Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle

Abstract

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512–530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys⁵²³ to Gln, Val⁵²⁴ to Leu, Ser⁵²⁶ to Thr, Pro⁵²⁷ to Cys, and Lys⁵²⁹ to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (−/−) animals did not develop, but heterozygous (+/−) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

caldesmon (cad), an actin/calmodulin-binding protein, was first isolated from chicken gizzard smooth muscle (52). The smooth muscle isoform of CaD (h-CaD) was found to be ubiquitous in all types of smooth muscles and associated with thin-filaments (16, 43), and the nonmuscle isoform (l-CaD) is associated with actin microfilaments (5, 6). The nonmuscle isoform of CaD has been shown to play a role in the regulation of the dynamics of podosome assembly, and with Ca²⁺/calmodulin, it has been suggested to be part of a regulatory mechanism in podosome formation in cultured aortic smooth muscle cells (14).

CaD binds to other smooth muscle contractile (13, 56, 57) and regulatory proteins (19, 25, 26, 29, 61, 63). Thus CaD has been thought to be an integral component of the thin filaments of the smooth muscle contractile apparatus (18, 47). It has been shown to have binding sites for myosin in the NH₂-terminal region (59) and actin (15, 52), calmodulin (60, 63), tropomyosin (25), and tubulin (35) in the COOH-terminal region.

Biochemical studies have shown that CaD inhibits actin-activated ATP hydrolysis by myosin (7, 24, 47) and that this inhibition is enhanced by tropomyosin (26, 46, 47, 53). The addition of CaD to actin filaments also has been shown to decrease the movement of actin filaments over the myosin heads in an in vitro motility assay (21, 27). The CaD-induced reduction in the proportion of motile filaments could be completely reversed by incorporation of an excess of calmodulin (CaM) at pCa 4.5 (16). The inhibition of the in vitro motility of actin filaments over myosin heads requires both the NH₂-terminal myosin-binding region and the COOH-terminal actin-binding region (59). Addition of synthetic peptides from the NH₂-terminal myosin-binding region of CaD to permeabilized vascular smooth muscle cells increases the basal tone but decreases the amplitude of subsequent phenylephrine-induced contractions (41). These results support a model whereby the tethering of actin to myosin by h-CaD may play a role in regulating tone by positioning the COOH-terminal domain of CaD so that it is capable of blocking the actomyosin interaction. Horiuchi and Chacko (27) showed that the inhibition of in vitro motility of actin over myosin heads is seen only at low levels of myosin light chain (MLC₂₀) phosphorylation, which is similar to the situation seen at the basal level. As MLC₂₀ phosphorylation increases, actin-activated ATP hydrolysis by myosin also increases, and at high ATP hydrolysis, the actin filament velocity of various types of myosin II has been shown to be proportional to actin-activated ATPase rates (22).

Wang and Chacko (57) made a series of COOH-terminal truncation and internal deletion mutants of chicken gizzard smooth muscle CaD using a site-directed mutagenesis approach and overexpressed these mutant proteins in a baculovirus expression system. The chicken CaD (NP_989489) COOH-terminal region that spans from amino acid (aa) 718–731 encompasses the core sequence of CaD-induced inhibition of actomyosin ATPase. However, the region required for maximal (60%) inhibition of CaD-induced inhibition of the actin-activated ATPase activity of myosin is located at the aa residues between 728–731(EKPA; Ref. 57), and this corresponds to aa residues between 522–525 (DKVT) in mouse CaD protein sequence (NP-663550). Using homologous recombination, we generated a transgenic mouse line with mutations in five aa residues (Lys⁵²³ to Gln, Val⁵²⁴ to Leu, Ser⁵²⁶ to Thr, Pro⁵²⁷ to Cys, and Lys⁵²⁹ to Ser) in the CaD functional domain in the COOH-terminal region, which encompasses the ATPase inhibitory determinants and the adjacent aa sequence located in exon 12. Homozygous animals failed to develop; however, heterozygous animals developed, matured, and reproduced normally. Awake cystometry of these CaD mutant mice revealed nonvoiding bladder contractions during the bladder filling phase. The detrusor smooth muscle (DSM) from the CaD mutant animals produced more force than from the wild type (WT). This is the first report to show in vivo organ dysfunction and a change in force generation due to mutations in the aa residues in the CaD functional domain of the COOH-terminal region. These results indicate the importance of CaD in the regulation of smooth muscle contraction.

MATERIALS AND METHODS

Cloning of the mouse CaD gene.

Mus musculus 6 BAC RP23–104H13 (C57BL/6J Female; GenBank: AC153627.2) was obtained from the Roswell Park Cancer Institute (Buffalo, NY). Restriction endonucleases, T4 DNA ligases, reagents, enzymes, and dNTPs were purchased from New England BioLabs. Enzymes and PCR master mixes were purchased from Invitrogen. A continuous sequence containing the left homology original ATPase inhibitory site (in a 1.81-kb target region) and the right arm was obtained from a mouse bacterial artificial chromosome (BAC) clone containing the CaD gene. The length of the homology arms was designed based on previous reports (17, 23, 34). The left (2.88 kb) arm, ATPase inhibitory site region, and right (2.46 kb) arm were amplified from BAC DNA by PCR.

Strategy for mutation.

