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Am J Physiol Regul Integr Comp Physiol 294: R58-R65, 2008. First published October 31, 2007; doi:10.1152/ajpregu.00477.2007
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GENETICALLY MODIFIED ANIMALS AND MODEL ORGANISMS

Deletion of one SERCA2 allele confers protection against bladder wall hypertrophy in a murine model of partial bladder outlet obstruction

Jenny Lassmann, Joanna Sliwoski, Andy Chang, Douglas A. Canning, and Stephen A. Zderic

The John W. Duckett Center for Pediatric Urology at The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

Submitted 2 July 2007 ; accepted in final form 25 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The sarco(endo)plasmic reticulum Ca2+-ATPase2 (SERCA2) is downregulated in cardiac hypertrophy with decompensation. We sought to determine whether mice heterozygous for the SERCA2 allele would develop greater bladder hypertrophy and decompensation than their wild-type littermates following partial bladder outlet obstruction (pBOO). We found that following 4 wk of surgically created pBOO, SERCA2 heterozygous murine bladders showed significantly less hypertrophy, improved in vitro cystometry performance, diminished expression of the slow myosin isoform A analyzed by RT-PCR, a significant drop in nuclear translocation of nuclear factor of activated T cells by EMSA, and decreased cell proliferation within the smooth muscle layer following 5-bromo-2'-deoxyuridine labeling compared with their wild-type littermates. Thus, in contrast to cardiac muscle, deletion of a SERCA2 allele confers protection against bladder hypertrophy in a murine model of pBOO. Compensatory mechanisms in heterozygous mice seem to be related to the calcineurin pathway. Further studies are underway to better define the molecular basis of this observation, which has potential clinical applications.

urinary bladder; obstruction; sarco(endo)plasmic reticulum Ca2+-ATPase2; nuclear factor of activated T cells

INTRACELLULAR CA2+ LEVELS are tightly regulated by a series of channels or ATP-dependent pumps that allow for transport across the plasma membrane or intracellular organelles such as the sarcoplasmic reticulum. This machinery has evolved in all cells given the key role of Ca2+ as a ubiquitous second messenger but assumes extra importance in muscle where Ca2+ release triggers contraction and sequestration is required for relaxation. Three categories of ion-transport ATPases transfer Ca2+ out of the cytosol and play a critical role in its homeostasis: the secretory pathway Ca2+-ATPase (SPCA), the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and the plasma membrane Ca2+-ATPase (PMCA) (28). Three SERCA isoforms are expressed in mammalian tissues (6, 7, 16, 21). SERCA1 is expressed in fast-twitch skeletal muscle (26). SERCA2-ATPases exist in three different isoforms: SERCA2a and SERCA2c are the Ca2+-ATPases of cardiac and slow-twitch skeletal muscle (13, 21), whereas SERCA2b is expressed in all tissues including heart and smooth muscle (17). SERCA3 can be found in different nonmuscle tissues as vascular and tracheal epithelium and pancreatic β-cells (1, 2).

Similar molecular mechanisms are involved in cardiac and bladder smooth muscle in response to mechanical stimuli and the subsequent development of hypertrophy and decompensation (10, 31). One pathway that has received attention in both organ systems is the calcineurin pathway, which is activated by sustained elevations of intracellular Ca2+ levels (25, 36). In response to a continued elevated Ca2+ concentration, calcineurin cleaves away a phosphate group from the nuclear factor of activated T cells (NFAT), which is found within the cytosol. Following this dephosphorylation, NFAT translocates into the nucleus and initiates muscle hypertrophy (12, 22). Inhibition of calcineurin with cyclosporine A (CSA) has been shown to prevent cardiac (35) as well as bladder hypertrophy in different animal models (11).

