In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction

Abstract

Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.

Key points

• Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities.

• MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro.

• Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo.

• Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction.

Introduction

The walls of hollow organs such as the gastrointestinal tract, circular blood vessels, urinary bladder, airways and uterus are composed of smooth muscle cells which serve vital homeostatic functions. An initial development of force enables organs to implement quick contractile responses, but they also may maintain force for an extended period of time related to specific physiological functions, e.g. vascular blood vessels for maintaining blood pressure, various sphincters for prolonged closure of an orifice and emptying of the urinary bladder. Abnormal contractile performance of smooth muscles contributes to different diseases, such as urinary incontinence, incomplete bladder emptying or retention of urine, hypertension, hypotension, asthma, gut dysmotility and various reproductive disorders (Uehata et al. 1997; Fernandes et al. 2007; Ohama et al. 2007; He et al. 2008; Fonseca et al. 2009; Meng et al. 2010; Satoh et al. 2011; Zhou et al. 2011; Zderic & Chacko, 2012; Fukumoto & Shimokawa, 2013; He et al. 2013; Hypolite et al. 2013; Stav et al. 2013; Vesterinen et al. 2013). Thus, force maintenance is a basic physiological property of smooth muscle that is important for diverse functions of different hollow organs.

Smooth muscle contraction is evoked by a network of signals involving ion channels or membrane receptors such as the voltage-operated Ca²⁺ channels or agonist-activated G-protein coupled receptors (GPCRs) (Somlyo & Somlyo, 2003). Depolarization of the smooth muscle cell membrane activates L-type Ca²⁺ channels, resulting in calcium influx (Hermsmeyer et al. 1988; Moosmang et al. 2003). The elevated intracellular calcium ([Ca²⁺]_i) in turn activates Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK), which phosphorylates the myosin light chain (RLC) to initiate myosin crossbridge movement on actin filaments (Kamm & Stull, 1985; Ito et al. 2004; He et al. 2008). Agonists of GPCRs also sequentially activate Gα_q/11 and phospholipase C, resulting in an increase in [Ca²⁺]_i by inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release from the sarcoplasmic reticulum (Garay, 2000; Wynne et al. 2009). After an initial elevation, [Ca²⁺]_i may subsequently decline, thereby reducing the extent of MLCK activation. However, other signalling modules are recruited to reduce the rate of RLC dephosphorylation by myosin light chain phosphatase (MLCP) (Somlyo & Somlyo, 2003; Hartshorne et al. 2004; Dimopoulos et al. 2007; Kitazawa, 2010; Grassie et al. 2011). CPI-17 (protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein of 17 kDa), a specific inhibitor protein for MLCP, is phosphorylated by sequential activation of Gα_q/11 and PKC, leading to inhibition of MLCP activity (Eto et al. 2004; Butler et al. 2013). Rho-associated protein kinase (ROCK) is also activated by Gα_12/13 activation induced by agonists, which then inhibits MLCP activity through myosin phosphatase targeting subunit-1 (MYPT1) and CPI-17 phosphorylation (Somlyo & Somlyo, 2000, 2013). Therefore, the signals converging on MYPT1 and CPI-17 for MLCP inhibition are central to enhancing RLC phosphorylation and force maintenance while [Ca²⁺]_i decreases (Ca²⁺ sensitization) (Kitazawa et al. 2000, 2003; Dimopoulos et al. 2007; Mori et al. 2011).

The MLCP holoenzyme is composed of three distinct subunits: a catalytic type 1 phosphatase subunit (PP1cδ), a regulatory subunit (MYPT1) and a 20 kDa subunit of unknown function (Alessi et al. 1992; Shirazi et al. 1994). MLCP dephosphorylation of RLC is involved in various biological processes involving different myosin II isoforms, including smooth muscle contraction, cell division, cell migration, morphogenesis and other developmental processes (Kawano et al. 1999; Xia et al. 2005; Yokoyama et al. 2005; Huang et al. 2008; Gutzman & Sive, 2010). Biochemical observations show that PP1cδ activity is increased by binding to MYPT1, and inhibited upon MYPT1 phosphorylation (Trinkle-Mulcahy et al. 1995; Tanaka et al. 1998; Terrak et al. 2004). MYPT1 also binds myosin and/or other proteins, thus serving as a scaffolding protein (Tanaka et al. 1998; Terrak et al. 2004).