Our strategy was to modify CaD functional domain, which is responsible for the highest percentage of CaD-induced inhibition of actin-activated ATP hydrolysis. Our earlier studies using CaD with internal deletions expressed in the baculovirus expression system (61, 63) showed two inhibitory determinants between aa residues 718–723 and 728–731 that are separated by four aa residues from Lys⁷²⁴ to Val⁷²⁷. The inhibitory determinant(s) of ATPase inhibitory site of CaD in mouse has not been reported. Thus, based on the aa sequence/position corresponding to chicken, we calculated the eight-aa region of mouse as the ATPase inhibitory site to be mutated. The chick CaD aa sequence from 728 to 731 (EKPA) was required for the maximal inhibition of ATPase and was independent of the actin binding. The sequence in mouse, corresponding to chick (EKPA), is DKVT (522–525). We mutated the aa from this region (523–529), which encompass the ATPase inhibitory site and the aas in the adjacent COOH-terminal region. The sequence in mouse, corresponding to chick is as follows: Chi: KPSDLRPGDVSGKRNLWEKQSVEKPAASSSKVTATGKKSETNGLRQFEKEP; and Mou: KPSDLRPGDVSGKRNLWEKQSVDKVTSPTKV.

The desired bases were introduced to replace the original bases by overlap PCR. Briefly, a PCR primer was designed to bear desired base(s). Then, the desired sequence of double stranded DNA was obtained after two additional PCR procedures. Most of the mutations were made to change the charge in the aa in the region following the procedure according to manufacturer's protocol (Mutagenesis Application Guide, Integrated DNA Technologies, Coralville, IA).

Generation of targeted embryonic stem cells.

The targeting vector was generated with a portion of the CaD gene (C57BL/6J female) spanning a 7.2-kb fragment from intron 11, exon 12, and intron 12 of the CaD gene. The DNA fragment that codes for the aa ⁵²³Lys-⁵²⁴Val-⁵²⁵Thr-⁵²⁶Ser-⁵²⁷Pro-⁵²⁸Thr-⁵²⁹Lys in exon 12 was changed to encode ⁵²³Gln-⁵²⁴Leu-⁵²⁵Thr-⁵²⁶Thr-⁵²⁷Cys-⁵²⁸Thr-⁵²⁹Ser by overlap PCR (3) and sequenced (51) to confirm the mutation, and then subcloned back into the targeting vector. A neocassette and a red fluorescent protein gene were introduced downstream of exon 12 of the CaD gene. The thymidine kinase (TK) gene was added to the 3′-end of the targeting vector.

Probes for Southern blotting.

The 5′ (probe 1, 121 bp)- and 3′ (probe 2, 133 bp)-probes were labeled with NEBlot Kit (New England BioLabs), as per the manufacturer's instructions. Briefly, a 25-ng template DNA in 20 μl nuclease free H₂O was denatured in a boiling H₂O bath for 5 min and immediately placed on ice for 5 min. The template DNA was amplified from the BAC DNA by PCR. After briefly spinning down at 4°C, reagents were added to the template DNA in the following order: 5 μl octadeoxyribonucleotides in 10× labeling buffer, 6 μl dNTP mixture (2 μl of dATP, dTTP, and dGTP), 5 μl dCTP (α-³²P; 3,000 Ci/mmol; 50 μCi), and 1 μl DNA polymerase I-Klenow Fragment (five units). The mixture was incubated at 37°C for 1 h, and the reaction was terminated by addition of 5 μl 0.2 M EDTA (pH 8.0). The probes in the reaction mixture were purified by passing through a Sephadex G-50 column.

The conventional Southern blotting procedure was applied to confirm recombination in embryonic stem (ES) cells. Briefly, genomic DNA from ES cells was digested with appropriate restriction enzymes (Xho1 and AatII) and separated by agarose gels. The DNA in the agarose gel was transferred to a nylon membrane; the membrane was baked at 80°C for 2 h. After an 8-h prehybridization, the membrane was incubated with a labeled probe that was a freshly denatured single-stranded DNA. Membranes were washed with specific buffers and scanned using a Typhoon Trio (GE Healthcare).

Generation of knockin transgenic mice.

The targeting vector (16.4 kb) was linearized using Not1 and electroporated into C57BL/6 ES cells by the Transgenic and Chimeric Mouse Facility of the University of Pennsylvania. Transfected ES cell clones were screened for the presence of red fluorescence protein. Two correctly targeted clones were identified by PCR and confirmed by Southern blot analysis of XhoI and AtaII-digested genomic DNA with probe 1. The 5′-probe (121 bp) was labeled using a NEBlot Kit and purified by a Sephadex G-50 column, as per the manufacturer's protocol. Southern blot analysis was performed as previously described (50) to identify the correctly targeted clones. Toward this goal, genomic DNA from ES cells was separated on an agarose gel after digestion with the indicated restriction enzymes and transferred to a nylon membrane. The membrane was hybridized to a labeled single-stranded DNA probe followed by a series of washing steps with specific buffers. The signals in the membrane were scanned by a Typhoon Trio, and blots were stripped and used for hybridization with 3′-probe following the same procedure. The presence of aa mutations in the ATPase inhibitory region of the CaD gene in the ES clones was confirmed by PCR. The correctly targeted ES cells were microinjected into embryonic day 3.5 blastocysts (C57BL/6 strain; Transgenic and Chimeric Mouse Facility at the University of Pennsylvania) and the embryos were surgically implanted into pseudo-pregnant foster mice by standard procedures. Research involving mice complied with all relevant federal and institutional policies as well as guidelines established by the Institutional Animal Care and Use Committee at the University of Pennsylvania, School of Medicine.

