension curve of bladder smooth
399
muscle: Bladder tissue strips were removed from organ culture after 3, 5, 7 or 9 days
400
and subjected to passive length-tension experiments as described in METHODS. The
401
results are shown in Figure 7. Tissues organ cultured for 3, 5, 7, or 9 days showed no
402
significant differences in the passive length-tension curve when compared to each
403
other. However the passive length-tension relationship of all tissues subjected to organ
404
culture was shifted to the left as compared to non-cultured tissues. The viscoelastic
405
properties of the bladder are primarily dependent on the elements contained within the
406
extracellular matrix (4, 64). Elastin, for example, is very compliant as compared to
407
collagen (32, 45). Elastin contributes mainly to the foot of the passive length-tension
408
curve while the sharp rising portion reflects the collagen component. As the content of
409
extracellular matrix proteins increases, the passive force curve shifts to the left (53).
410
Consistent with our immunohistochemical results showing an increase in collagen
411
content, these results provide mechanical data consistent with an increase in collagen
412
content in the organ cultured tissues.
21
413
Quantification of smooth muscle specific marker proteins with organ culture:
414
Similar to other types of smooth muscle cells, BSM cells phenotypically modulate when
415
subjected to cell culture (30, 42).
416
undergo a transition from a contractile to a migratory/secretory state. Biochemically,
417
expression levels of smooth muscle specific-isoforms of contractile and regulatory
418
proteins as well as cell adhesion proteins, ion channels, and receptors decrease when
419
smooth muscle cells are placed in culture, while concurrently non-muscle isoforms of
420
several of the same proteins increase (28). In order to verify that the BSM cells in our
421
organ culture model retained the contractile phenotype, we measured the expression
422
levels of several smooth muscle-specific proteins. α-Smooth muscle actin, h-caldesmon,
423
calponin, smooth muscle myosin heavy chain, and the ratio of SM1/SM2 myosin
424
isoforms are specifically down-regulated in dedifferentiated or synthetic smooth muscle
425
cells (2, 29, 30, 44, 65). The results shown in Figure 8A-E are from experiments to
426
quantify expression levels of these proteins in non-cultured and organ cultured smooth
427
muscle strips. To normalize for any potential loading differences, we first measured the
428
expression levels of several commonly used house-keeping proteins, GAPDH, β-actin,
429
and α-actin. The results suggested the most stably expressed proteins during 9 days of
430
organ culture are α-actin and β-actin (data not shown). We used α-actin as the loading
431
control in all experiments. Quantified and averaged data based on several Western
432
blots and gels are shown to the right of the respective blot or gel.
Following exposure to culture media, BSM cells
22
433
h-Caldesmon levels were not significantly changed during the 9 days of organ
434
culture (Figure 8A).
435
expression levels noted in smooth muscle cells subjected to cell culture (46, 59). The
436
basic isoform of calponin is a smooth muscle-specific marker that is significantly
437
depressed in cultured smooth muscle cells (7, 24). Figure 8B shows that subjecting
438
bladder strips to 9 days of organ culture has no effect on calponin expressions levels.
This is in contrast to the significant decreases to near zero
439
BSM cells subjected to culture or to conditions that induce hypertrophy exhibit
440
decreased levels of total myosin and an increase in the ratio of the SM1/SM2 myosin
441
isoforms (9, 62). Representative Western blots for total myosin and Coomassie blue
442
stained gel for the ratio of SM1/SM2 myosin isoforms from bladder strips subjected to
443
organ culture for up to 9 days are shown in Figure 8C and Figure 8D, respectively.
444
Neither total myosin nor the ratio of the SM1/SM2 myosin isoforms was changed by
445
subjecting BSM strips for 9 days of organ culture.
446
Quantification of ROCK-I and PKG in organ cultured bladder smooth muscle
447
tissue:
448
culture or tissues to protocols that produce hypertrophy result in changes in the
449
expression level of ROCK (6, 41). To exclude the possibility that our organ culture
450
model might have similar changes on ROCK-I, we examined ROCK-I protein
451
expression levels in bladder tissues subjected to 0 - 9 days of culture. The results of
Several studies have demonstrated that subjecting smooth muscle cells to
23
452
Figure 8E demonstrates that 3 - 9 days of organ culture had no significant effect on
453
levels of ROCK-I expression.
454
Expression of PKG has been suggested to positively correlate with the phenotype
455
of smooth muscle, meaning that higher expression levels of PKG correlate with the
456
contractile phenotype and lower expression levels suggest a secretory phenotype.
457
Vascular smooth muscle cells undergoing in vitro culture show decreased levels of PKG
458
expression over time.
459
rescues the vascular smooth muscle cells from the secretory phenotype and reverts
460
them back to a contractile phenotype (15, 18, 33). We investigated the expression levels
461
of PKG in our organ cultured BSM strips.