The SERCA2 plays a pivotal role in regulating cytosolic calcium levels. Studies have shown that SERCA2 levels are decreased in experimental and human heart failure (33). SERCA2 expression and function is also impaired in decompensated experimental (32, 37) and human (20) bladders following partial outlet obstruction. To better understand what role downregulation of SERCA2 might play in the pathophysiology of congestive heart failure, a mouse was generated with deletion of one SERCA2 allele; deletion of both alleles resulted in early embryonic death (27). At baseline, there was no evidence of cardiac hypertrophy in the heterozygous group compared with their wild-type littermates, despite a reduced mRNA SERCA2 expression by 45% and reduced SERCA2 protein expression by 35%. However, in response to transverse aortic occlusion, a substantial difference was noted between these two genotypes; the heterozygous mice developed greater cardiac hypertrophy, demonstrated diminished cardiac performance, and had an increased mortality (29).

We sought to determine the role of SERCA2 expression in the development of bladder wall hypertrophy following partial outlet obstruction. Our initial hypothesis was that mice heterozygous for the SERCA2 allele would undergo greater hypertrophy and be more likely to develop bladder decompensation than their wild-type littermates after partial bladder outlet obstruction (pBOO).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Operative procedure obstruction. Mice were housed and handled in accordance with standard use protocols and animal welfare regulations with the approval of the Children's Hospital of Philadelphia Institutional Animal Care and Use Committee. Breeder pairs of heterozygous SERCA2 mice were used to establish a colony. A male murine model of surgically created pBOO was used as previously described (3). Briefly, under isoflurane anesthesia administered by nose cone, mice underwent a 1-cm laparotomy to expose the bladder. The ureters were identified, and under x4.5 optical magnification, a 8-0 Prolene suture was passed around the bladder neck, above the prostate, and below the ureters. The Prolene suture was tied down over a 22-gauge angiocatheter, and the abdomen was closed in two layers of 7-0 Maxon. Eight-week-old male (+/+) and (+/–) SERCA2 mice of unknown genotype were subjected to operatively created pBOO. After 4 wk the mice were killed, animal weight was measured, and the bladder was excised just below the obstructing ligature. The bladder was emptied, weighed, and then prepared for in vitro physiology or molecular studies. Tail snips were obtained for genotyping at the time of death.

In vitro whole bladder cystometry. For in vitro whole bladder cystometry (3, 18), the bladders were canulated with a blunt 30-gauge needle inserted at the urethral stump and secured in place with a 5-0 Vicryl water-tied suture around the bladder neck; the ureters were ligated with 7-0 Vicryl sutures to prevent leakage. The needle was mounted onto a three-way stopcock connected to a pressure transducer and a microperfusion pump (KD Scientific, Holliston, MA). The bladder was immersed into a 5-cc organ chamber containing Tyrode's solution (in mM: 125 NaCl, 2.7 KCl, 1.8 NaCL2, 0.5 MgCL2, 23.8 NaHCO3, 0.4 NaHPO4, and 5.6 glucose at 37°C) and bubbled with 95% O2-5% CO2 gas. Pressure transducer output was directed to a digital data acquisition system (Daisy Lab Data Acquisition Software; Kent Scientific, Litchfield, CT). After a 20-min equilibration period, the bladders were filled with saline at 37°C at a rate of 10 µl/min. Field stimulation (70 V, 32 Hz, delivered by an S-70 neurostimulation unit; Grass Instruments, Quincy, MA) was delivered during filling at intervals of 2 min until the bladder achieved an optimum filling volume (Vo) for pressure generation (pressures within ± 5% of adjacent values). At Vo the infusion was stopped. The bladders were then stimulated by immersion in high potassium buffer (in mM: 127 KCl, 1.8 CaCL2, 0.5 MgCL2, 23.8 NaHCO3, 0.4 NaHPO4, and 5.6 glucose). The peak pressure and rate of pressure generation (dP/dt) were measured.

Genotype analysis. Genotyping (27) was performed by RT-PCR analysis of tail-snip DNA isolated according to the manufacturer's instructions (REDExtract PCR-Kit; Sigma, St. Louis, MO) using a combination of three primers that amplify both wild-type and mutant allele in the same reaction diluted to 100 µM: primer 1 (5'-CGG CCT TCT AGA GCC GGC TG-3'), primer 2 (5'-CTT ACG AAA GAT ATA CAT GCT GCC AGC AG-3'), and primer 3 (5'-GCA TGC TCC AGA CTC CCT TG-3'). PCR conditions for simultaneous amplification of wild-type (250-bp product) and mutant (250 and 120-bp product) alleles were a 3-min denaturation at 94°C, 40 cycles with 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C with a final extension to 72°C for 5 min. PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized with ethidium bromide. Heterozygote mice were identified by the appearance of two bands, whereas homozygous wild-type mice showed only one PCR product. Homozygous recessive mice are never seen with this particular deletion as it is embryonically lethal.