Genotyping of the knockin transgenic mice.

There are eight aa residues in the ATPase inhibitory domain of CaD as follows: WT: - - -CTGTG-GAT-AAG-GTC-ACT-TCC-CCC-ACT-AAG-GTACT- - -and- - - -Val-Asp–Lys—Val—Thr—Ser-Pro—Thr—Lys-Val-(intron)- - -; mutant: - - -CTG-TG G-AT-CAG-CTC-ACT-ACA-TGC-ACG-TCT-GTACT- - - and - - -Val-Asp–Gln—Leu–Thr—Thr–Cys–Thr—Ser-Val-(intron)- - - -.

Five of eight aa were changed in the mutant mice. We designed the primers specific for WT and mutant CaD DNA. The primer for WT CaD was expected to amplify WT and heterozygous CaD mutants but not the homozygous mutant. The mutant primer was expected to amplify the heterozygous and homozygous CaD mutants but not the WT sequence. The genomic DNA isolated from WT and mice with knockin mutations of CaD ATPase inhibitory domain was amplified by PCR using the WT- and mutant-specific sense and appropriate antisense primers. The primer sequences used for the PCR are as follows: WT: forward 5′-TCTGTGGATAAGGTCACTT-3′ and reverse 5′-AAACCTGCAGTGTCAA-3′; and mutant: forward 5′-TCTGTGGATCAGCTCACTA 3′ and reverse 5′-CAAACCTGCAGTGTCAA-3′.

The PCR products were analyzed on 2% agarose gel electrophoresis. The mutations were confirmed by DNA sequencing using the purified PCR products (51).

Protein extraction and Western blot analysis.

Smooth muscle tissue devoid of mucosa and serosa from WT and CaD mutant murine bladder was frozen in liquid nitrogen and pulverized into a fine powder and protein was extracted using 1 ml extraction buffer, which contained 50 mM Tris (pH 6.8), 20% glycerol, 1% SDS, and a protease inhibitor cocktail (Sigma, St. Louis, MO). For CaD phosphorylation studies the carbachol (10 μM) stimulated smooth muscle from WT and CaD mutant murine bladder was rapidly frozen in liquid nitrogen and then homogenized using 1 ml extraction buffer, which contained 50 mM Tris (pH 6.8), 20% glycerol, 1% SDS, phosphatase inhibitor, and a protease inhibitor cocktail (Sigma). Equal amounts of protein (50 μg) samples were separated on SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membrane was subjected to immunoblot analysis using CaD (dilution 1:3000; Sigma) and phosphoserine⁷⁸⁹ CaD (dilution 1:1,000; Sigma) antibodies, and the immunoreactive proteins were visualized as described before (49). Equal loading between lanes was confirmed by probing the membranes with anti-GAPDH antibody (dilution 1:3,000; Abcam, Cambridge, MA).

Cystometry.

WT and heterozygous male mice were used for cystometry. Mice were placed in an anesthesia induction chamber and anesthetized using isofluorane inhalation (1.5–3% isofluorane with 2 l/min of oxygen). After they were anesthetized, mice were transferred to a warmed table with a nose cone for isoflorane delivery. Following betadine and alcohol skin preparation, a vertical midline incision was made. The peritoneum was entered and a subcutaneous tunnel was created back towards the animal's neck. Through this tunnel, a 3–0 French feeding tube was passed from the neck into the peritoneal cavity. At this point, a catheter end was cut and a 1-mm flange was glued in place to serve as a cuff to hold the catheter within the bladder. The bladder was grasped with ring forceps, and a 7–0 prolene purse string suture was placed at the dome of the bladder. In the middle of this purse string, a small cystotomy was made using iris scissors. The feeding tube with its flange was passed into the bladder, and the purse string suture was tied down so as to allow for a water tight seal that was tested by gentle infusion of saline into the bladder. The abdomen was closed in layers, and the incision at the neck was also closed. The catheter was anchored in position by a 5–0 prolene suture placed at the neck. The mouse was returned to its cage to recover.

After the catheters were in place for 3 days, mice were placed in a cystometry chamber free of any restraint. The bladder catheter was hooked up to the extension tubing attached to an infusion pump and pressure manometer. Mice were allowed to roam freely throughout the chamber. Warm saline was infused (10 μl/min) and pressures were recorded when mice voided. The cystometry chamber emptied out onto a digital scale that was used to record the voided volume of each micturition. The voiding cycle was repeated in this manner until a stable pattern was reached. Usually, this was seen within five cycles over a maximum of a 120-min examination period. Mice were euthanized at the end of these experiments.

Force measurements.