462
Lincoln’s lab (15, 18), if our organ cultured BSM strips maintained the contractile
463
phenotype, then PKG expression levels should not be different from the non-cultured
464
control tissues. The results shown in Figure 8E demonstrate that 3 - 9 days of organ
465
culture had no significant effect on PKG expression levels as compared to non-cultured
466
strips of BSM, supporting the conclusion that our strips maintain their contractile
467
phenotype.
468
469
Restoration of PKG expression to near physiological levels
Based on the hypothesis proposed by
Effect of stretch and daily contraction/relaxation on bladder smooth muscle
protein expression:
470
To determine if either stretch and/or daily contraction/relaxation were important
471
in the maintenance of the contractile phenotype during organ culture of BSM, strips
24
472
were subjected to organ culture with stretch and daily contraction/relaxation, stretch
473
only, contraction/relaxation only, or neither for 9 days.
474
caldesmon, calponin, total myosin heavy chain, myosin SM1/SM2 ratio, ROCK-1, and
475
PKG were measured in control, non-cultured BSM and BSM subjected to the various
476
organ culture conditions listed above.
477
results from several such blots are provided in Figure 9. h-Caldesmon expression was
478
significantly decreased when organ cultured in the absence of both stretch and daily
479
contraction/relaxation compared to both control, non-cultured BSM strips and those
480
organ cultured for 9 days with stretch and daily contraction/relaxation (Fig 9A).
481
Similarly, ROCK-1 expression was also significantly decreased when organ cultured in
482
the absence of both stretch and daily contraction/relaxation (Fig 9E). Neither calponin,
483
PKG, nor α-actin expression levels were decreased by organ culture in any condition
484
(Fig 9B and 9F). Total myosin expression was increased by organ culture in the absence
485
of stretch but subjected to daily contraction/relaxation (Fig. 9C). No change was noted
486
in the ratio of the SM1/SM2 isoforms of myosin (Fig. 9D).
Expression levels of h-
Representative Western blots and averaged
487
Effect of organ culture on bladder smooth muscle tissue membrane ion channel
488
function: Calcium and potassium channels play indispensable roles in establishing the
489
cell membrane potential and regulating smooth muscle contraction. Calcium channel
490
function in organ cultured smooth muscle tissues and cell cultured smooth muscle cells
491
have been shown to significantly decrease as time in culture increases (20, 26).
25
492
However, consistent with previous studies from our laboratory using organ culture of
493
vascular smooth muscle tissues (21, 48, 49), functional voltage dependent calcium
494
channels are retained. This is based on the observation that both vascular and BSM
495
tissues subjected to organ culture exhibit rapid and robust contractions in response to
496
KCl-PSS (21, 48, 49, and Figures 1 and 2).
497
A second class of ion channels important in the regulation of smooth muscle
498
contraction and therefore suggestive of a contractile phenotype are potassium channels.
499
Among them, the large-conductance calcium-activated potassium channels (BKCa) are
500
particularly important in modulation of the membrane potential of urinary BSM and
501
regulating tissue repolarization and relaxation (25). We used the reasonably specific
502
BKCa channel inhibitor paxilline to test whether or not BKCa channels are present and
503
active in our organ culture BSM preparation.
504
channel activity as that is outside the scope of our study, but it was important to
505
determine if BKCa channels are functional after 9 days of organ culture.
506
representative tracings depicted in Figure 10A - C show that the addition of 10 µM
507
paxilline to resting tissues subjected to organ culture for 0, 7, or 9 days increased the
508
frequency and amplitude of spontaneous contractions. This is consistent with previous
509
studies showing that inhibition of BKCa channels with paxilline increased spontaneous
510
contractions of resting smooth muscle strips (11, 17). Our BSM preparation does not
511
typically exhibit spontaneous activity (55, 61).
26
Our goal was not to quantify BKCa
The
This is presumably due to the
512
predominance of smooth muscle cells and the lack of pacemaker cells or other
513
modulatory cells of smooth muscle function in our BSM preparation. The presence of
514
spontaneous activity with inhibition of the BKCa channel inhibitor in a tissue normally
515
devoid of such activity, suggests the presence of active BKCa potassium channels.
516
27
517
DISCUSSION
518
Our understanding of the mechanism(s) by which an increase in intracellular
519
Ca2+ initiates contraction of smooth muscle has increased dramatically since the
520
discovery of MLC phosphorylation in the 1970s (3, 50).
521
information is needed on the changes in the regulatory mechanisms during many
522
disease states (63). Cell culture systems have been used to investigate basic cellular
523
mechanisms that are observed in vivo in disease processes.
