Altered detrusor contractility and voiding patterns in mice lacking the mechanosensitive TREK-1 channel

Adult male and female C57BL/6 J wild-type (WT, N = 32) and TREK-1 knockout (Kcnk2^−/−, TREK-1 KO, N = 33) mice were used in this study. Wild type mice (10–12 weeks old) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). TREK-1 KO mice, a generous gift from the laboratory of Dr. Min Zhou (Department of Neuroscience, The Ohio State University, Columbus, OH), were bred in the Breeding Core Barrier Facility at the University of Colorado Anschutz Medical Campus. Originally, the Kcnk2 ^−/− mice were generated as described in [45]. Briefly, the TREK-1 gene, Kcnk2, spans approximately 136 kbp of the M. musculus genome and includes eight exons. The knockout mice were generated by replacing 4 kbp of the Kcnk2 gene including the second exon (excluding the first 23 bp), the second intron, the third exon and the first 23 bp of the third intron with a β-galactosidase/Neomycin selection cassette producing a truncated, nonfunctional protein.

All animals were housed in a regulated environment on a 12:12-h light/dark cycle with free access to food and water. There were no overt differences in feeding behavior, litter size, growth rate and body weight between the WT and TREK1 KO mice. All protocols were approved by the University of Colorado Institutional Animal Care and Use Committee.

Genomic DNA was extracted using the REDExtract-N-Amp Tissue PCR Kit (Sigma-Aldrich, St Louis, MO. USA) from the urinary bladders of WT and TREK-1 KO mice. Genotyping was performed as previously described [45]. Briefly, the WT Kcnk2 allele was detected by using the following primer pairs: forward 5′-GCTGGGTGAAGTTCTTCAGC-3′, and reverse: 5′- CATTACCTGGATGAGTTCGTC-3′. The Kcnk2 KO allele was detected using the primer pairs: forward 5′-GCAGCGCATCGCCTTCTATC-3′ and reverse 5′-AGGAGATGAAGACCTCTGCAAAGG-3′. End-point RT-PCR products from WT and TREK-1 KO specimens were subsequently run on 2% agarose gels. Gel images were taken and analyzed using the Doc-It LS Image Acquisition & Analysis System (UVP, LLC, Upland, CA, USA).

To compare the gene expression levels of several protein kinase C (PKC) isoforms expressed in the bladder, whole bladder tissues were removed from WT and TREK-1 KO mice, and snap-frozen in liquid nitrogen. Posteriorly, total RNA was extracted and purified using TRIzol® Plus RNA Purification Kit (Ambion, Thermo Fisher Sci. Waltham, MA, USA) according to the manufacturer’s instructions. RNA concentrations and purity were determined spectrophotometrically on a Nanodrop device (Thermo Fisher Sci. Waltham, MA, USA). RNA integrity was evaluated by formaldehyde agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. Isolated RNAs were stored at − 80 °C until use. First-strand complementary DNA (cDNA) was synthesized using the Qiagen OneStep RT-PCR kit (Qiagen, Valentia, CA. USA) by the manufacturer’s guidelines. Products were stored at − 20 °C until the use. End-point PCR was performed using the Qiagen Multiplex PCR plus kit (Qiagen, Valentia, CA. USA) following the manufacturer’s instructions. The following PKC isoforms were selected for comparison due to their significant level of expression in the urinary bladder: PKC-α (alpha), PKC-β (beta), PKC-γ (gamma), PKC- δ (delta), PKC-ε (epsilon), PKC-μ (mu) and PKC-τ (tau). Primer sequences for each isoform, gene accession numbers and predicted RT-PCR product sizes are listed in Table 1. Non-template control reactions were included in each reaction to test for possible RT-PCR contamination and were run in parallel with the experimental samples. End-point PCR products were analyzed by electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light.