RT-PCR. Upon completion of the physiological studies, the bladders were stored at –80°C. Total RNA was isolated according to the manufacturer's instructions (Trizol; Invitrogen, Carlsbad, CA), and the final pellet was air dried and dissolved into a ribonuclease-free storage buffer (RNA Storage Solution; Ambion). The RNA concentration was determined spectrophotomically. cDNAs were produced using 1 µg total RNA with a commercially available kit (Retroscript; Ambion). cDNA (2.5 µl, 0.25 µg) was used for PCR with 1 µg each of the upstream and downstream primers. The total volume was brought to 12.5 µl with PCR grade water and then 12.5 µl PCR Master Mix (PCR Master; Roche Diagnostics Mannheim, Germany) were added. The primers for the myosin heavy chain (MHC) isoforms smooth muscle (SM)A and SM-B were: upstream, 5'-CCA CAA GGG CAA GAA AGA CAG C-3' and downstream, 5'-TCC GGC GAG CAG GTA GTA GAA GA-3'. Conditions for the simultaneous amplification of SM-B (289 bp) and SM-A (268 bp) PCR products were as follows: following a 5-min denaturation at 95°C, 30 cycles consisting of 95°C for 50 s, 55°C for 1.5 min, and 72°C for 2.5 min with a final extension of 7 min at 72°C. PCR products were resolved by 2% agarose gel electrophoresis and visualized with ethidium bromide, and images were then digitized. Individual band density was determined using image analysis software (ImageJ, version 1.36), and SM-A-to-SM-B ratios were compared among wild-type and heterozygote bladders (3, 14). The ratio of SM-A/SM-B was compared without the use of an 18S or other standard because of the nature of primer design around the MHC mRNA splice site. These primers lead to amplification of a longer segment of myosin mRNA that contains within it the smaller mRNA fragment that is produced after the alternative splicing takes place. This allows for measurement of the SM-A-to-SM-B ratio with accuracy (3, 14). All bladders subjected to this molecular analysis had undergone physiological studies.

Determination of nuclear NFAT by EMSA. Nuclear protein extracts were prepared from whole bladders following instructions from the manufacturer's kit with a minor modification of the suggested buffer volume to account for the small tissue size (Panomics, Fremont, CA). The nuclear protein levels were quantified by a modified Lowry assay (Bio-Rad, Hercules, CA). All binding reactions were performed at room temperature for 20 min in a volume containing 3 µg nuclear protein and 5 ng Biotin-NFATc oligonucleotide probe (AY1027P; Panomics, Fremont, CA); and total volume was adjusted to 10-µl with binding buffer. For the competition reaction, 990 ng of cold nonbiotinylated probe was also added. In another group of experiments, protein extracts were also preincubated for 10 min with the Biotin-NFATc probe and 1 µg of NFATc3 or NFATc4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for 10 min on ice followed by 10 min at room temperature. Upon completion of the binding reaction, all samples were run on a precast 10% Tris-borate EDTA gel (Bio-Rad) at 100 V for ~1.5 h. Electrophoretic transfer and detection were accomplished using the LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, IL). Shifted bands were analyzed with image analysis software (ImageJ, version 1.36) with comparisons made among the (+/+) and (+/–) groups.