524
problems that have hindered progress in this area is the fact that smooth muscle cells
525
when subjected to culture phenotypically modulate from a contractile to a non-
526
contractile migratory/secretory phenotype (5, 10, 33). This presents a roadblock to
527
progress toward understanding the biology and pathobiology of smooth muscles. In
528
this study we provide an extensive qualitative and quantitative characterization of a
529
novel organ culture model of BSM. The advantage of organ culture as compared to cell
530
culture is that the contractile phenotype is maintained and the cell to cell associations
531
are similar to that of intact smooth muscle. Organ culture preparations of other smooth
532
muscle tissues have been reported (20, 40, 48, 49) but to the best of our knowledge this
533
is the first demonstration of BSM subjected to organ culture as well as a near complete
534
characterization of the preparation in terms of contractility, isoform protein expression,
535
and qualitative information on ion channels. Our primary finding is that BSM tissue
536
can be organ cultured for up to 9 days with maintenance of the contractile phenotype.
28
However, much more
One of the technical
537
With this organ culture model grams force of the cultured tissues remained similar to
538
the values obtained from non-cultured tissues in response to stimulation, sensitivity to
539
both membrane depolarization and agonist activation was unchanged, membrane ion
540
channels remained functional, and all contractile proteins were retained in their smooth
541
muscle specific isoform and at expression levels similar to non-cultured tissues.
542
Smooth muscle contractility: The organ culture model of BSM developed in this
543
study retained a high degree of contractility over the 9 day experimental period. This
544
was demonstrated by the presence of robust contractions in response to KCl and
545
carbachol. We were at first disappointed that stress values appeared to be depressed in
546
tissues subjected to organ culture.
547
secretory responses to organ culture these changes would increase the weight of the
548
tissue thus increasing the calculated cross-sectional area and decrease stress values
549
calculated in N/m2. Therefore, we re-analyzed the contractions in terms of grams force.
550
When presented as grams force, which does not take into account changes in tissue
551
weight, it was apparent that the carbachol contraction was unaffected by organ culture
552
except at 7-9 days of culture and KCl contractions were only slightly but significantly
553
reduced. This conclusion is supported by the significant increase in cross-sectional area
554
and increase in collagen content indicative of an increase in non-muscle tissue weight.
555
If non-muscle tissue weight increased in the absence of smooth muscle hypertrophy,
556
but force levels remained similar, then it is reasonable to suggest that force as a function
However, we realized that if there were any
29
557
of smooth muscle mass remained similar in the organ cultured as compared to non-
558
cultured tissues. These results are important because smooth muscle cells subjected to
559
cell culture produce little to no contraction and several organ culture preparations have
560
significantly depressed contractions in response to membrane depolarization (20, 26,
561
30).
562
maintain BSM contractility using in vitro culture methods. Moreover, compared to the
563
complete or near complete loss of contractility following standard cell culture
564
techniques, the small losses in grams force noted in this study should be considered
565
minimal.
To our knowledge these results demonstrate for the first time the ability to
566
Our hypothesis that the BSM strips maintained the contractile phenotype after
567
organ culture is, we believe, strengthened by the finding that the sensitivity as
568
determined by EC50 to both membrane depolarization (KCl) and agonist activation
569
(carbachol) were unaffected by 9 days of organ culture. It should be noted however
570
that although the EC50 for carbachol was not statistically shifted to the right after 9 days
571
in organ culture while stretched, there was a non-significant, qualitative decrease in
572
sensitivity. If the BSM tissues were organ cultured in the absence of longitudinal
573
stretch, the EC50 to carbachol was significantly higher denoting a shift to the right in
574
the concentration-response curve and therefore a decrease in sensitivity to the agonist.
575
In our opinion, the key elements in our new organ culture protocol that
576
contributed to the maintenance of contractility were the slight longitudinal stretch
30
577
applied to the tissues during organ culture and daily contraction/relaxation protocol
578
using a depolarizing solution of KCl. Slightly stretching the tissue is apparently the
579
more important of the two conditions, consistent with the results of a recent study using
580
organ cultured vascular smooth muscle (58). However when both stretch and daily
581
contraction/relaxation were employed, the organ cultured BSM tissues retained
582
phenotypic characteristics most similar to the non-cultured control tissues. Stretching
583
the tissues may have also enhanced an integrin-mediated increase in extracellular
584
matrix proteins (60) resulting in an increased weight of the tissue and therefore a
585
decrease in the calculated levels of stress.
586
Smooth muscle contractile pattern and extracellular matrix content:
587
contraction is characterized by an initial phasic-like increase in force followed by a
588
smaller tonic-like phase of force maintenance.
589
defined not only by the total amount of force developed but also the temporal profile of
590
the contraction. In this study, the time course of contraction in response to both KCl
591
and carbachol stimulation were measured. Organ culture caused no significant changes
592
in the temporal pattern of the contraction in response to KCl-induced membrane
593
depolarization. In response to carbachol, the transition from the phasic-like portion to
594
the tonic-like portion of the contraction was slower in organ cultured tissues as
595
compared to non-cultured tissues. The mechanism responsible for the slower change to
596
the tonic-like portion of the contraction is not known. This change is similar to the
31
BSM
A typical BSM contractile event is
597
change seen in hypertrophied bladder such as that which occurs in response to partial
598
bladder outlet obstruction (6, 19, 70). The hypertrophied bladder, however, exhibits this
599
change in response to both membrane depolarization and carbachol stimulation,
600
whereas, in our organ cultured preparation the slower transition from the phasic-like to
601
the tonic-like contraction occurs only in response to carbachol stimulation, suggesting
602
that receptor activation or receptor activated pathways are involved. It is possible that
603
the muscarinic receptor population or sub-type may be altered following organ culture;
604
a question beyond the scope of this current study.