Total protein was isolated from the urinary bladder by conventional tissue lysis with T-PER Tissue Protein Extraction Reagent (ThermoFisher Sci, Waltham, MA. USA) containing protease and phosphatase inhibitors (Sigma Aldrich, St Louis, MO. USA). Western blot experiments were performed with equal loads of the total protein per lane (20 μg). WT and KO samples were size fractionated by SDS-PAGE electrophoresis using 4–10% polyacrylamide Mini Protein gels (BioRad, Hercules, CA. USA). Proteins were then electrotransferred to polyvinylidene difluoride membranes (LICOR Biotechnology, Lincoln, NW. USA) and incubated for one hour at room temperature (RT) in Odyssey Blocking Buffer (TBS, LICOR Biotechnology, Lincoln, NW. USA). Membranes were subsequently washed three times in PBST for 10 min each and incubated with Anti-TREK-1 (F6, 1:1000, Santa Cruz Biotechnology, Dallas, TX. USA) and anti-Histone H3 as a house-keeping gene (1:2000, Abcam, Cambridge, MA. USA) in blocking buffer under constant agitation. We also tested other commercially available antibodies from the same company which included the C20, E-19, and H-75 anti-TREK-1 variants. The C20 and E19 anti-TREK-1 antibodies did not work for Western blotting, while H-75 antibody (aa352–426 within the C-terminus) showed two bands in WT animals and one band in TREK-1 KO (data not shown). Therefore, we used F6 anti-TREK-1 for Western blotting. The next day, membranes were rinsed three times with PBST for 10 min each time, and incubated with anti-mouse and anti-rabbit VRDye secondary antibodies (1:10000, LICOR Biotechnology, Lincoln, NW. USA) for 2 hrs at RT. Afterwards, membranes were rinsed three times with PBST for 10 min each and imaged using the Odyssey CLx imaging system (LICOR Biotechnology, Lincoln, NW. USA).

WT and TREK-1 KO animals were anesthetized with isofluorane (2.5%) and perfused via the left cardiac ventricle with phosphate-buffered-saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS (PFA) for 10 min. The colon, bladder, and kidneys were removed and placed in PFA for two hours at 4^o C. Subsequently; the tissues were placed in 70% alcohol for at least 2 days before being dehydrated and embedded in paraffin. 4 μm -thick sections were cut and mounted on “frosted” slides (Fisher Scientific Co, Waltham, MA. USA) at the Morphology and Phenotypic Core of the University of Colorado Anschutz Medical Campus. Before immunolabeling, sections were washed in Xylene (Sigma-Aldrich, St Louis, MO. USA) to remove paraffin, re-hydrated in a graded ethanol series, washed with distilled water, and rinsed in 50 mM Tris-buffered saline (TBS, pH 7.4) for 5 min. Heat-induced epitope retrieval was performed by incubating the slides at 85–90⁰ C for 15 min in Target Retrieval Solution (DAKO, Agilent Pathology Solutions, Santa Clara, CA. USA). Once cold down to room temperature, the sections were washed twice in TBS containing 0.1% Tween 20 (TBST, Sigma-Aldrich, St Louis, MO. USA), and incubated for 30 min in casein blocking solution (CBS, DAKO, Agilent Pathology Solutions, Santa Clara, CA. USA). Slides were incubated overnight at 4⁰ C (ON) in CBS with mouse monoclonal antibodies against the TREK-1 protein (F6, 1:300; Santa Cruz Biotechnology, Dallas, TX. USA). The next day, sections were rinsed three times with TBST for 5 min each time, and then incubated for 2 h at RT with a secondary antibody conjugated with Alexafluor 488-labeled anti-mouse IgG (1:500; Fisher Scientific Co, Waltham, MA. USA) in CBS. After incubation with secondary antibody, sections were washed three times with TBST for 5 min each, rinsed with TBS and counterstained with 200 nM 4′6-diamidino-2-phenylindoledihydrochloride (DAPI, Fisher Scientific Co, Waltham, MA. USA) in TBS for 3–5 min at RT and rinsed twice in TBS. Coverslips were mounted with Fluoromount-G (SoutherBiotech, Birmingham, AL. USA). Imaging was performed on an Olympus FV1000 confocal microscope with 40X plan-apo/1.4 numerical aperture objective and FV-viewer software (Olympus, Tokyo, JP). For visualization, three-dimensional z-stack images of x-y sections at 0.5 μm steps were collected. Two-dimensional average intensity projection images were generated for analysis in FIJI (Fiji Is Image J, [50].