Longitudinal muscle strips (2 × 5 mm) devoid of mucosa and serosa were obtained from WT and CaD mutant mice and suspended in 7 ml Tyrode's buffer (124.9 mM NaCl, 2.5 mM KCl, 23.8 mM NaHCO₃, 0.5 mM MgCl₂, 0.4 mM NaH₂PO₄, 1.8 mM CaCl₂, and 5.5 mM dextrose) in an organ bath (Radnoti) to equilibrate for 1 h in 95% O_2, 5% CO₂ and at 37°C. The force generated in response to KCl (80 mM) and electrical field stimulation (EFS; 40 V, 32 Hz for 1 ms) was measured using the AD Instruments Power Lab computerized system and Lab charts analysis program 7.0 (AD Instruments), as described previously (31, 32, 33). Force measurements were normalized to the wet weight of the muscle strips.

Analysis of MLC₂₀ phosphorylation.

MLC phosphorylation in muscle strips was measured as described previously (32). Briefly, strips were frozen rapidly at rest (with no stimulation) or at peak force in response to KCl stimulation by clamp freezing in liquid N₂ followed by immersion in dry ice-acetone slurry and stored in liquid nitrogen. Frozen muscle strips were pulverized while immersed in liquid nitrogen, and the resulting powder was added to a mixture of dry ice-acetone. The acetone was removed by centrifugation and the pellet was mixed with isoelectric focusing (IEF) sample buffer containing 9.5 M urea, 1.6% ampholyte (pH 5–7), 0.4% ampholyte (pH 3–10), 2% NP-40, and 5% β-mercaptoethanol, and homogenized with a mini electric homogenizer. After centrifugation, the supernatant was applied to IEF gels and IEF was carried out at 350 V overnight and gels were subjected to a 14% SDS-PAGE and stained with Coomassie blue. Identification and measurement of MLC₂₀ phosphorylation using two-dimensional (2D) gel electrophoresis were performed as described previously (32). Briefly, the identity of these spots in a 2D PAGE was confirmed by Western blotting with an anti-MLC₂₀ monoclonal antibody and using pure ³²P-phosphorylated myosin. When ³²P-phosphorylated myosin was added to the frozen powder, ∼95% was recovered in the phosphorylated MLC₂₀ spots. Spots corresponding to phosphorylated and unphosphorylated MLC₂₀ in the 2D PAGE were scanned and analyzed with a Bio-Rad GS-700 imaging densitometer and 2D-PAGE Molecular Analyst Software (Bio-Rad, Hercules, CA).

Histology.

Bladders were fixed in 10% phosphate buffered formalin and 5-μ thick paraffin sections were prepared. Sections were stained with Masson's trichrome (51a). Photographs were taken under a photomicroscope (Nikon).

Statistical analysis.

Where appropriate, comparisons between experimental groups were performed using Student's t-test. One-way ANOVA was performed using Prism Graph Pad software for multiple sample comparisons. P < 0.05 was considered statistically significant.

RESULTS

Generation of ES cells for CaD ATPase inhibitory site mutation.

To identify the role of the ATPase inhibitory site in the COOH-terminal region of CaD in force generation, a gene knockin strategy was used to mutate the aa from the functional domain that are required for the CaD-induced inhibition of actin-activated ATPase activity of myosin. The ATPase inhibitory site located in exon 12 of the CaD gene -CTGTG-GAT-AAG-GTC-ACT-TCC-CCC-ACT-AAG-GTACT- was mutated to -CTG-TG G-AT-CAG-CTC-ACT-ACA-TGC-ACG-TCT-GTACT- by overlap PCR (3). This corresponds to the aa ⁵²¹Val-⁵²²Asp-⁵²³Lys-⁵²⁴Val-⁵²⁵Thr-⁵²⁶Ser-⁵²⁷Pro-⁵²⁸Thr-⁵²⁹Lys-⁵³⁰Val-(intron)- in the WT and ⁵²¹Val-⁵²²Asp-⁵²³Gln-⁵²⁴Leu-⁵²⁵Thr-⁵²⁶Thr-⁵²⁷Cys-⁵²⁸Thr-⁵²⁹Ser-⁵³⁰Val-(intron)- in the mutant (Fig. 1A). The strategy for the homologous recombination is shown in Fig. 1A. Two sets of probes, 5′ (probe 1) and 3′ (probe 2) to the mutational region, were designed for Southern analysis to detect the recombinant DNA fragment (Fig. 1B).

Fig. 1.

Cloning of a mutant ATPase inhibitory region of the murine caldesmon (CaD) gene and construction of a knockin targeting vector. A: left arm (2.8 kb) of the ATPase inhibitory domain of CaD (1.81 kb) and right (2.5 kb) arm were amplified from BAC DNA by PCR. The PCR amplified arms were cloned to left and right regions of the pPNT vector. Exon 12 of the CaD gene, which codes for the ATPase inhibitory domain, was chosen for the site-directed mutagenesis. The wild-type (WT) CaD ATPase inhibitory site GAT-AAG-GTC-ACT-TCC-CCC-ACT-AAG was changed to GAT-CAG-CTC-ACT-ACA-TGC-ACG-TCT, and the DNA fragment with a mutated site on the CaD gene was cloned downstream of the left arm in the targeting vector. Mutations were designed to change 5 aa residues (Lys⁵²³ to Gln, Val⁵²⁴ to Leu, Ser⁵²⁶ to Thr, Pro⁵²⁷ to Cys, and Lys⁵²⁹ to Ser) in the CaD ATPase inhibitory domain as described in materials and methods. A red fluorescent protein gene was introduced downstream of the Neo gene in the targeting vector to permit the selection of embryonic stem (ES) cells. The homologous recombination made between the targeting vector and the endogenous WT CaD gene to generate a mutated CaD gene is shown by dotted lines. The negative selection marker thymidine kinase (TK) gene was introduced into the targeting vector for negative selection of a random integrant. B: positions and the sequences of 5′ (probe 1)- and 3′ (probe 2)-probes for Southern blot analysis to detect the recombinant are shown.