605
The decrease in stress with only a small change in grams force following culture
606
implies an increase in tissue weight. This would suggest that organ cultured tissues
607
had increased extracellular matrix content or the cells have undergone hypertrophy
608
during in vitro growth with frequent stimulation. To determine if this resulted in a
609
functional change, we measured the passive length-tension relationship in the organ
610
cultured and non-cultured BSM tissues. The shape of the passive length-tension curve
611
is dependent on the type and amount of extracellular matrix proteins (28, 39). Our
612
results showed a leftward shift of the passive length-tension curve, which is suggestive
613
of an increase in extracellular matrix content, specifically collagen content (53).
614
Interestingly, although the passive length-tension curve was shifted to the left after 3
615
days of organ culture, no further changes were noted with up to 9 days of organ
616
culture. This would suggest that the increase in extracellular matrix is a rapid response
32
617
to organ culture and that maintained organ culture either does not further increase
618
matrix content or does so very slowly.
619
Histological examination of the tissues subjected to organ culture was consistent
620
with the results of the passive length-tension results. Masson’s Trichrome staining of
621
both longitudinal and circular BSM layers subjected to 0, 5, or 9 days of organ culture
622
showed an increase in extracellular matrix proteins while the smooth muscle cells
623
appeared to not be affected. Although Masson’s Trichrome stain does not differentiate
624
the type of extracellular matrix proteins, the overall increase in matrix shown is
625
consistent with a decrease in stress but not grams-force and our interpretations based
626
on the passive length-tension experiments. The immunohistochemistry experiments
627
demonstrated that the increase in matrix shown in the Masson's Trichrome stained
628
sections is, at least in part, collagen.
629
One concern of using whole tissue strips in any cultured environment is the
630
possibility that the central core of the tissue became hypoxic and the cells in the core
631
dying. However, one advantage of our predominantly smooth muscle strip preparation
632
is that due to removal of the urothelium and mucosa hypoxia should be less of a factor.
633
To determine the viability of the smooth muscle cells within the organ cultured strips,
634
we introduced a recombinant adenovirus encoding GFP into our organ cultured smooth
635
muscle tissues and observed the fluorescence. The cells in the smooth muscle tissue
636
strips showed a strong and evenly distributed green fluorescent signal. This confirms
33
637
our hypothesis that the smooth muscle cells within the cultured strips retained viability,
638
both in the periphery and in the core.
639
confirmation of our observation that tissues subjected to organ culture for up to 9 days
640
are fully functional.
The tissue viability also provides another
641
Expression of smooth muscle specific proteins: One of the primary changes that
642
occur in smooth muscle cells subjected to cell culture is loss of smooth muscle-specific
643
isoforms of several contractile related proteins and the increase in the non-muscle
644
specific isoform of those same proteins (37, 51). In terms of thick filament proteins,
645
subjecting BSM strips to organ culture resulted in the maintenance of total myosin
646
levels, the ratio of SM1 to SM2 myosin isoforms, and the predominance of the SMB
647
myosin isoform (data not shown). These indices are altered in cell culture or in bladder
648
subjected to a stimulus that induces hypertrophy (9, 19, 62). Surprisingly, the lack of
649
either stretch or daily contraction/relaxation increased total myosin expression. Tissues
650
that were organ cultured in the absence of stretch but subjected to daily
651
contraction/relaxation had statistically significant elevation in myosin expression. In
652
terms of thin filament proteins, the smooth muscle specific isoform of calponin was not
653
altered nor was the smooth muscle specific α- smooth muscle actin isoform.
654
smooth muscle specific h-caldesmon was also not significantly decreased in our model
655
in contrast to results obtained in smooth muscle cells subjected to either cell culture or
656
organ culture (16, 29, 69). It should be noted however that in one report of cultured
34
The
657
smooth muscle cells, h-caldesmon levels were shown to be reasonably well maintained
658
(14). Maintenance of h-caldesmon in a rabbit smooth muscle cell line required frequent
659
stimulation of cultured cells with carbachol or transfection of the cultured cells with h-
660
caldesmon cDNA (47). Our repeated daily stimulation of the organ cultured tissues
661
may therefore account for the maintained levels of h-caldesmon in our preparation.
662
Stretch alone or daily contraction/relaxation alone did not result in a decrease in h-
663
caldesmon expression in the organ cultured BSM tissues. If the tissues were organ
664
cultured in the absence of both manipulations, h-caldesmon content was significantly
665
decreased. The loss of the contractile isoform of caldesmon is a good indicator of the
666
phenotypic modulation from a contractile to a non-contractile phenotype, suggesting
667
again that stretch and daily contraction are important in maintaining the contractile
668
phenotype. Overall, these results support our hypothesis and interpretation that our
669
organ cultured BSM maintains the contractile phenotype.