Bladder smooth muscle (BSM) strips were isolated from WT (N = 17) and TREK-1 KO (N = 17) mice following the previously described procedures [23, 34]. Briefly, BSM strips (∼8–10 mg each, 2–3 mm wide and 7–8 mm long) were placed in individual organ baths (Radnoti LLC, Monrovia, CA, USA), containing 7 ml of normal Tyrode Buffer (TB, in mM: NaCl 130.0, KCl 5.0, CaCl₂ 1.7, MgCl₂ 1.0, NaH₂PO₄ 1.3, NaHCO₃ 17.0, Glucose, 10.0. pH 7.4) maintained at 37 °C and equilibrated with a constant supply of 95% O₂–5% CO₂. Strips were subjected to 1 h equilibration followed by determination of the length of optimal force development (L_o). 1 μM of tetrodotoxin (TTX, Sigma-Aldrich) was added to fresh TB to minimize neural effects on BSM contractility. The strips were subjected to different pharmacological treatments including either arachidonic acid (AA; 10 μM), a TREK-1 channel agonist, or L-methionine (LM, 1 mM), a TREK-1 channel blocker [5, 34, 42]. Untreated muscle strips served as controls. At the end of the incubation period, in order to assess the effects of TREK-1 channels on stress-relaxation, all strips were subjected to an additional 30% stretch to the initially established L_o, and allowed for complete relaxation. Following the stretch protocol, a KCl test (bath solution was replaced with TB containing 125 mM KCl) was performed to ensure that the BSM could maintain the force after stretch [23, 24]. After KCl stimulation, muscle strips were washed three times (10 min each time) in fresh TB and allowed to recover for 30 min. After recovery, all strips were incubated with a non-specific PKC activator, Phorbol 12,13-Dibutyrate (PDBu, 1 μM: Sigma; Greenwood Village, CO), for 45 min.

A separate set of experiments was performed to evaluate the contribution of active and passive basal tone components in BSM from WT and TREK-1 KO bladders. After establishing L₀ in normal Ca²⁺ TB, the buffer was changed to Ca²⁺ free TB followed by 30 min incubation and measurements of the basal tone and spontaneous contractile activity of BSM strips. Some strips were subjected to an additional 30% stretch to evaluate stress-relaxation under Ca² free conditions. At the end of the testing protocol, all strips were treated with 2 μM of nifedipine, an L-type Ca²⁺ channel blocker, to assess the role of Ca²⁺ entry via voltage-gated Ca²⁺ channels on BSM tone and spontaneous activity.

All BSM experiments were recorded and analyzed using LabChart 8 Pro (AD Instruments, Colorado Springs, CO, USA), pClamp 10 (Molecular Devices, LLC. San Jose CA, USA) and Matlab R2014b (MathWorks, Natick, MA, USA). The following parameters were measured and analyzed: basal muscle tone (in g/g, g of force per g of muscle strip weight), amplitude of spontaneous contractions (g/g, measured as amplitude from the baseline level of each strip and normalized to its weight), peak force (PF; in g or g/g when normalized to the weight of the muscle strip), and integral force (IF) which reflects how long a muscle strip can maintain the contractile force before it starts relaxing [33].