Transfection of ES cells, analysis of DNA from ES cell clones, and generation of knockin mice.

After the linearized targeting construct was transfected into mouse ES cells, the cells were selected by neomycin (non-Neo gene-containing cells were killed). DNA isolated from ES cell clones was subjected to PCR amplification with the following primers: forward primer-ES-F2: 5′-GATAGGGTTGAGTGTTGTTCC; and reverse primer-ES-R2: 5′-AGATCAGTGGTAGACTAGTG.

PCR was also performed using another set of primers located in the Red Fluorescent Protein expression box of the knockin vector (forward primer-Red3: 5′-TCACGGTCGACGCAGCTTATAATGGTTACAAAT; and reverse primer-Red4: 5′-GCAATGGTACCCCCCGAAAAGTGGCACCT). The results from the PCR (Fig. 2A) and fluorescence microscopy (data not shown) analysis revealed that the successfully targeted ES cells express the red fluorescent protein.

Fig. 2.

Identification of correctly targeted ES cells. The targeting vector was electroporated into ES cells and the indicated ES cell clones were used for genomic DNA preparation. A and B: genomic DNA samples isolated from ES cells were amplified using the primers described in materials and methods by PCR and analyzed on a 1% agarose gel. C: genomic DNA isolated from the selected ES clones (this was identified as positive for the recombinant by PCR) was digested with the indicated restriction enzymes. Digested genomic DNA fragments were separated on a 0.7% agarose gel and transferred to a nylon membrane for Southern blot analysis. After 8 h of prehybridization, the membrane was incubated with a labeled 3′-probe (probe 2, which is a freshly denatured single-stranded DNA). After a series of washes using specific buffers, the membrane was scanned by a variable mode fluorescence imager (Typhoon Trio, GE Healthcare). Predicted sizes for WT after restriction digestion with AatII and Xho1 are shown. Scanned Sothern blotting picture is from the recombined genome, which gives a fragment of ∼25 kb after AatII. D: genomic DNA prepared from a successfully identified ES cell (clone B6) was amplified with forward and reverse primers by PCR and the PCR product was separated on a 1.2% agarose gel. The gel purified PCR product was sequenced using forward and reverse primers according to Sanger et al. (51), using an ABI automated sequencer.

Successfully targeted ES cells were also screened using primers that are inside and outside of the targeting allele. ES cells that were devoid of the targeted allele failed to amplify the DNA (Fig. 2, A and B). The presence of mutations in the targeted allele in selected ES cell clones was confirmed by Southern blotting using 5′- and 3′-probes (Fig. 2C) and confirmed by sequencing (Fig. 2D). Successfully targeted ES cells clones A3 and B6 were selected for microinjection to generate the transgenic mice. Chimeras obtained after injection of these cells to blastocysts were back-crossed with C57BL/6J-Tyrc-2J (Stock No. 000058) mice. The F1 black mice were bred with C57BL/6 mice, and the pups obtained were genotyped to identify the mice with the mutated ATPase inhibitory site by genomic PCR using WT- and mutant-specific forward and reverse primers. The mutation in the ATPase inhibitory site of CaD in the transgenic mice was confirmed by sequencing. Offspring derived from the clone B6 were found to generate more CaD mutants. Via genotyping, offspring were found to be only heterozygous and WT. Therefore, homozygosity of the CaD ATPase inhibitory domain mutation appeared to be lethal, and examination of the uterine endometrium in early stages of pregnancy revealed areas with resorption of undeveloped early stage embryos.

Identification of heterozygous animals with a mutation of the CaD functional domain containing the actomyosin ATPase inhibitory site.

Genomic DNA was isolated from tail samples of the F2 litter obtained from inbreeding F1 mice. There are eight aa residues in the ATPase inhibitory domain of CaD. Five of eight aa were altered in the mutant mice. We designed primers specific for WT and mutant CaD DNA. The strategy for PCR analysis is shown in Fig. 3A. In this strategy, the WT primer amplifies the WT and heterozygous (+/−) CaD gene, whereas the mutant primer amplifies the heterozygous (+/−) and homozygous (−/−) CaD gene. Figure 3, B and C, depicts the PCR products from a typical genotype analysis. All of the samples expressing the mutant PCR product also expressed the WT PCR product, indicating the absence of homozygous offspring. The presence of mutations was confirmed by sequencing the PCR product, as shown in Fig. 4. The CaD mutant revealed the sequence for CaD mutation and WT, whereas the WT revealed only the WT sequence.

Fig. 3.

Genotyping transgenic mice expressing the CaD mutant ATPase inhibitory domain. A: schematic demonstrates the screening strategy for identifying CaD mutant transgenic mice. B and C: genomic DNA isolated from WT and transgenic mice was PCR amplified using WT- and mutant-specific forward and reverse primers and the PCR product was separated on a 1.2% agarose gel.

Fig. 4.