670
The organ culture BSM model was further evaluated by quantification of the
671
expression levels of the signaling protein PKG. The fact that PKG levels were not
672
affected by 9 days of organ culture is one of the best lines of evidence supporting our
673
hypothesis that this tissue model retains the contractile phenotype. This is based on the
674
work from Lincoln's lab, which clearly demonstrated that loss of PKG correlates with
675
the loss of the smooth muscle contractile phenotype while increasing expression levels
676
of PKG rescues the cells from the secretory phenotype back to the contractile phenotype
35
677
(18, 34).
678
subjecting BSM tissues to organ culture in the absence of stretch, daily contraction, or
679
both had no effect on PKG expression.
Based on this work from Lincoln's lab (18, 34), we were surprised that
680
Potential for smooth muscle hypertrophy: One concern related to subjecting
681
smooth muscle to cell or organ culture is the possibility of inducing smooth muscle cell
682
hypertrophy. If the smooth muscle cells undergo hypertrophy, it could complicate the
683
interpretation of experimental results in terms of signaling and contractility. Based on
684
the literature, there are several characteristics seen after in vitro cell culture suggestive
685
of smooth muscle hypertrophy, such as: increased expression levels of ROCK-I (6), a
686
shift of the ratio of the myosin SMA/SMB isoforms from a predominance of SMB to a
687
predominance of SMA (9), overexpression of l-caldesmon levels (8, 70), and a shift in
688
contractile pattern from a phasic-like to a more tonic-like contraction (8). Our results
689
showed that organ culture did not produce any significant change in the expression
690
levels of either l-caldesmon (data not shown) or ROCK-I. The absence of both stretch
691
and daily contraction decreased ROCK-1 expression after 9 days in organ culture.
692
ROCK-1 expression in BSM tissues not stretched but subjected to daily contraction was
693
also lower than non-cultured tissues, albeit not significant, suggesting that stretch is an
694
important parameter in maintaining ROCK-1 expression. Moreover, preliminary data
695
not presented here demonstrated that organ culture did not alter the ratio of the myosin
696
isoforms, SMB/SMA. The SMB isoform remained the predominant isoform present in
36
697
the organ cultured tissues. While organ culture did alter the phasic component of a
698
contraction in response to carbachol, no change in contractile profile was evident in
699
response to KCl-induced membrane depolarization. Based on those observations, we
700
suggest that our organ culture technique did not induce a significant hypertrophic
701
response in the strips of BSM.
702
Ion channels:
Several groups have presented evidence that smooth muscle
703
subjected to culture, even organ culture, showed significant loss of membrane
704
associated ion channels (5, 20, 25). The results presented in this study suggest that our
705
BSM organ culture model maintains functional membrane associated ion channels. The
706
fact that membrane depolarization induced contractions are maintained at near control
707
levels clearly demonstrates the presence of voltage gated calcium channels. Another
708
important smooth muscle ion channel is the BKCa channel. The BKCa channel is a key
709
regulator of BSM contractility.
710
depolarization and local increases in cytosolic free Ca2+ to hyperpolarizing K+ outward
711
currents, thereby limiting smooth muscle contractility (25, 31, 54). Our results showed
712
that inhibition of BKCa channels with the specific BKCa channel inhibitor paxilline had
713
similar effects in both organ cultured and non-cultured tissues. In both cultured and
714
non-cultured tissues, inhibition of the BKCa channel increased spontaneous basal
715
activity which is consistent with the loss of a hyperpolarizing outward K+ current that
716
limits contractility. This finding is consistent with a recent report from Nelson's group
It functions through connecting membrane
37
717
showing that activation of the BKCa channel decreased contractility of BSM (31). Taken
718
together, these results support the idea that our organ culture model maintains at least
719
these two ion channels in terms of functionality.
720
Summary: In conclusion, the results of this study provide a near complete
721
characterization and verification of a novel organ culture model of BSM. In contrast to
722
most cell culture models for BSM, our new organ cultured model maintains near control
723
levels of contractility, normal sensitivity to both membrane depolarization and agonist
724
stimulation, functional ion channels, and control levels of smooth muscle specific
725
isoforms of contractile and signaling proteins. Our results also demonstrate that this
726
model maintains cell viability across the entire cross-section of the tissue. It is evident
727
that some secretory function is generated due to the increase in extracellular matrix,
728
specifically collagen, and a decrease in calculated stress values most likely due to
729
increased tissue weight. However, this increase in secretory activity does not appear to
730
be deleterious to the overall contractile functionality of this model of BSM. The ability
731
to culture strips of BSM while maintaining the contractile phenotype will now allow the
732
use of molecular biology techniques, such as siRNA or lentiviral knock-down of specific
733
proteins, which should provide new information concerning the regulation of both
734
normal and diseased BSM.