MYPT1 may be phosphorylated by different kinases at multiple sites, including S432, S445, S472, S473, S601, S668, S692, S695, S852, S910, T696 and T853 (numbering based on human MYPT1) (Feng et al. 1999; Totsukawa et al. 1999; Wooldridge et al. 2004; Yamashiro et al. 2008; Zagorska et al. 2010; Yuen et al. 2011; Butler et al. 2013). Among these, S668, S692, S695 and S852 are phosphorylated by protein kinase G or protein kinase A with S695 and S852 inhibiting phosphorylation of T696 and T853 in smooth muscles (Wooldridge et al. 2004; Nakamura et al. 2007). T696 and T853 can be phosphorylated biochemically by ROCK, zipper interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) (Kimura et al. 1996; Feng et al. 1999; MacDonald et al. 2001; Muranyi et al. 2002). Phosphorylation of T696 or T853 inhibits MLCP activity towards phosphorylated RLC in vitro (Trinkle-Mulcahy et al. 1995; Ichikawa et al. 1996; Muranyi et al. 2005; Khromov et al. 2009). However, the role of MYPT1 phosphorylation at these sites in isolated tissues containing intact smooth muscle cells has been deduced primarily by pharmacological approaches with inhibitors of the multifunctional kinase ROCK. These data indicate that unidentified protein kinases (not PKC) phosphorylate T696 while ROCK is primarily responsible for the phosphorylation of T853. Investigations of these signalling pathways need additional approaches to rule out off-target effects where pharmacological inhibitors may act non-selectively on other protein kinases that affect contractile responses in different signalling modules. Additionally, inhibition of ROCK would result in inhibition of other proteins phosphorylation that affects contractile responses in addition to inhibition of T853 phosphorylation. Because ROCK is considered a potential therapeutic target related to its ability to inhibit force maintenance in different smooth muscle tissues (Fernandes et al. 2007; Fonseca et al. 2009; Satoh et al. 2011; Zhou et al. 2011; Fukumoto & Shimokawa, 2013; Vesterinen et al. 2013), it is important to understand its mechanistic importance relative to MYPT1 T853 phosphorylation.

Previous investigations show ileal and urinary bladder smooth muscles contract and relax in MYPT1-depleted tissues although RLC phosphorylation and force maintenance may be enhanced (He et al. 2013; Tsai et al. 2014). However, these studies did not address specifically the role of MYPT1 T696 and T853 phosphorylation. To test the hypothesis that MYPT1 regulates force maintenance through phosphorylation at these two sites in vivo, we mutated MYPT1 individually in mice with alanine substitution at T694 and T852 (corresponding to human T696 and T853, respectively) to inhibit phosphorylation. Analyses of contractile and biochemical responses of isolated urinary bladder smooth muscle from mice containing the T694A mutation suggest that phosphorylation of MYPT1 T694 is required for force maintenance, and the underlying mechanism involves constitutive phosphorylation without dependency on ROCK activity. In contrast, results obtained with bladder smooth muscles from mice containing the MYPT1 T852A mutation suggest that phosphorylation induced by G_12/13/RhoA/ROCK has no significant effect on RLC phosphorylation and contractile responses. Based on these results, we propose a novel model regulating force maintenance of urinary bladder smooth muscle.

Methods

Ethical approval and bladder isolation

All experiments were conducted in accordance with the Animal Care and Use Committee of Model Animal Research Center of Nanjing University. Pregnant mice were killed by cervical dislocation, and the pups at embryonic day (E)18.5 were collected by caesarian section. The pups were immersed in ice to induce hypothermia. Once deep hypothermia was achieved, animals were decapitated, and the bladders removed and transferred to cold Hepes-Tyrode (H-T) buffer (137 mm NaCl, 2.7 mm KCl, 1.0 mm MgCl₂, 1.8 mm CaCl₂, 10 mm Hepes, 5.6 mm glucose, pH 7.4) for force measurement and protein preparation. The bladders isolated from postnatal day 0 pups were prepared as for E18.5 pups.