5-Bromo-2'-deoxyuridine application and staining. In a subset of mice Alzet (model 2004; Durect, Cupertino, CA) osmotic minipumps filled with 5-bromo-2'-deoxyuridine (BrdU; 50 mg/ml in 0.1 M carbonate solution) (9) for labeling cell populations undergoing DNA synthesis were implanted subcutaneously via a small incision in the back on the day of obstruction as well as in age-matched nonobstructed controls. BrdU was delivered at 0.3 mg/day (10 µg/g animal wt) over the course of 4 wk. At the death of the animals, the bladders were filled with 150 µl normal saline, the urethral stump was closed with 5-0 Vicryl, and the specimens were fixed in formalin. Following paraffin embedding, 5-µm sections were cut and stained with a monoclonal antibody directed against BrdU (Abcam, Cambridge, MA) followed with a secondary antibody coupled to horseradish peroxidase and β-napthol staining for colorimetric detection. A BrdU incorporation index was calculated by microscopic evaluation of three random sites per sample within the urothelium and muscle layer by counting the BrdU-positive cells per area. Seven heterozygous and four wild-type bladders after a 4-wk course of obstruction, as well as four heterozygous and four wild-type bladders of age-matched nonoperated controls were investigated.

Localization of BrdU synthesis within the bladder wall. A set of paraffin-embedded bladders were analyzed with dual-immunofluorescence labeling using a sheep polyclonal antibody to BrdU (Capralogics, Hardwick, MA) with a secondary antibody coupled to Texas Red and a mouse monoclonal antibody against smooth muscle MHC (Abcam) with a secondary antibody coupled to fluorescein. All nuclei were visualized by staining with 4,6-diamidino-2-phenylindole (DAPI; Sigma). Following deparaffinization, sections were incubated at 4°C overnight with the primary antibody (1:400 dilution) and washed three times with PBS. Secondary antibodies were applied at 4°C overnight, followed by an additional three washes with PBS. DAPI staining of the nuclei was carried out as per the supplier's instructions. Sections were imaged at the respective excitation wavelengths for DAPI (360 nm), FITC (480 nm), and Texas Red (595 nm) using the Leica DM400B microscope with a digital imaging camera and software (Diagnostic Instruments, Sterling Heights, MI). Images acquired from all three wavelengths were imported into Adobe Photoshop and merged.

Controls. No difference has been shown in regard to bladder weight, pressure generation to field stimulation, and KCl depolarization and myosin heavy isoform expression between nonoperated and sham-operated controls using applied male murine model for pBOO as previously described (3). Nonoperated, age-matched, 12-wk-old male mice were used as controls and the same protocols were applied to this population of bladders.

Statistical analysis. Comparison between groups was performed using the Student's t-test with P < 0.05 considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Bladder and body mass. SERCA2 (+/–) mice developed less bladder wall hypertrophy, as measured by bladder weight, than their (+/+) littermates (46.8 ± 7.3 mg vs. 79.2 ± 30.3 mg, P = 0.006, Fig. 1). While mean total body weight was significantly less in (+/–) compared with +/+ mice after a 4-wk course of obstruction (34.4 ± 4.6 g vs. 41.2 ± 7.3 g, P = 0.005), the ratio of bladder weight/animal weight was significantly lower in (+/–) compared with (+/+) mice (1.4 ± 0.6 vs. 1.9 ± 0.6, P = 0.04). There was no significant difference in age-matched nonoperated (+/–) and (+/+) controls in mean values for bladder weight, animal weight, and ratio of bladder/animal weight.


Figure 1
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  		Fig. 1. (+/–) Sarco(endo)plasmic reticulum Ca2+-ATPase2 (SERCA2) mice developed significantly less bladder hypertrophy after partial bladder outlet obstruction (pBOO; 79.2 vs. 46.8 mg; P < 0.01). This difference remained significant even after correction for differences in body mass. NS, not significant.

 
In vitro whole bladder cystometry. SERCA2 (+/–) bladders showed significantly improved in vitro bladder cystometry performance compared with (+/+) bladders. Bladders from SERCA2 (+/–) mice generated significantly higher pressures in response to both field stimulation (Fig. 2A) and KCL (Fig. 2B) depolarization than their (+/+) littermates. In addition, SERCA2 (+/–) mice showed a significantly increased rate of pressure generation in response to field stimulation or KCl compared with their (+/+) littermates (7.5 ± 4.5 vs. 3.6 ± 1.6 for electrical field stimulation, and 3.4 ± 1.5 vs. 1.7 ± 1.2 for KCl in both instances [P < 0.05 for +/–) vs. (+/+)]. Figure 2C shows representative tracings of in vitro whole organ cystometry with higher generated peak pressure in (+/–) than in (+/+) bladders during field stimulation and KCl depolarization. There were no significant differences between age-matched nonoperated (+/–) and (+/+) nonoperated controls in mean values for peak pressure generation during field stimulation and KCl depolarization, as well as velocity of pressure change over time for field stimulation and KCl depolarization.