735
38
736
ACKNOWLEDGEMENTS
737
This study was supported, in part, by funds from NIH grants DK 57252 and DK 85734
738
(to RSM), DK 69898 (to SC), and an O'Brien Urology Center Grant P50 DK 52620 to the
739
University of Pennsylvania. DM Kendig was supported by a predoctoral fellowship
740
from the Drexel University College of Medicine Aging Initiative. The authors would
741
like to acknowledge the excellent technical assistance of Stephanie Costa, Hillevi K. Ets,
742
and Bailey Sylvester.
743
744
39
745
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746
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747
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748
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749
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955
956
51
957
FIGURE LEGENDS:
958
959
Figure 1: Force development in BSM strips after 0 - 9 days of organ culture. Strips were
960
mounted for isometric force recording and stimulated with either 110 mM KCl-PSS
961
(black bars) or 30 µM carbachol (gray bars). Panel A: Values are expressed as stress at
962
the point of maximal response to either KCl-PSS or carbachol. Stress is calculated as
963
force divided by the cross-sectional area of the tissue. Shown in the far right are stress
964
measurements in tissues organ cultured for 9 days but not subjected to stretch, daily
965
contraction/relaxation, or both. Panel B: Cross-sectional area of BSM strips after 0 - 9
966
days of organ culture. Cross-sectional area was calculated as [(wet weight, in kg) x
967
(1,050 kg/m)]/length at Lo, in m. Panel C: Values are expressed as grams-force at the
968
point of maximal response to either KCl-PSS or carbachol. Shown in the far right are
969
force measurements in tissues organ cultured for 9 days but not subjected to stretch,
970
daily contraction/relaxation, or both. *Denotes statistically different from control non-
971
cultured strips (Day 0). # Denotes statistically different from 9 days of culture. Values
972
shown are the means ± SE from at least 6 bladder strips (n = 3 for not stretched, not
973
contracted, or both non-stretched and not contracted); each strip was taken from a
974
different animal.
975
976
52
977
978
Figure 2: Time course of force development in BSM strips after 0 - 9 days of organ
979
culture. Strips were mounted for isometric force recording and stimulated with either
980
110 mM KCl-PSS or 30 µM carbachol for 5 minutes. Panel A: Representative tracings
981
of non-cultured BSM tissues and those subjected to 9 days of organ culture. Left panel:
982
Carbachol stimulation; Right panel: KCl stimulation. Panel B: The time course of
983
isometric force development in response to 30 µM carbachol in muscle strips subjected
984
to organ culture for 0 - 9 days. Force is expressed as a percentage of the maximum
985
response to 30 µM carbachol.
986
Panel C: The time course of isometric force development in response to 110 mM KCl-
987
PSS in muscle strips subjected to organ culture for 0 - 9 days. Force is expressed as a
988
percentage of the maximum response to 110 mM KCl-PSS.
989
Day 5;
990
strips at the corresponding time point. Values shown are the means ± SE from at least 6
991
bladder strips; each strip was taken from a different animal.
■ = Day 7; □ =
● = Day 0; ○ = Day 3; ▼= Day 5; ■ = Day 7; □ = Day 9.
● = Day 0; ○ = Day 3; ▼=
Day 9. * Denotes statistically different from cultured muscle
992
53
993
Figure 3: Concentration response curves of organ cultured BSM strips to KCl and
994
carbachol.
995
carbachol (B). BSM strips were either not cultured (●), organ cultured for 9 days while
996
stretched and subjected to daily contraction/relaxation (), organ cultured for 9 days
997
not stretched but subjected to daily contraction/relaxation (), organ cultured for 9
998
days stretched but not subjected to daily contraction/relaxation (), or organ cultured
999
for 9 days neither stretched nor subjected to daily contraction/relaxation (). A. The
1000
concentration-response relationship to KCl was unaffected by any of the various organ
1001
culture conditions. B. The carbachol sensitivity of BSM strips organ cultured with
1002
stretch alone or stretch and daily contraction were not significantly different from
1003
control, non-cultured BSM strips. Strips placed in organ culture in the absence of
1004
stretch but subjected to daily contraction/relaxation or organ cultured in the absence of
1005
both manipulations significantly shifted the concentration-response curve to carbachol
1006
to the right. Values shown are the means ± SE from 3-4 bladder strips; each strip was
1007
taken from a different animal.
BSM strips were subjected to the cumulative addition of KCl (A) or
1008
1009
1010
1011
54
1012
Figure 4: Masson’s Trichrome staining of BSM strips subjected to 0, 5, or 9 days of
1013
organ culture (40× magnification). Longitudinal BSM: images on the left. Circular BSM:
1014
images on the right. Red: smooth muscle. Blue: extracellular matrix. These slides are
1015
representative of an n of at least 4 experiments. Each experiment was performed on
1016
BSM strip taken from a different animal.