DNA sequence analysis. PCR-amplified DNA from WT and transgenic mice (CaD mutant) was gel purified and sequenced according to Sanger et al. (51), using an ABI automated sequencer. The WT revealed WT sequence whereas the CaD mutant (+/−) showed both WT and the mutant sequence.

Effects of mutation of the CaD ATPase inhibitory site on the morphology and the expression of CaD in the bladder wall.

The bladder-to-body weight ratio of CaD mutant mice is significantly higher than that of WT mice (Fig. 5A). A hypertrophy of the bladder wall is also evident on histologic examination (Fig. 5B). The smooth muscle bundles in bladder sections from CaD mutant mice appear to be larger compared with that of WT mice. The level of expression of h-CaD in the DSM of the CaD mutant and WT is not significantly different (Fig. 5, C and D). Since we mutated Ser⁵²⁶ site (in mouse CaD), a site that is phosphorylated by extracellular signal-regulated kinase 1/2 (ERK 1/2) (9), we stimulated the DSM from CaD mutant and WT mice with carbachol (10 μM). The muscle was freeze clamped at the peak force and the amount of CaD Ser⁵²⁶ in the protein extracts was compared in CaD mutant and WT by Western blotting using the human CaD phosphoSer⁷⁸⁹ antibody. The expression levels of total and phospho h-and l-CaD in WT and CaD mutant are shown in Fig. 5, C–F. As shown in Fig. 5D, the relative amount of total h-CaD compared with GAPDH is similar in WT and CaD mutant. However, the relative amount of phospho h-CaD compared with total h-CaD (Fig. 5E) and the relative amount of phospho h-CaD compared with GAPDH (Fig. 5F) were less in CaD mutant (CaD mutant does not contain this phosphorylation site) compared with WT.

Fig. 5.

Morphology and CaD protein expression in WT and CaD mutant mice bladders. Bladder to body weights from CaD (Bl.W/B.W) mutant mice were significantly higher compared with WTs (A). Quantitative data are means ± SE from 8 different animals from each group, and asterisks indicate P < 0.05. Masson's Trichrome stained histological sections reveal detrusor smooth muscle (DSM) layers in the bladders of WT and CaD mutant mice (B). Muscle layer and the bundles are thicker in CaD mutant mice. Scale bar in B = 50 μm. C: immunoblot analyses of total and phospho-caldesmon protein (h- and l-CaD) from WT and CaD mutant DSM tissue. GAPDH was probed as a loading control. D: CaD protein expression relative to GAPDH. E: Relative amount of phosphorylated h-CaD relative to total h-CaD. F: phosphorylated h-CaD relative to GAPDH in CaD mutant vs. WT. Total CaD protein expression was unaffected (C and D). Notice that the phosphorylated CaD is less in mutant CaD, which is devoid of the ERK1/2 phosphorylation site at Ser⁵²⁶. Quantitative data are means ± SE of 5 experiments using 5 different animals from each group. **P < 0.05.

Effects of mutation of the CaD ATPase inhibitory site on in vivo cystometry.

In vivo cystometry was performed on both WT and CaD mutant (+/−) mice under an unanesthetized condition. Although the WT showed a relatively quiet tracing during bladder filling and micturition contractions accompanied by void volumes, CaD mutant mice reveal several nonvoiding contractions during the filling phase of the bladder with no accompanying volume emptied before voiding contractions that resulted in bladder emptying (Fig. 6).

Fig. 6.

In vivo cystometry of WT and CaD mutant bladders. Representative traces of the cystometrograms recorded in awake and freely moving animals are shown. In vivo cystometry was performed for 3 micturition cycles in both WT and CaD mutant mice. Saline was infused at a rate of 10 μl/min using a 19.13 mm syringe for both WT and mutant mice. Top: tracing for pressure. Bottom: tracing for the volume emptied. WT shows a relatively quiet tracing with micturition contractions accompanied by 3 void volumes. The average number of nonvoiding contractions (NVCs) for WT and CaD mutant mice was 2.2 ± 0.2 and 5.4 ± 0.51 per 3 micturition cycles, respectively (n = 5 P < 0.05). Arrows indicate several NVCs in the mutant mouse with no accompanying volume emptied.

Effects of mutation of the CaD ATPase inhibitory site on DSM force generation.

Force profiles of DSM strips in response to KCl and FS from WT and CaD mutants are shown in Fig. 7. The peak forces developed in response to EFS (Fig. 7A) and KCl (Fig. 7B) were higher in CaD mutants (Fig. 7, A and B, bottom) than WTs (Fig. 7, A and B, top). Field stimulation and KCl caused 57 ± 16 and 64 ± 17.3% increase in contractile force, respectively, for CaD mutants compared with WTs (Fig. 7C). The increased force was produced without a significant difference in the level of MLC₂₀ phosphorylation (Fig. 7D). Tetrodotoxin caused an ∼97% decrease in the response to EFS compared with the untreated muscle strip indicating that this effect is substantially due to intramural nerve activation in the DSM (Fig. 8).

Fig. 7.