Figure 2
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  		Fig. 2. A: (+/–) bladders generated significantly higher pressures during field stimulation after pBOO (32.3 vs. 16.8 mmHg). B: (+/–) bladders generated significantly higher pressures during KCl depolarization after pBOO (32.3 vs. 16.8 mmHg). Bars in A and B represent means ± SD of 6–15 preparations per category. C: representative tracings of in vitro whole organ cystometry. Higher pressure was generated during field stimulation with relatively less volume in heterozygous mice vs. wild-type mice.

 
MHC isoform expression. RT-PCR analysis for mRNA expression for MHC isoforms SM-B and SM-A revealed predominant expression of fast SM-B in nonobstructed controls independent of the genotype (an SM-A-to-SM-B ratio of 0.1 ± 0.05). However, following 4 wk of obstruction, the bladders of (+/–) mice demonstrated predominantly SM-B with minimal expression of SM-A; in contrast for (+/+) bladders, the SM-A isoform expression increased significantly (Fig. 3A). With pBOO, the ratio of SM-B/SM-A mRNA isoform expression was 0.39 ± 0.17 in (+/–) bladders (n = 11) vs. 0.94 ± 0.32 in (+/+) bladders (n = 7, P < 0.0016) (Fig. 3B).


Figure 3
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  		Fig. 3. A: representative RT-PCR analysis for myosin heavy chain (MHC) SM-B/SM-A isoform mRNA expression shows that fast SM-B predominates in unobstructed controls and (+/–) bladders after pBOO, while a switch to slow SM-A occurs in (+/+) mice. B: quantification reveals the increase in the SM-A isoform to be much greater in bladders from the (+/+) mice compared with their (+/–) littermates. Bars represent means ± SD of 5–7 preparations per category.

 
Obstruction-induced nuclear translocation of NFAT detected by EMSA. NFAT nuclear translocation was significantly higher in (+/+) compared with (+/–) bladders after 4 wk of pBOO (1.99 ± 0.6 vs. 1.31 ± 0.5, P < 0.02) (Figs. 4 and 5) . NFAT nuclear translocation was significantly lower in unobstructed controls, and no difference was seen between (+/+) and (+/–) bladders (Figs. 4 and 5). These shifted bands corresponded to an interaction between NFAT and the labeled oligonucleotide probe, because 1) the bands disappeared in the presence of "cold" nonbiotin-labeled probe, and 2) the bands were diminished in the presence of NFATc3 (Fig. 4) or NFATc4 antibody (gel not shown).


Figure 4
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  		Fig. 4. EMSA showing the obstruction-induced nuclear translocation of nuclear factor of activated T cells (NFAT) takes place to a greater degree in (+/+) compared with (+/–) bladders. Ctl, control. *Probe alone.

 

Figure 5
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  		Fig. 5. Quantification of NFAT activity in unobstructed control (+/+) and (+/–) bladders and following a 4-wk course of pBOO. Notice the significantly higher NFAT activity in (+/+) than in (+/–) bladders following obstruction. Bars represent means ± SD of 7 preparations per category.

 
BrdU-staining estimating the cell proliferation index. After a 4-wk course of obstruction, there was a significantly higher BrdU index within the muscle layer in (+/+) compared with (+/–) bladders (0.0046 ± 0.001 vs. 0.0023 ± 0.0006 density per area, P = 0.02) but not within the urothelium (0.01 ± 0.0008 vs. 0.01 ± 0.0007 density per area, P = 0.76) (Figs. 6 and 7). The BrdU index in (+/+) and (+/–) control bladders without obstruction did not differ within the muscle layer or the urothelium (Figs. 6 and 7).