Animals assigned for urodynamic evaluation of urinary bladder function (awake cystometry) underwent a survival surgical procedure to insert bladder catheters as previously described [35]. Briefly, the animals were anesthetized with isofluorane (VEDCO, St.. Albans, VT) and the bladder was exposed through a lower midline abdominal incision. A polyethylene tubing (PE-50, I.D. 0.58 mm, O.D. 0.96 mm; Intramedic, Becton Dickinson. Parsippany, NY) with a flared end was inserted through a puncture at the bladder dome, and sutured in place with purse string suture and 7.0 silk (Ethicon, Somerville, NJ). The catheter was then tunneled subcutaneously and exteriorized at the scapular region where it was sutured to the skin and filled with sterile saline solution. After confirming no leakage in the bladder, the catheter was plugged with a metal rod, and the muscle and skin layers were closed with a 5.0 silk suture (Ethicon, Somerville, NJ). Particular care was taken not to stretch the bladder during the procedure or to restrict the normal bladder movement once the catheter was in place.

Mice were allowed to recover from surgery (4–5 days) before the urodynamic study. Unanesthetized animals were placed in the cystometry cage, and allowed to acclimate for 30–40 min. Saline was slowly infused into the urinary bladder at a rate of 10 μl/min, and micturition cycles were recorded using the MED-CMG Small Animal Cystometry acquisition software (Catamount Research and Development). The following cystometric parameters were evaluated in this study: bladder capacity, pressure at the start of micturition, micturition rate, intravesical pressure, inter-micturition interval, and number of non-voiding contractions. Non-voiding contractions were defined as the increased values in detrusor pressure not associated with voiding, equal or larger than twice the mean value of baseline. The number of non-voiding contractions was measured for each micturition cycle, and then averaged per cycle based on the number of recorded cycles (e.g. voiding episodes) per animal. Normal voiding contractions had amplitudes of at least a third of maximal pressure recorded during a single micturition event.

In TREK-1 KO mice used for this study, the four transmembrane domains and two-pore forming regions were genetically truncated by replacing most of exon 2 and all exon 3 by a LacZ/Neo cassette [45]. We confirmed the deletions by performing end-point RT-PCR in mRNA extracted from TREK-1 KO and WT bladder tissue samples (N = 5 in each group). The WT primers were designed, as previously described, to include parts of intron 2 and exon 3 sequences, while the KO primers included part of the LacZ/Neo cassette and intron 3 [14]. The results of RT-PCR genotyping from TREK-1 KO and WT mice are shown in Fig. 1a. The sizes of the bands corresponding to TREK-1 KO and WT alleles were around ~ 200 and ~ 450 bp, respectively. Figure 1b represents a Western Blot image of the total protein isolated from WT, and TREK-1 KO mouse bladders probed with an anti-TREK-1 antibody showing one band ~ 50 kDa in WT but not in TREK-1 KO specimens (N = 5 in each group). As previously reported [14, 45], no significative changes were observed in fertility, number of the offspring or growth rate between the WT and TREK-1 KO groups.

Immunofluorescent tissue labeling was used to compare the presence and distribution of TREK-1 proteins in the visceral organs of WT and TREK-1 KO mice including the urinary bladder, colon, and kidneys. As shown in Fig. 2a (left panel), TREK-1 was expressed in the colon of WT animals with stronger labeling present in the colonic mucosa in comparison to the muscle staining. Intense TREK-1 immunoreactivity was also found in the muscularis mucosa layer. In contrast, TREK-1 KO animals showed a significantly reduced TREK-1 immunoreactivity across the colonic tissue (Fig. 2a, right panel). Confocal images of the bladder sections from WT group (Fig. 2b, left panel) displayed a homogeneous TREK-1 like staining across the detrusor with faint signal in the urothelium and serosa. In contrast, TREK-1 KO animals showed a significant reduction in TREK-1 immunofluorescence. Histologically, the urinary bladders showed no visible morphological alterations (Fig. 2d), and no significant differences were observed in bladder weight between WT and TREK-1 KO animals (9.3 ± 0.7 vs. 10.2 ± 0.6 mg, respectively). The body/bladder weight ratio (g/mg) in WT and TREK-1 KO mice was also similar between the groups (Fig. 2e). The kidneys were bean-shaped, ~ 1.0 cm in length and weighted individually 0.50 ± 0.1 g and 0.46 ± 0.1 g (WT and TREK-1 KO groups, respectively). Histological images taken at the cortex level showed no visible histological changes in TREK-1 KO mice compared with WT aminals. In WT mice, most of the tubules in the kidney nephrons were positively labeled with TREK-1 antibody (Fig. 2c, left panel) with very faint or no positive reaction at the glomerular level. A substantial reduction in TREK-1 immunoreactivity was observed in the kidneys from TREK-1 KO animals (Fig. 2c, right panel).