Generation of Mypt1 mutant mice

The Mypt1 targeting vector was constructed by use of bacterial artificial chromosome (BAC) retrieval methods (Liu et al. 2003). In brief, genomic fragments containing exons 15–19 of the mouse Mypt1 gene was retrieved from a 129/sv BAC clone bMQ 218k07 (provided by the Sanger Institute, Cambridge, UK) by a retrieval vector containing two homologous arms. For the T694A targeting vector, the codon encoding T694 (ACA) in exon 15 was mutated to GCA to encode alanine. Simultaneously, a loxp-Neo-loxp cassette was introduced as a positive selection marker. Similarly, the codon encoding T852 (ACT) in exon 18 was mutated to encode alanine with the additional introduction of a loxp-Neo-loxp cassette. The respective restriction sites at 694 (HincII) or at 852 (AccI) were lost after mutation, which enabled identification of mutated DNA by enzymatic digestion. Embryonic stem (ES) W4 cells were electroporated with the linearized targeting vectors by _NotI, screened and then expanded for Southern blot analysis. Injecting ES cells into C57BL/6 blastocysts followed by transfer to pseudopregnant mice generated chimeric mice. Neonates of T694A heterozygous mice did not survive, and thus chimeric males were crossed with female Mypt1^+/− mice to obtain offspring with different genotypes, including Mypt1^T694A/−, Mypt1^T694A/+, Mypt1^+/+ and Mypt1^+/−. T852A heterozygotes were crossed with female Mypt1^+/− mice or Mypt1^T852A/+ mice after deletion of the neo-cassette by crossing with EIIA-Cre mice. All mice used in this study were of a mixed 129S6/SvEvTac:C57BL/6 J background.

Genotyping by Southern blotting and PCR analyses

Genomic DNA of ES cells was extracted and screened for positive clones by long distance PCR analyses and Southern blotting. For identifying the T694A mutation, PCR primers 5′-CAGTTATTAGCAGTTAGGCGTTTTAGTC-3′ and 5′-CGATTGTCTGTTGTGCCCAGTCATAG-3′ amplify the left homologous arm, and primers 5′-ATCTCCTGTCATCTCACCTTGCTCCTG-3′ and 5′-AATCCCTCCACTTAGCCATTTTCTTA-3′ amplify the right homologous arm. For identifying the T852A mutation, PCR primers 5′-CAGTTATTAGCAGTTAGGCGTTTTAGTC-3′ and 5′-CGATTGTCTGTTGTGCCCAGTCATAG-3′ amplify the left homologous arm and primers 5′-ATCTCCTGTCATCTCACCTTGCTCCTG-3′ and 5′-AATCCCTCCACTTAGCCATTTTCTTA-3′ amplify the right homologous arm. For Southern blotting assays, the probes located outside the target region were prepared by PCR using the folowing primer pairs: for the 5′ region, 5′-TGAAATGGGATATTGTCATAATCG-3′ and 5′-CTTCTACCAAAGGAGCAACTAAA-3′; for 3′ region, 5′-CACAAATTGATAATACCTTACAGAG-3′ and 5′-ATAGGAAGACTGTGTCTAAACAGC-3′. Genomic DNA was digested with BamHI overnight and blotted on a Nylon membrane followed by probing with α-³²P-labelled 577 bp (5′ region) and 637 bp (3′ region) probe.

Genotyping of tail DNA was conducted with PCR and Southern blot assay. Primers 5′-AGAGGGACCCAATGGTCATG-3′ and 5′-GCCCAGAAAGCGAAGGAG-3′ were used for T694A mice genotyping; primers 5′-GATTACAGTCATCCTGCGAAAA-3′ and 5′-ACAGTGCCACCCCACATTA-3′ were used for T852A mice genotyping; and primers 5′-AGCCCAAGGTTTGAGGTTTC-3′ and 5′-TTCCCTTATCAGATTCACTACCAA-3′ were used for detection of Mypt1 gene deletion. Homologous recombinants were further verified with Southern blot assays as mentioned above.

Measurement of smooth muscle contractility

Smooth muscle strips (1 mm wide and 2.8–3 mm long, dissected from the mid-portion of the urinary bladder in the circular direction) (Ekman et al. 2005) were tied to one end of a steel rod with the other end tied to a force transducer (MLT0201; ADInstruments, Colorado Springs, CO, USA) and placed in an organ bath (5 ml) at 37 °C. The strips were stretched to 0.1 g resting force and equilibrated for 45 min in Krebs solution (119.0 mm NaCl, 4.7 mm KCl, 1.17 mm MgSO₄, 2.5 mm CaCl₂, 20 mm NaHCO₃, 1.18 mm KH₂PO₄, 0.027 mm EDTA and 11 mm glucose) continuously gassed with 95% O₂ and 5% CO₂. After the resting tension declined, the strips were re-stretched to maintain 0.1 g resting force. After the stable force responses evoked by KCl were obtained, the muscle strips were stimulated with 30 μm carbachol (CCh) or 200 μm bethanechol (BCh) to evaluate initial and sustained contraction.