Force production of DSM strips from CaD mutant mice compared with that of WT mice in response to stimulation as shown. A and B: force profiles of muscle strips in response to electrical field stimulation (FS) and KCl are shown for WT (top) and CaD mutant (bottom) mice. Summary information for the maximal contractile response of DSM from WT and CaD mutant mice is shown in C. EFS (40 V, 32 Hz, 1 ms) and KCl (80 mM) caused respectively 57 ± 16 and 64 ± 17% increased force in CaD mutants vs. WT. Quantitative data are mean ± SE of 5 experiments using 5 different animals from each group; *P < 0.05. D: protein extract prepared from muscle strips freeze-clamped at the peak of maximal force in response to KCl was analyzed by 2-dimensional gel (2D) electrophoresis. Quantization of the bands from 2D gels showed similar low levels of myosin light chain (MLC₂₀) phosphorylation in CaD mutants and WT at peak KCl-induced contraction. Quantitative data are means ± SE of 5 experiments using 5 different animals from each group.

Fig. 8.

Effect of tetrodotoxin (TTX; 1 uM) on electrical FS-induced contraction of DSM from CaD mutant. A: control in the absence of TTX. B: effect of 30-min preincubation with 1 uM TTX. Data from 5 experiments using detrusor muscle strips from 5 mutant mice is depicted in C. TTX caused an ∼97% decrease in the response to EFS compared with the untreated muscle strip indicating that this effect is substantially due to intramural nerve activation in the DSM. *P < 0.05, significantly different from control; n = 5 separate animals.

DISCUSSION

It is well known that thick filament (myosin)-mediated regulation via MLC₂₀ phosphorylation by a Ca²⁺-dependent MLC kinase (MLCK) activates actomyosin ATPase and force generation in smooth muscle (2, 37). However, there is also evidence for thin filament-mediated regulation, which is probably mediated by CaD and which complements myosin/thick filament-mediated regulation via MLC₂₀ phosphorylation by Ca²⁺/CaM-dependent MLCK. Thin filament-associated CaD has been postulated as a second regulatory system (4, 45, 54, 56). The support for CaD-mediated thin filament regulation comes from in vitro biochemical studies on isolated proteins (24, 28, 40, 52, 59) and from physiological studies using permeabilized muscle fibers (39, 44, 55). The exact role of CaD in the regulation of contraction can be better understood using smooth muscle that lacks whole CaD or a selected functional domain of CaD. Ablation of smooth muscle CaD (h-CaD) in a previous study showed overexpression of nonmuscle CaD (l-CaD), and while the Ca²⁺ sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different (20).

In a recent study, transgenic zebra fish that express inhibitory peptides derived from the h-CaD, myosin, and actin-binding domains and their effect on peristalsis in WT zebra fish larvae and sox10 mutant larvae that lack enteric nerves was examined (1). This study showed that the disruption of the normal inhibitory function of h-CaD enhances intestinal peristalsis in both WT zebrafish larvae and mutant larvae that lack enteric nerves, thus giving evidence for a physiologic role for CaD-mediated regulation of smooth muscle contraction at the actin filament.

In our earlier study (60), we used a site-directed mutagenesis approach to generate internal deletion mutants of chicken gizzard smooth muscle CaD, in which 4–18 aa residues in the region from Lys⁷¹⁸ to Ser⁷³⁵ were progressively nest deleted and these mutants were used to identify the core sequence necessary for inhibition of actomyosin ATPase activity using reconstituted actomyosin system. These results demonstrated that there are two inhibitory determinants between residues 718–723 and 728–731 that are separated by four aa residues from Lys⁷²⁴ to Val⁷²⁷.

In this study, we generated a transgenic mouse with a mutation in a CaD COOH-terminal functional domain that is involved in the CaD-induced inhibition of actin-activated ATPase activity. The effect of this mutation on force generation by intact smooth muscle fibers and the in vivo bladder function was analyzed. The presence of the transfected fragment in the ES cell clone used for creating the transgenic mouse was identified by PCR analysis and confirmed by Southern blot analysis and sequencing of the DNA extracted from the clone. Genotyping of the mice showed the presence of a mutation in the CaD ATPase inhibitory site. An important finding from genotypic analysis of the offspring is the absence of pups homozygous for this mutation, indicating that homozygosity for this mutation is lethal. This is the first evidence of the need for CaD functional domains for the development of the early embryo. Lack of homozygous mice with the knockin constructs indirectly shows that the CaD COOH-terminal functional domain is critical for embryonic development. This is not surprising, considering the importance of CaD in actin filament assembly in test tube experiments (8) and the lack of actin-containing microfilaments and intermediate size filaments with down regulation of CaD expression via CaD small interfering RNA (10). Cytoplasmic intermediate size filaments form a dense filament network radiating from the nucleus and extending to the plasma membrane (11). Stable expression of a CaD mutant in Chinese hamster ovary cells has been shown to disrupt assembly of stress fibers and focal adhesions and contains altered cell morphology and slowed cell cycle progression suggesting a role for CaD regulation by Ca²⁺/CaM in cell migration (42). CaD also induces filament formation of dephosphorylated myosin in the presence of Mg²⁺-ATP, and this may maintain the stability of myosin filaments in relaxed smooth muscle (38).

An increased bladder-to-body weight ratio for the CaD mutant and increased smooth muscle bundle size in the bladder wall in the CaD mutant compared with the WT indicate hypertrophy of the bladder wall smooth muscle. The level of expression of CaD protein in the DSM of a CaD mutant is not different than the expression in a WT mouse. Basal smooth muscle tone is important in viscous organs, such as the urinary bladder, to prevent the bladder from overdistension upon filling with urine. One of the important phenotypes of the heterozygous animal with mutation of 50% of the CaD ATPase inhibitory site is the increase in phasic contraction of the DSM during the bladder filling phase, causing detrusor overactivity. This is common in bladder dysfunctions including bladder outlet obstruction, which is associated with an overactive bladder, a clinical condition for which there is no satisfactory therapy.