1017
1018
1019
1020
1021
1022
1023
1024
1025
1026
1027
1028
1029
55
1030
Figure 5: Organ cultured BSM immunostained for collagen I. Panel A: Fluorescent
1031
image of BSM strips organ cultured for 1 day. Green fluorescent signal shows collagen I
1032
staining. Panel B: Fluorescent image of BSM strips organ cultured for 9 days. Note the
1033
enhanced green fluorescent signal denoting an increase in collagen I.
1034
Fluorescent image of the negative control using non-immune mouse serum in place of
1035
the primary antibody, showing the specificity of the collagen I antibody in bladder
1036
tissue subjected to 9 days of organ culture.
1037
56
Panel C:
1038
Figure 6: GFP-containing adenoviral infection of BSM subjected to 9 days of organ
1039
culture.
1040
subjected to 7 days of organ culture and then co-cultured with GFP-adenovirus for an
1041
additional 2 days for a total of 9 days of organ culture. Fluorescence of 5 µm frozen
1042
sections was observed using epifluorescence microscopy. Panel A: DAPI staining
1043
showing nuclei of smooth muscle cells in the organ cultured strips of BSM. Panel B:
1044
Fluorescence of organ cultured BSM infected with GFP-containing adenovirus,
1045
indicating the viability of cells in the organ cultured muscle strips.
GFP-containing adenovirus was introduced into BSM strips that were
1046
1047
1048
1049
1050
1051
1052
1053
1054
1055
57
1056
Figure 7: Passive length-tension curve in BSM strips subjected to 0 - 9 days of organ
1057
culture. Strips were mounted between a force transducer and an adjustable clip for
1058
passive length-tension measurement as described in Methods. The experimental points
1059
for each strip were plotted using stress as the Y-axis and the length increments
1060
normalized to cross-sectional area as the X-axis. ● = Day 0; ○ = Day 3; ▼= Day 5; ■ =
1061
Day 7; □ = Day 9.
1062
determinations, each from a different animal.
Values shown are individual data points from at least 4
1063
1064
1065
1066
1067
1068
1069
1070
1071
1072
1073
1074
1075
58
1076
Figure 8: Representative Western blot or Coomassie blue stained electrophoretic gel of
1077
smooth muscle specific protein isoforms in BSM strips subjected to organ culture for 0 -
1078
9 days. Smooth muscle α-actin was used as the loading control in all blots. Figures
1079
showing the cumulative results of several blots or gel are shown to the right of each
1080
respective blot or gel. Panel A: Representative Western blot showing h-caldesmon
1081
content in non-cultured control tissues (Day 0) and tissues subjected to organ culture for
1082
3, 5, 7 or 9 days. Panel B: Representative Western blot showing calponin content in
1083
non-cultured control tissues (Day 0) and tissues subjected to organ culture for 3, 5, 7 or
1084
9 days. Panel C: Representative Western blot showing myosin heavy chain content in
1085
non-cultured control tissues (Day 0) and tissues subjected to organ culture for 3, 5, 7 or
1086
9 days. Panel D: Representative electrophoretic gel stained with Coomassie blue of
1087
SM1 and SM2 myosin isoforms in non-cultured tissues (Day 0) and tissues subjected to
1088
organ culture for 3, 5, 7, and 9 days. Panel E: Representative Western blot showing
1089
ROCK-I (upper band) and PKG (lower band) content in non-cultured control tissues
1090
(Day 0) and tissues subjected to organ culture for 3, 5, 7 or 9 days. Organ culture of
1091
BSM for up to 9 days had no effect on the cellular content of any of the smooth muscle
1092
specific isoforms shown in this figure. Values for all panels shown to the right of the
1093
respective blots or gel are the means ± SE of 5 - 7 determinations.
1094
1095
59
1096
Figure 9: Representative Western blots or Coomassie blue stained electrophoretic gel of
1097
smooth muscle proteins in BSM strips subjected to various organ culture conditions for
1098
9 days. Protein expression was determined in: BSM strips not cultured (Control); organ
1099
cultured for 9 days with stretch and daily contraction/relaxation (+K+S); organ cultured
1100
for 9 days without stretch but with daily contraction/relaxation (+K-S); organ cultured
1101
for 9 days with stretch but without daily contraction/relaxation (-K+S); or organ
1102
cultured for 9 days without either stretch or daily contraction/relaxation (-K-S). Panel
1103
A: h-Caldesmon. Nine days of organ culture in the absence of both stretch and daily
1104
contraction/relaxation significantly reduced h-caldesmon expression.
1105
Calponin. Calponin expression levels were not affected by any of the various organ
1106
culture conditions. Panel C: Total myosin heavy chain. Nine days of organ culture
1107
without stretch but with daily contraction/relaxation increased myosin expression
1108
levels. Panel D: Myosin SM1/SM2 ratio. SM1/SM2 ratio was not affected by any of the
1109
various organ culture conditions. Panel E: ROCK-1. Nine days of organ culture in the
1110
absence of both stretch and daily contraction/relaxation significantly reduced ROCK-1
1111
expression levels. Panel F: PKG. PKG expression levels were not affected by any of the
1112
various organ culture conditions. * Denotes statistically different from control, non-
1113
cultured muscle strips.