Protein sample preparation and Western blots

For measurements of contractile and regulatory protein expression, whole bladder tissues were homogenized in a sample buffer solution (2% SDS, 10 mm dithiothreitol (DTT), 10% glycerol, a trace amount of bromophenol blue and 50 mm Tris HCl, pH 7.4), heated at 85°C for 5 min, and incubated at room temperature for 60 min. After centrifugation at 10,600 g for 10 min, the supernatant fraction was subjected to SDS-PAGE and either stained with Coomassie blue or transferred to PVDF membranes for subsequent Western blotting. The following antibodies used were: smooth muscle myosin heavy chain (SM-MHC; 1:1000, Abcam, Cambridge, MA, USA), MLCK (1:10,000, Sigma, St Louis, MO, USA), PKC (1:1000, Millipore, Billerica, MA, USA), PP1cδ (1:1000, Millipore), RhoA (1:500, Cell Signaling Technology, Danvers, MA, USA), ROCKII (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-smooth muscle actin (α-SMA; 1:1000, Thermo Fisher Scientific, Waltham, MA, USA), myosin phosphatase-rho interacting protein (M-RIP; 1:500, Signalway Antibody, College Park, MD, USA) and prostate apoptosis response-4 (Par-4; 1:1000, Signalway Antibody).

To measure MYPT1, CPI-17 and RLC phosphorylation, tissue strips were rapidly frozen by immersion in a frozen slush of 10% trichloroacetic acid (TCA) and 10 mm DTT in acetone pre-cooled by liquid nitrogen. The strips were then slowly thawed at room temperature followed by a thorough homogenization in 10% TCA and 10 mm DTT. The samples were centrifuged and the pelleted protein was washed with diethylether. The protein samples were dissolved completely in an 8 m urea solution (He et al. 2008, 2013). Equal amounts of protein were mixed with Laemmli buffer (2% SDS, 10% glycerol, 0.002% bromophenol blue, 0.125 m Tris HCl, pH 6.8), and boiled for SDS-PAGE with one of three polyacrylamide gradients (6, 7.5 or 12%). The resolved proteins were blotted to PVDF membranes. The membranes were blocked with 5% non-fat milk and then incubated individually with primary antibodies at 4°C overnight. The corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies were added after washing. The primary antibodies include a Phospho-MYPT1-T694 antibody, a Phospho-MYPT1-T852 antibody (1:1000, MBL, Inc., Denver, CO, USA), an MYPT1 antibody (1:2000, Upstate Biotechnology, Lake Placid, NY, USA), a Phospho-RLC-S19 antibody (1:1000, Cell Signaling Technology), an RLC antibody (1:1000, Cell Signaling Technology), a Phospho-CPI-17 antibody (1:5000, Bio-World) and a CPI-17 antibody. The blots were visualized by using the enhanced chemiluminescence (ECL) method (Pierce, Rockford, IL, USA) and quantified using a Jieda 801 Image Analysis System 3.3.2 (JEDA Science-Technology Development Co., Ltd, Nanjing, China).

Myosin RLC phosphorylation was also measured alternatively by urea/glycerol-PAGE as previously described (He et al. 2008, 2013). Briefly, protein was prepared from the tissue samples by using TCA precipitation as described above and, after dissolving in urea sample buffer, subjected to PAGE in glycerol and then transferred to a PVDF membrane. The RLC protein was visualized following Western blotting with RLC antibody. The percentage of phosphorylated RLC relative to the total RLC protein level was quantified using a Jieda 801 Image Analysis System 3.3.2 (JEDA Science-Technology Development).

Histology

Bladder tissues were fixed in 4% paraformaldehyde overnight at 4°C, dehydrated with a graded series of ethanol solutions and embedded in paraffin. The embedded tissues were transversely sectioned at a thickness of 6 μm and slides were dewaxed with xylene, rehydrated with descending grades of ethanol and then rinsed with distilled water. Tissue sections were then stained with haematoxylin and eosin to examine the morphology and the smooth muscle areas of the cross-sections were calculated from light microscopy images (Dotslide, Olympus, Tokyo, Japan).

Reverse transcription-PCR

Total RNA from bladders or other tissues was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then, 500 ng RNA from each sample were used for reverse transcription and amplification. Primers 5′-TCCACCACCACCCTGACTACAAC-3′ and 5′-CTTGTTCTCTGGTTCTCGTAGAAC-3′ were used for amplification of Mypt1 mRNA containing the 694 site; primers 5′-TCGTCCTCACTCTCTACTCTAGG-3′ and 5′-AACTGTATGAAGCAGAGCGACCC-3′ were used for Mypt1 mRNA containing the 852 site; and primers 5′-ATTCCTTGCTGGGTCGCTCTGC-3′ and 5′-ATCAAGGCTCCATTTTCATCC-3′ were used for splice variants of Mypt1 exon 23 (E23) (Fu et al. 2012) and PCR products were separated by 2% agarose gel electrophoresis.