Figure 6
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  		Fig. 6. A: 5-bromo-2'-deoxyuridine (BrdU) index within the urothelium was significantly higher after 4 wk of obstruction compared with unobstructed controls. However, there was a difference between (+/+) and (+/–) mice. B: BrdU index within the muscle was significantly higher after 4 wk of obstruction in (+/+) compared with (+/–) bladders. There was no difference in unobstructed controls independent from genotype. Bars in A and B represent means ± SD of 4–7 preparations per category.

 

Figure 7
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  		Fig. 7. Representative sections of BrdU immunostaining in (+/+) and (+/–) control bladders and after a 4-wk course of obstruction. Notice the increased number of BrdU-stained cells within the urothelium following obstruction in (+/+) and (+/–) bladders cells and within the muscle layer in (+/+) bladders. Brown nuclei, sites of BrdU uptake; blue nuclei, normal nuclear staining.

 
Localization of BrdU synthesis within the bladder wall. The dual immunohistochemistry studies revealed that the overwhelming amount of DNA synthesis was taking place outside the cell population identified as smooth muscle by virtue of their green staining with the FITC marker. When viewed as a single merged image, the blue nuclei reflect areas in which there is no DNA synthesis. In contrast, pink nuclei reflect areas in which there has been BrdU uptake (due to the merging of the blue nuclei due to DAPI and the Texas Red due to BrdU). Scanning of multiple fields revealed only an occasional pink nucleus within the smooth muscle beds as defined by the smooth muscle myosin stain (Fig. 8).


Figure 8
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  		Fig. 8. Representative sections showing merged smooth muscle myosin (FITC), BrdU uptake (Texas Red), and nuclei stained with 4,6-diamidino-2-phenylindole (magnification, x200). Pink nuclei reflect those nuclei undergoing DNA synthesis as opposed to those staining a pure blue, which have been quiescent. There is baseline DNA synthesis in control tissues within the lamina propria. However, with pBOO, there is a marked increase in DNA synthesis within the urothelium and the serosal edges. The overwhelming preponderance of nuclei seen within the green-staining smooth muscle bundles are blue. Pink nuclei predominated between the smooth muscle bundles. Shown above are sections representative for control tissues and those taken following pBOO from a SERCA2 (+/–) mouse. The preference of BrdU uptake by the nonsmooth muscle cell population was the same when compared with (+/+) and (+/–) populations.

 
Genotypes of postoperative deaths. Depending upon the cohort of obstructions performed, perioperative mortality ranged from 10 to 20%. Tail snips and a genotypic analysis were performed for all mice who did not survive to the 4-wk mark. The postoperative deaths were evenly distributed between the (+/+) and (+/–) group.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In contrast to cardiac muscle, where transverse aortic occlusion leads to hypertrophy, congestive heart failure, and increased mortality, deletion of a SERCA2 allele confers protection against the bladder hypertrophy that develops following an increase in outlet resistance. Contrary to our starting hypothesis, we observed that mice missing one SERCA2 allele developed significantly less bladder hypertrophy and showed improved in vitro whole organ performance compared with their wild-type littermates following pBOO. These results were independent of the obstructions created by surgeons (J. Lassmann and S. A. Zderic), who were unaware of the genotype at the time of surgery. We considered that mortality might be higher for (+/–) mice due to a survival disadvantage following obstruction, which could potentially affect the data; however, a genotypic analysis of tail snips from mice who expired postoperatively revealed an even distribution of deaths among the two genotypes. It is also important to note that in age-matched, unobstructed control mice, we could not identify any phenotypic differences between (+/–) and (+/+) litters, leading us to conclude that the differences in the response to pBOO do not reflect baseline differences in genitourinary anatomy or physiology between the heterozygous and wild-type mice.