Another physiologic difference observed in the DSM from CaD mutants (compared to WTs) is the increased force produced in response to EFS and KCl. This increased force in a CaD mutant is not associated with an increase in MLC₂₀ phosphorylation, which is correlated with cross-bridge cycling rate and force production (36).

CaD has been shown to contain two high-affinity CaM binding sites in its COOH-terminal region that bind calcium and CaM and (30, 60, 62, 63). The CaM-binding site is expected to be intact and function normally in our CaD mutant mouse. CaD-induced inhibition of force in permeabilized smooth muscle is reversed by CaM in the presence of Ca²⁺ (55) and by the motility of actin over myosin heads in the in vitro motility assay (16).

Based on studies using organ-cultured arterial smooth muscle made deficient in CaD by antisense oligodeoxynucleotides, Earley et al. (12) suggested that in resting vascular smooth muscle, active cross bridges are inhibited by CaD and that the regulation of smooth muscle tone involves a thin filament-mediated disinhibition component. We believe the results from the present study may represent the first direct piece of evidence to support the importance of CaD in the maintenance of the quiescent state of the DSM tone and of its role in complementing myosin-mediated calcium regulation in force development. Our results suggest that CaD induces the inhibition of force development at the resting level during the bladder filling phase, when the Ca²⁺/CaM-mediated MLCK-based phosphorylation level is low. CaD has functional domains that bind to several smooth muscle proteins involved in force development. In many cases, there are multiple binding sites for muscle and regulatory proteins, but their binding affinity to CaD varies (57). Mutating a specific site in the functional domain of CaD allowed us to generate a knockin mouse model with alteration of five aa from the functional domain, which is involved in the CaD-mediated inhibition of actin-activated ATP hydrolysis by myosin. Interestingly, in the entire CaD molecule, the ATPase inhibitory site is adjacent to the determinants that are important for tropmyosin binding to CaD and tropomyosin-mediated enhancement of the CaD-mediated inhibition of actin-activated ATP hydrolysis by myosin (57, 58).

Our previous studies (60) using deletion mutants of avian CaD suggests that a 10-aa stretch from Lys⁷¹⁸ to Val⁷²⁷ covers the strong tropomyosin-actin-binding motif in the COOH-terminal end of CaD. Analysis of actin binding and inhibition of actomyosin ATPase activity using deletion mutants of avian CaD identified a strong actin-binding motif of six aa residues (from Lys⁷¹⁸ to Glu⁷²³), which also form the core sequence for CaD-induced inhibition of actomyosin ATPase. However, maximal inhibition by CaD requires the presence of residues 728–731, which are not associated with actin binding. Although our goal was to affect the ATPase inhibitory site, we realized that the tropomyosin-actin binding site may also be affected since the ATPase inhibitory site is adjacent to the tropomyosin-binding domain as we indicated in our previous publication (60). In addition, we mutated S⁵²⁶ site (in mouse CaD) since this site is one of the aa in the region that was important for maximal inhibition of actin-activated ATPase by CaD. The S⁵²⁶ is also the ERK1/2 phosphorylation site in mammals (9), and our data indicate that the S⁵²⁶ phosphorylation is diminished in the CaD mutant compared with that of WT. Phosphorylation of h-CaD by ERK1/2 has been implicated in the myometrium in the development of phasic contractions during labor. Their phospho-regulation is dynamic, in that h-CaD and ERK1/2 are phosphorylated and dephosphorylated in phase with contraction and relaxation respectively (48). Future studies using a mouse model with mutation of specific phosphorylation sites on CaD should address whether these point mutations affect the CaD-induced inhibition of actomyosin ATPase activity and force.

In conclusion, the result from this study provides the first direct evidence for CaD-mediated thin filament regulation in smooth muscle in vitro using intact smooth muscle and in vivo based on data from cystometry. CaD inhibits cross-bridge cycling and force generation at the resting level, and a mutation of the ATPase inhibitory site on CaD causes increased phasic contractions at the basal level in the filling phase of the bladder. Mutational changes in the CaD ATPase inhibitory domain appear to remove the CaD-induced repression of actomyosin ATPase and force. The CaD mutant model would help to extend further studies to determine whether CaD in association with CaM may confer calcium sensitivity of smooth muscles.

GRANTS

This study was supported by a grant from Pfizer-AUA and National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-069898 and P50-DK-052620.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: M.D. and S. Chacko conception and design of research; M.D., E.B., J.A.H., T.R., S. Chang, and S.A.Z. performed experiments; M.D., E.B., J.A.H., S. Chang, S.A.Z., and S. Chacko analyzed data; M.D., E.B., and S. Chacko drafted manuscript; M.D., E.B., and S. Chacko approved final version of manuscript; E.B., J.A.H., T.R., S. Chang, S.A.Z., A.J.W., and S. Chacko interpreted results of experiments; E.B. and J.A.H. prepared figures; E.B., A.J.W., and S. Chacko edited and revised manuscript.