Statistical analyses

Data are presented as the mean ± SEM with the number of observations shown in parentheses. The statistical significance of the differences between groups was determined by using Student's t-test.

Results

MYPT1 T694 and T852 phosphorylation in bladder smooth muscle

As the phosphorylation patterns of MYPT1 are dependent on the kind of smooth muscle as well as animal species (Dimopoulos et al. 2007; Neppl et al. 2009; Maki et al. 2010; Hudson et al. 2012), we first examined the MYPT1 T694 and T852 phosphorylation responses to KCl and the muscarinic receptor agonist BCh in isolated bladder tissues. We detected only a weak phosphorylation of T852 under resting conditions. However, the extent of T852 phosphorylation was increased rapidly after treating with KCl, BCh or CCh (Fig. 1A, B, F and J). To identify the signalling module responsible for T852 phosphorylation, we tested the effect of kinase inhibitors. The increase in T852 phosphorylation was inhibited 60–80% by the ROCK inhibitor Y27632, but less so (KCl, 10–40%; CCh, 10–50%) by the PKC inhibitor GF-109203 (Fig.1D, F, H and J). This result indicates that the induced phosphorylation of T852 is predominantly catalysed by ROCK. In contrast, we detected a strong signal for T694 phosphorylation under resting conditions, and this phosphorylation did not increase significantly after treatment with KCl, BCh or CCh (Fig. 1 A, B, E and I). Addition of calyculin A, a potent PP1 and PP2A phosphatase inhibitor capable of preventing MYPT1 dephosphorylation (Hirano et al. 1992; Kiss et al. 2008; Cho et al. 2011; Gao et al. 2013), did not increase the extent of T694 phosphorylation, while it led to a marked and significant increase in T852 phosphorylation (Fig. 1C). We also examined the effect of ROCK and PKC inhibitors on T694 phosphorylation. Neither Y27632 nor GF-109203 affected T694 phosphorylation (Fig. 1D, E, H and I). These results indicate that T694 was highly phosphorylated constitutively and the lack of an increase with calyculin A suggests it may be 100% phosphorylated in bladders from E18.5 tissue (Tsai et al. 2014). We also tested T694 phosphorylation of postnatal tissue (P0) and found a similar result. By contrast, T852 phosphorylation was inducible by both KCl and cholinergic agonists and increased greatly with calyculin A treatment.

Figure 1.

Phosphorylation pattern of MYPT1 at T694 and T852 in bladder smooth muscle

Bladder smooth muscle strips from P0 or E18.5 mice were treated with different substances. The resultant muscle samples were subjected to Western blot assay for MYPT1, CPI-17 as well as RLC phosphorylation. A and B, time courses of phosphorylation of MYPT1, RLC and CPI-17 of bladder smooth muscle (from P0 mice) that received stimulation of 124 mm KCl or 200 μm BCh. Total MYPT1, CPI-17 and RLC proteins were used for their corresponding loading control. At least three independent experiments were performed and showed similar results. C, MYPT1 phosphorylation in response to calyculin A (CLA), which causes a maximal increase in MYPT1 phosphorylation by preventing dephosphorylation. Bladder strips from mice (E18.5 or P0) were treated with 1 μm calyculin A for 20 min and then quickly frozen followed by sampling for Western blot analysis. D and H, Western blot analysis showing effects of ROCK and PKC inhibitors on phosphorylation of MYPT1 and CPI-17. Bladder strips from P0 mice were preincubated for 15 min with 10 μm Y27632 or 3 μm GF109203X followed by 124 mm KCl or 30 μm CCh containing Y27632 or GF109203X as indicated. Total actin was used as a loading control. Quantification is shown in E, F, G, I, J and K, and the data are analysed with Student's paired t test; n = 3–5, *P < 0.05, **P < 0.01, ***P < 0.001 (compared with vehicle at the same time), ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001 (compared with value at rest).

In addition to RLC, CPI-17 phosphorylation was also increased with both KCl and BCh treatments (Fig. 1A and B) and inhibited with the PKC inhibitor, but not the ROCK inhibitor (Fig. 1D, G, H and K).