However, we were able to unmask substantial phenotypic differences following a 4-wk course of partial outlet obstruction between (+/–) and (+/+) bladders using the in vitro whole bladder model as described previously (3). Bladders from (+/–) bladders generated significantly higher peak pressures in response to field stimulation as well as in response to direct membrane depolarization with KCl (Fig. 2). Furthermore, a significantly higher rate of pressure generation during field stimulation or KCl depolarization, measured as dP/dt (pressure change/elapsed time), was observed in (+/–) bladders. This physiologic finding correlated with the observed shift to the faster MHC mRNA-B isoform distribution (Fig. 3). Prior studies have demonstrated that phasic muscle, such as the bladder, which must be able to contract rapidly, contain predominantly the SM-B MHC isoform; in contrast, the SM-A isoform predominates in tonic smooth muscle as seen in the aorta (34). The SM-B isoform contains a 7-amino acid insert on its NH2 terminus near the ATP-binding site, resulting in an increased shortening velocity of smooth muscle in vitro and in vivo (5, 19). Our results demonstrate significantly increased expression of the SM-B MHC isoform mRNA in (+/–) bladders, which can be correlated with their improved rate of pressure generation (dP/dt) compared with their (+/+) littermates (Fig. 3). This shift from fast-acting SM-B to slow-acting SM-A has been described previously in murine as well as in rabbit bladders following partial outlet obstruction (3, 15). This relationship between bladder performance and the SM-A-to-SM-B isoform ratio is being further explored using this model of pBOO in a murine SM-B knockout model.

In both rabbit (11) and murine (Chang AY, Sliwoski J, Lassmann J, Chacko S, Canning DA, Zderic SA, unpublished observation) models, we have shown that the calcineurin pathway is activated following pBOO. Furthermore, inhibition of the calcineurin pathway with CSA resulted in diminished bladder wall hypertrophy and a pronounced shift in MHC isoforms back toward the fast (MHC SM-B) isoform (11). Similar observations have been recorded in a murine model of pBOO (Chang AY, Sliwoski J, Lassmann J, Chacko S, Canning DA, Zderic SA, unpublished observation). The findings in SERCA2 (+/–) mice were similar to those we had observed in rabbits (11) or mice with an NFAT-luciferase reporter transgene (Chang AY, Sliwoski J, Lassmann J, Chacko S, Canning DA, Zderic SA, unpublished observation) who had received CSA following pBOO. Given the importance of SERCA2 in regulating intracellular Ca2+ homeostasis, we suspected that our observations might be related to differential activation of the calcineurin pathway. Using the EMSA technique allowed us to demonstrate that there was less nuclear uptake of NFAT in the (+/–) population compared with their (+/+) littermates (Figs. 4, 5). Such a finding suggests that with pBOO there is less of a rise in basal cytosolic Ca2+ in the (+/–) mice, and as a result, there is less activation of calcineurin's phosphatase activity, and subsequently, less nuclear translocation of NFAT.

These findings lead us to speculate that with deletion of a SERCA2 allele, compensatory changes might occur within other channels or pumps that regulate cytosolic Ca2+ homeostasis, although so far, we have not identified such changes in the expression of the plasma membrane Ca2+ ATPase, the ryanodine channel isoforms, or the voltage-operated dihydropyridine-sensitive calcium channel. Alternatively, these secondary changes may occur in other cell compartments or pathways. The identification of where within the cell such protein shifts occur would allow for testing of small molecular compounds, which could block the development of bladder wall hypertrophy in the presence of pBOO. This would offer an alternative to the use of CSA, which can prevent hypertrophy in the presence of pBOO, but does so at the expense of toxicity.