We next evaluated the role of TREK-1 channels in the maintenance of the basal smooth muscle tone and spontaneous contractions in vitro. The weight of BSM strips isolated from WT and TREK-1 KO mice and used for contractile function studies was similar between the groups (8.0 ± 1.5 mg and 9.1 ± 2.1 mg, respectively, N = 17 and n = 34 in each group). The basal muscle tone of the strips at their optimal length (L0) was 50.7 ± 6.1 g/g in WT and 71.2 ± 8.5 g/g in TREK-1 KO mice (Fig. 3 a, N = 17; n = 34 in each group, p ≤ 0.01). Application of AA caused 24% reduction in basal tone of WT muscle strips (from 50.7 ± 6.1 g/g to 38.5 ± 4.6 g/g, p = 0.025) whereas the strips from TREK-1 KO bladders had a similar tendency in response to AA, however, did not reach a level of statistical significance (Fig. 3a). In a different set of experiments, BSM strips underwent 30% stretch in addition to L₀ in order to evaluate stress-relaxation of the detrusor when the bladder undergoes a significant stretch upon reaching its maximal capacity. After the stretch protocol, BSM strips from WT mice experienced an increase in the basal tone up to 81.5 ± 7.3 g/g (58.3% to L₀ level, p ≤ 0.05, Fig. 3b), whereas the strips from TREK-1 KO mice had an increase of 50% (from 71.2 ± 8.5 g/g to 107.2 ± 9.6 g/g, p ≤ 0.05 to L₀ level). Application of LM did not significantly affect the basal muscle tone of the stretched strips in either group, however, incubation with AA led to 32% decrease in WT group (N = 8, n = 8, p ≤ 0.05 to WT) without causing significant changes in TREK-1 KO group (N = 8, n = 9, Fig. 3 b). Analysis of spontaneous activity of BSM strips at L₀ revealed the presence of spontaneous contractions in TREK-1 KO group (2.2 ± 0.2 g/g, Fig. 3c, p ≤ 0.05 to WT) in comparison to lower contractile activity in WT group (1.3 ± 0.1 g/g, Fig. 3 c, an insert shows representative raw traces of the spontaneous activity in both groups). Additional stretch increased the amplitude of spontaneous contractions by 61% in WT group (Fig. 3d, p ≤ 0.05 to respective L₀ level) with the amplitude of spontaneous contractions in BSM strips being doubled in TREK-1 KO group (109% increase to respective L₀, Fig. 3d).

The differences observed in the basal muscle tone and frequency of spontaneous activity between WT and TREK-1 KO BSM strips were further tested in Ca²⁺ free TB to evaluate the role of active contractile processes due to Ca²⁺ influx into the cells. First, we incubated BSM from WT and TREK-1 KO animals in Ca-free solution for 30 min followed by either 30% additional stretch or incubation with nifedipine, a general L-type calcium channel blocker (2 μM, 30 min). Representitave recordings from WT and TREK-1 KO BSM strips are included in Fig. 4. Substitution of normal TB by Ca²⁺-free TB reduced the basal muscle tone in both WT and TREK-1 KO BMS strips when compared to normal Ca²⁺ solution (Fig. 4a, middle traces). However, BSM from TREK-1 KO mice showed a ~ 2.0 fold increase in the amplitude of spontaneous contractions (from 2.2 ± 0.1 g/g in normal Ca²⁺ to 4.1 ± 0.1 g/g in Ca²⁺-free TB, N = 4,n = 8 in each group, p ≤ 0.05 to normal Ca TB). Application of additional stretch under Ca²⁺ free conditions showed decreased values of PF in both WT and TREK-1 KO groups by ~ 30–32% when compared to normal Ca²⁺ solution. The contractile reponses also had a significantly faster initial decline phase of stress-relaxation in both groups (Fig. 4b).