Generation and characterization of MYPT1 T694A and MYPT1 T852A mutant mice

To assess the role of T694 and T852 phosphorylation in smooth muscle contraction in vivo, we established two lines of knockin mice with replacement of T694 or T852 with alanine, in which an ACA codon of a threonine at 694 or 852 of MYPT1 was mutated to a GCA encoding alanine. We introduced these mutations into the mouse genome through a standard homologous recombination method including ES electroporation, microinjection to blastocysts with the correctly targeted ES cell clones (Fig.2B) and establishment of chimeric founders. The detailed scheme is represented in Fig. 2A. To obtain a germline of the T694A mutant, we crossed male chimeric T694A mice with C57BL/6 mice. Unfortunately, we failed to obtain an Mypt1^T694A/+ heterozygote among 232 pups. We injected alternative ES clones for generating chimeric mice, but still could not obtain an Mypt1^T694A/+ heterozygote. Thus, loss of the heterozygotes was probably not caused by abnormal ES clones, because karyotyping analysis showed normal chromosomes of the mouse embryo fibroblast cells from Mypt1^T694A/+ heterozygotes (data not shown). We then examined the embryos at E18.5 and found that T694A mutant embryos appeared in an expected Mendelian ratio. All of these mutants had severe omphalocoele, whereas body sizes and survival appeared normal (Fig. 2C). Loss of Mypt1^T694A/+ heterozygotes after birth was due to cannibalization by mother mice. We confirmed the mutation with multiple analyses (Fig. 2D–F). PCR products of cDNA containing the 694 site from Mypt1^T694A/+ and Mypt1^+/+ mice could be cut by HincII as expected; sequencing for this PCR product showed a correct mutation at T694; Western blot assay with anti-phosphorylated T694 antibody showed absence of T694 phosphorylation in tissues from Mypt1^T694A/− mice (Fig.2D–F). All lines of evidence suggested that we introduced a T694A mutation into mice successfully, and the omphalocoele phenotype and perinatal lethality were attributable to the heterozygous T694A mutation. To test possible effects on MYPT1 expression by the T694A mutation, we measured total MYPT1 protein and MYPT1 leucine zipper (LZ)+/LZ– isoforms with Western blot assay or RT-PCR. The results showed no significant alterations of the MYPT1 proteins between Mypt1^T694A/+ and Mypt1^+/+ mice (Fig.2D, G and H).

Figure 2.

Generation of Mypt1 knock-in mutant mice

A, schematic representation of Mypt1 knock-in strategy of T694A (top) and T852A (bottom). B, Southern blot assay for positive ES clones with homologous recombination. The extracted genomic DNA digested with BamHI, electrophoresed on a 1% agarose gel, transferred to nitrocellulose and probed with ³²P-labelled 5′ and 3′ probe as indicated in A. The wild-type allele generates a 18 kb fragment while the targeted allele generates a 7.7 kb (T694A) or 12.5 kb (T852A) fragment for the 5′ probe; and a 10.7 kb (T694A) or 7.7 kb (T852A) fragment for the 3′ probe. C, gross appearance of mutant embryos (E18.5). Top panel (left): 100% of T694A heterozygous littermates have the severe omphalocoele phenotype. Top panel (right): magnification of the area containing umbilicus from T694A and control pups. The appearance of T852A homozygotes was varied: mice with severe (50%) and moderate (7.1%) omphalocoele and normal appearance (42.1%) were observed as shown in the bottom panel. Arrows indicate omphalocoele phenotypes. D, PCR products of cDNA containing the T694 site or T852 site from the mutant mice were cut by HincII (T694A) or AccI (T852A) in an expected pattern. The arrows show the bands containing mutation that cannotbe cut by the enzymes. ‘1/2’ indicates half loading. E, sequencing for PCR products from cDNA isolated from T694A heterozygous (T694A/+) mice, T852A homozygous (homo) mice and wild-type (+/+) mice. Correct mutations at T694 and T852 are shown. F, Western blot assay showing disappearance of MYPT1 phosphorylation at the T694 site and T852 site in smooth muscle tissues from Mypt1^T694A/− mice and Mypt1^T852A/− mice, respectively. G, Western blot assay showing the T694A mutation does not affect MYPT1 expression but reduces T694 phosphorylation of MYPT1. β-Actin was used as an internal control. H, RT-PCR for Mypt1 LZ– (with exon 23) and Mypt1 LZ+ (without exon 23) transcripts of bladder tissues from E18.5 mice. Smooth muscle tissues such as aorta and jejunum from adult mouse were used as positive controls which respectively enriched LZ+ and LZ– isoforms.