We also sought to understand which cell populations were undergoing DNA synthesis in the bladder wall following pBOO and how these might differ between the (+/+) and (+/–) genotypes. Several studies have looked at [H3]thymidine incorporation into the bladder wall using either short-term in vivo (24–48 h) or in vitro methods (23, 24). Given the problems associated with in vivo use of radioisotopes for prolonged periods, there is a paucity of data looking at DNA synthesis in the bladder wall over a prolonged course of pBOO. The short-term studies cited offer evidence for intense DNA synthesis in the lamina propria with virtually no evidence of DNA synthesis within the smooth muscle layers. As with the other reported studies (30), we observed an intense uptake of BrdU within the lamina propria following pBOO in both (+/+) and (+/–) mice compared with their nonoperated controls. Consistent with Sjuve et al. (30), we observed sporadic BrdU staining in control bladders without obstruction and an increased index within the muscle layer observed with an extended course of obstruction. However, after an initial increase, they noted a marked decrease of submucosal staining with prolonged obstruction, whereas our results suggest a continued high-proliferation index within the submucosa. Such differences might be related to an intermittent, rather than the continuous application of BrdU throughout the 4-wk course of obstruction employed in our present study. Our approach gave differing results in that we observed increased BrdU incorporation into what appeared to be the smooth muscle layer of SERCA2 (+/+) bladders following 4 wk of obstruction compared with nonoperated (+/+) controls. Our study is also unique in that we found a much lower rate of BrdU incorporation was observed within the smooth muscle layer of the (+/–) obstructed bladders (Figs. 6 and 7).

An additional question posed by these initial BrdU experiments was which cell fraction within the bladder wall was undergoing hyperplasia. Our immune fluorescence colocalization data suggests that this DNA synthesis is occurring predominately within the fibroblasts found within the connective tissue septae that are interspersed among the bundles of smooth muscle (Fig. 8). This issue will be studied in further detail by using additional antibodies targeting the fibroblast cell population, a similar double-labeling immunofluorescent technique, and confocal microscopy to enhance spatial accuracy. One possibility is that with the application of bladder wall stress, proliferation of fibroblasts occurs, which subsequently produce the abundant extracellular matrix deposition that is observed following pBOO. This deposition of fibroblasts and extracellular matrix, in turn, diminishes the efficiency with which the smooth muscle bundles can contract; as a result, the bladder loses its ability to generate pressure.

There are several possibilities for why the bladder and heart differ in their response to outflow occlusion in the SERCA2 (+/–) mice. The SERCA2 transcript is alternatively spliced into SERCA2a and SERCA2b, which differ in the carboxyl terminus; SERCA2a is expressed in the heart and SERCA2b, within the smooth muscle (17, 21). The loss of SERCA2b may well trigger a different set of compensatory mechanisms in bladder than in the heart, which would not be surprising, given the differences in their performance. The heart is cycling continuously, and rapid return of cytosolic calcium to its proper storage sites is critical. In contrast for the bladder, which contracts once every 1 to 3 h, rapid calcium cycling is less important. Our current efforts are focused on identifying these secondary compensatory changes, which we feel will help us target new drug therapy for the prevention of bladder wall hypertrophy in the presence of outlet obstruction. Such an approach would find clinical applicability in the setting of the fetus with posterior urethral valves where surgical intervention carries significant risk or in managing the hypertrophied neurogenic bladder that results from spina bifida.

Conclusion

In contrast to cardiac muscle, deletion of a SERCA2 allele confers protection against bladder hypertrophy after a 4-wk course of obstruction along with improved in vitro organ function. This phenotypic observation correlated with higher expression of the fast-MHC isoform SM-B. Diminished DNA synthesis is seen within the smooth muscle bundles and is actually taking place within the fibroblasts that are interspersed in septae between the muscle bundles. Nuclear translocation of NFAT is decreased, suggesting that compensatory mechanisms in (+/–) mice are related to decreased Ca2+-levels and its role in Ca2+-signaling in relation to the calcineurin pathway. Further studies are underway to better define the molecular basis for this protective effect, which has potential clinical applications.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by The Leonard and Madlyn Abramson Endowed Chair in Pediatric Urology, The Academic Development Fund of the John W. Duckett Center for Pediatric Urology at The Children's Hospital of Philadelphia, and a grant from the American Foundation for Urologic Disease (to A. Chang).


   ACKNOWLEDGMENTS
 
We thank Dr. Gary Shull at the University of Cincinnati School of Medicine for his kind gift of breeder mice to establish our colony of SERCA2 knockout mice and for valuable discussions. We also thank Dan Martinez and the pathology core lab of the Joseph Stokes Jr. Research Institute for their help with the BrdU immunohistochemistry and immune fluorescence experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. A. Zderic, Division of Pediatric Urology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104 (e-mail: zderic{at}email.chop.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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