1114
cultured for 9 days in the presence of stretch and daily contraction/relaxation.
1115
Statistical significance was taken as P < 0.05. Values for all panels shown to the right of
Panel B:
# Denotes statistically different from muscle strips organ
60
1116
the respective blots or gel are the means ± SE of 4 determinations, each from a different
1117
animal.
1118
1119
1120
61
1121
Figure 10: Representative force tracings of the effect of the BKCa channel inhibitor,
1122
paxilline (10 µM), on BSM strips subjected to organ culture for 0 (Panel A), 7 (Panel B),
1123
or 9 (Panel C) days. The arrow denotes time of addition of paxilline to the muscle
1124
baths. The addition of paxilline induced basal spontaneous activity in unstimulated
1125
strips of BSM. Organ culture for either 7 or 9 days did not alter the effects of paxilline
1126
demonstrating that functional BKCa channels were still present. The large contraction at
1127
the end of the force tracings is in response to the addition of 30 µM carbachol to ensure
1128
tissue viability.
62
Table 1: EC50 values for KCl and carbachol-induced contraction of control and cultured rabbit bladder smooth muscle strips.
EC50 values were calculated from individual cumulative concentration-response curves to KCl or carbachol. Values shown are the
means ± SE for 3-4 determinations, each from a different bladder. * P < 0.05 as compared to Day 0, uncultured bladder smooth
muscle strips. # P < 0.05 as compared to Day 9 cultured bladder smooth muscle strips.
Figure 1
14
A
110 mM KCl
30 uM CCh
12
*
4
Stress, x 10 N/m
2
10
*
*
8
*
*
*
6
*
*
#
4
#
#
2
0
Day 0
Day 3
Day 5
Day 7
Day 9
S
ly
er
nly
On
ith
nO
Ne
o
i
t
ac
ntr
Co
tc h
tre
B
16
*
*
12
2
Cross-sectional in mm x 10
7
*
*
8
4
0
Day 0
Day 3
Day 5
Day 7
Day 9
Days in Culture
C
10
110 mM KCl
30 uM CCh
*
8
Force (grams)
*
*
*
6
*
#
4
#
#
#
2
0
Day 0
Day 3
Day 5
Day 7
Day 9
S
#
ly
ly
er
On
On
ith
Ne
on
i
t
ac
ntr
Co
t ch
tre
Figure 2
A
Carbachol
KCl-PSS
B
Force, % of maximum carbachol contraction
5g
1 min
5g
1 min
120
100
80
*
60
*
*
40
20
0
0
50
100
150
200
250
300
350
C
Force, % of maximum 110 mM KCl-PSS contraction
Time, in sec
120
100
80
60
40
20
0
0
50
100
150
200
Time, in sec
250
300
350
Figure 3
A
% Maximal Response
100
80
60
40
20
0
3
10
30
100
[KCl], in mM
B
% Maximal Response
100
80
60
40
20
0
0.01
0.03
0.1
0.3
1
3
[Carbachol], in μM
10
30
100
Figure 4
Figure 5
A
B
C
Figure 6
A
B
Figure 7
16
14
4
Stress x 10 N/m2
12
10
8
6
4
2
0
0
20
40
60
3
Length increase/cross-section area x 10
Figure 8
A
120
Caldesmon, % of Day 0
100
80
60
40
20
0
Day 0
Day 3
Day 5
Day 7
Day 9
Days in Culture
B
140
Calponin, % of Day 0
120
100
80
60
40
20
0
Day 0
Day 3
Day 5
Day 7
Day 9
Day 7
Day 9
Days in Culture
C
Myosin heavy chain, % of Day 0
200 KDa
140
120
100
80
60
40
20
0
Day 0
Day 3
Day 5
Days in Culture
0.7
D
SM1/SM2 Ratio
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Day 0
Day 3
Day 5
Day 7
Days in Culture
140
E
160 KDa
ROCK-1, % of Day 0
120
100
80
60
40
20
78 KDa
0
Day 0
Day 3
Day 5
Day 7
Day 9
Days in Culture
140
PKG, % of Day 0
120
100
80
60
40
20
0
Day 0
Day 3
Day 5
Day 7
Days in Culture
Day 9
Day 9
Figure 9
A
B
160
Calponin, % of Control
140
120
100
80
60
40
20
0
Control
C
+K+S
+K-S
-K+S
-K-S
D
0.8
SM1/SM2 Ratio
0.6
0.4
0.2
0.0
Control
+K+S
+K-S
-K+S
Control
+K+S
+K-S
-K+S
-K-S
E
160
F
140
PKG, % of Control
120
100
80
60
40
20
0
-K-S
Figure 10
A Day 0
5g
5 min
B Day 7
C Day 9