Using a similar strategy, we introduced the T852A mutation of MYPT1 into mice via germline transmission (Fig. 2A). The mutation of the resultant heterozygous (Mypt1^T852A/+) or homozygous (Mypt1^T852A/T852A) mice was confirmed by restriction enzyme digestion, Southern blot, sequencing and Western blot (Fig. 2D–F). Western blot analysis with anti-phosphorylated T852 antibody showed abolished phosphorylation of T852, but not T694 (Fig. 2F). We crossed male Mypt1^T852A/+ with female Mypt1^+/− or Mypt1^T852A/+ mice and obtained littermates with various genotypes (Mypt1^T852A/+, Mypt1^T852A/−, Mypt1^T852A/T852A, Mypt1^+/− and Mypt1^+/+). All pups appeared in the expected Mendelian ratio. Nevertheless, 50% of the mice with homozygous T852A mutations showed severe omphalocoele while 7.1% showed moderate forms of omphalocoele and 42.1% had a normal appearance and reached adulthood without detectable abnormalities. Due to this perinatal lethality, we characterized bladder smooth muscle tissues by first examining bladders from mutant embryos with different genotypes. Gross examination showed comparable morphology, body weight and ratio of bladder/body weight among Mypt1^T694A/−, Mypt1^T694A/+, Mypt1^+/−, Mypt1^+/+, Mypt1^T852A/+, Mypt1^T852A/− and Mypt1^T852A/T852A littermates, except for a minor decreased ratio of bladder/body weight in Mypt1^+/− mice (Figs 3A and B and 4A and B). Additionally, the thickness of the smooth muscle layer was not altered (Figs 3D and 4D). Histological examination showed normal mucosa, submucosa and smooth muscle layers in the bladder walls among all lines. Typical histology is presented in Figs 3C and 4C. Therefore, we concluded that mutation of T694A or T852A did not affect bladder development.

Figure 3.

Morphology and weight of bladders from T694A mice

A, gross morphology of bladders from Mypt1^+/+, Mypt1^+/−, Mypt1^T694A/− and Mypt1^T694A/+ mice at E18.5; no obvious alteration was found among these bladders. Scale bars = 1 mm. B, the body weight (left) (n = 24–42), bladder wet weight (middle) (n = 14–27) and bladder to body weight ratio (right) (n = 12–20) did not alter in Mypt1^T694A/+ mice, but slightly decreased in Mypt1^+/− compared to Mypt1^+/+ mice. The T694A mutation significantly rescued the bladder to body weight ratio in Mypt1^+/−. Asterisks, Mypt1^T694A/– vs. Mypt1^+/−; daggers, Mypt1^+/− vs. Mypt1^+/+. ***P < 0.001; ^†††P < 0.001 (Student's t test). C, haematoxylin & eosin-stained sections of bladders showed normal mucosa layer, submucosa and smooth muscle layers comprising the bladder walls among all lines with different genotypes. D, comparable thickness of smooth muscle layers from all four genotypes (Mypt1^+/+, n = 8; Mypt1^+/−, n = 6; Mypt1^T694A/−, n = 4; Mypt1^T694A/+, n = 8). Scale bars = 400 μm.

Figure 4.

Morphology and weight of bladders from T852A mice

A, gross morphology of bladders from Mypt1^+/+, Mypt1^+/− and Mypt1^T852A/− mice at E18.5. Scale bars = 1 mm. B, quantification of body weight (left), bladder wet weight (middle) and bladder to body weight ratio (right) from T852A mutant and control mice. The T852A mutation slightly rescued bladder weight and bladder to body weight ratio decreased in Mypt1^+/−. Asterisks, Mypt1^T852A/– vs. Mypt1^+/−; daggers, Mypt1^+/– vs. Mypt1^+/+. *P < 0.05, **P < 0.01; ^†††P < 0.001 (Student's t test). C, haematoxylin & eosin-stained sections of bladders showed normal bladder morphology among all lines with different genotypes. D, comparable thickness of smooth muscle layers from Mypt1^+/+, Mypt1^+/− and Mypt1^T852A/− mice (all n = 4). Scale bars = 400 μm.

To assess compensatory expressions of contraction-related proteins after mutation, we measured SM-MHC, MLCK, PKC, PP1cδ, RLC, CPI-17, α-SMA, M-RIP and Par-4 proteins in mutant bladder smooth muscles, and found no apparent alteration of these proteins among Mypt1^+/+, Mypt1^+/−, Mypt1^T694A/− and Mypt1^T694A/+ mice (Fig. 5A–C). Similar results were obtained in the T852A mutant smooth muscle (Fig. 5D–F).