Bladder smooth muscle strips from TREK-1 KO mice generated more contractile force in response to EFS (32 Hz) in comparison to WT group (222.2 ± 21.2 g/g and 162.2 ± 16.2 g/g, p ≤ 0.05, Fig. 5a, middle panel). The IF calculated as ratio of the area under the curve (AUC) of a single contraction divided by its PF was also significantly higher for TREK-1 KO group in comparison to WT (250 ± 30.1 vs 163.1 ± 19.6, p ≤ 0.05, Fig. 5a, right panel). Similar to the effects of EFS, TREK-1 KO strips also revealed an increased contractile response to high KCl stimulation (Fig. 5b). However, application of TREK-1 inhibitor (LM) and activator (AA) did not substantially change the contractile responses to high potassium in both WT and TREK-1 KO groups in comparison to baseline values (Fig. 5b).

Previous studies from our laboratory established that PKC activation with general agonist phorbol-12,13-dibutyrate (PDBu) significantly reduced the amplitude and increased the frequency of spontaneous BSM contractions at low concentrations (10 nM) while causing an increase in muscle force generation at higher concentrations (1 μM). Although several previous studies provided evidence that PKC mostly affects large conductance Ca²⁺-activated potassium channels (BK), TREK-1 channels could also be modulated by PKC phosphorylation. Therefore, we tested the reponses to a PKC activator in TREK-1 KO bladders. Application of PDBu (1 μM) significantly increased PF (by 50%, p ≤ 0.05, Fig. 6a) and the amplitude of spontaneous contractions in TREK-1 KO muscle strips when compared to WT group. Protein kinase C conveys both calcium-dependent, and calcium-independent effects on DSM contractility in vitro mediated via inhibition of myosine light chain phosphatase. However, it is still unknown if these separate effects are mediated by different PKC isoforms. Therefore, we aimed to test if elevated reponses to PKC stimulation in TREK-1 KO bladders may be due to compensatory changes in expression levels of the PKC isoforms present in the detrusor. The results of RT-PCR presented in Fig. 6b did not reveal any significant changes in the expression levels of 7 tested PKC isoforms including (PKC-α (alpha), PKC-β (beta), PKC-γ (gamma), PKC- δ (delta), PKC-ε (epsilon), PKC-μ (mu) and PKC-τ (tau)) between the WT and TREK-1 groups. This data suggests that the effects are likely due to the secondary regulatory changes in PKC–related signaling pathways.

The analysis of cystometrograms recorded under control conditions in WT (N = 5) and TREK-1 KO (N = 6) mice showed an approximately 2-fold increase in bladder capacity in TREK-1 KO animals in comparison to WT mice (210.0 ± 8.1 μl vs. 108.2 ± 8.0 μl, respectively.Figure 7 a, b and c. p ≤ 0.05), and a significant prolongation of the intermicturition interval (540.0 ± 43.8 s vs 1188.4 ± 166.4 s, WT and KO mice, respectively. Figure 7d. p ≤ 0.05). The substantially elevated volume of infused saline during storage phase, which is reflective of increased bladder capacity, with the prolonged duration of the voiding cycle point toward the development of an underactive phenotype in TREK-1 KO mice. However, although no differences were observed in bladder pressure at voiding (32.1 ± 3.2 mmHg vs. 39.0 ± 2.7 mmHg, TREK-1 KO, and WT mice, respectively), BMS from TREK-1 KO animals showed a 2-fold increase in the number of non-voiding contractions (TREK-1 KO: 4.5 ± 0.6 vs WT: 2.0 ± 0.5, p ≤ 0.05, Fig. 7e) per micturition cycle. Other urodynamic characteristics were not different between the WT and TREK-1 KO animals. Therefore, the overall TREK-1 KO bladder phenotype displays the characteristics of both bladder under- and overactivity.