Figure 5.

Expression of contractile and regulatory proteins in MYPT1 mutant bladder

Bladder smooth muscle samples were prepared from E18.5 mice and subjected to SDS-PAGE (A and D) and Western blot assay (B and E). There were no apparent differences in the whole protein expression patterns, including the contraction-related proteins myosin heavy chain (MHC), caldesmon (CaD), RLC and 17 kDa essential light chain (LC17), between T694A mutant mice and control mice (A) and T852A mutant mice and control mice (D). Western blot assays (B and E) and quantitative results (C and F) are shown for contractile and regulatory proteins for bladder tissue from Mypt1^T694A/− (B and C) and Mypt1^T852A/− (E and E) and their control bladders. Total actin stained with Coomassie brilliant blue was used as a loading control and the values are expressed relative to those obtained in tissues from Mypt1^+/−; n = 3–4, P > 0.05.

Reduced force responses of isolated tissue from MYPT1 T694A mutant mice

As we could not obtain adult smooth muscle tissues from T694A mutant mice, we analysed contractile responses of the bladder smooth muscle from E18.5 mice. We mated T694A chimeric founders with Mypt1^+/− mice and produced Mypt1^T694A/−, Mypt1^T694A/+, Mypt1^+/− and Mypt1^+/+ littermates. Compared with Mypt1^+/− smooth muscle, Mypt1^T694A/− muscle displayed a comparable maximal force with KCl treatment (Mypt1^T694A/−: 2.77 ± 0.26 g mm⁻² vs. Mypt1^+/−: 2.72 ± 0.21 g mm⁻², P > 0.05), but a significantly reduced sustained contraction (Fig. 6A, B and E). One minute after KCl treatment, the sustained force had declined to 38.8 ± 2.8% of maximal force in muscles from Mypt1^T694A/− mice, which was significantly lower than the value (49.2 ± 3.1%; P < 0.05) from Mypt1^+/− control mice. The forces for both kinds of smooth muscles continued to decline gradually, but the muscles from Mypt1^T694A/− mice had values that were significantly lower than those obtained from Mypt1^+/− mice at all times (e.g. 2, 4, 5 and 8 min; P < 0.01). We also compared muscles from Mypt1^T694A/+ and Mypt1^+/+ mice, and obtained similar results. Mypt1^T694A/+ muscle displayed a comparable maximal force to Mypt1^+/+ control (2.81 ± 0.23 g mm⁻² vs. 2.63 ± 0.36 g mm⁻², P > 0.05), and less sustained force than control values at different times (P < 0.01) (Fig. 6B and E). Note that the sustained force of Mypt1^T694A/+ and Mypt1^+/+ smooth muscle was respectively lower than those of Mypt1^T694A/− and Mypt1^+/−. This is due to less MYPT1 protein in the latter groups with absence of an Mypt1 allele. The amount of MYPT1 in Mypt1^+/− muscle was 49.8% of that in Mypt1^+/+ muscle (data not shown). We also determined the effect of the T694A mutation in CCh-evoked smooth muscle contraction. The bladder smooth muscles from Mypt1^T694A/− and Mypt1^T694A/+ mice showed significantly reduced tension during the sustained contractile phase in comparison with Mypt1^+/− and Mypt1^+/+, respectively (Fig. 6C and F). To further confirm the effect of the T694A mutation on force maintenance, we measured force responses at 5 min after different doses of CCh. The force responses were normalized to the maximal forces induced by 100 μm CCh. As shown in Fig. 6G, there was a reduction of force associated with the MYPT1 T694A mutation at different concentrations of CCh. Taken together, our results showed that the MYPT1 T694A mutation led to reduction of force maintenance in bladder smooth muscle.

Figure 6.

Bladder smooth muscle contraction of MYPT1 T694A mutants evoked by KCl and CCh

Bladder smooth muscle strips from E18.5 mice were isolated and subjected to force measurement responding to different stimuli. A and C, representative force traces evoked by depolarization or 30 μm CCh in Mypt1^T694A/− and Mypt1^+/− bladder smooth muscles. B and D, maximal force evoked by KCl or CCh in bladder smooth muscle from E18.5 mice of Mypt1^T694A/− (KCl, n = 10; CCh, n = 6), Mypt1^+/– (KCl, n = 16; CCh, n = 10), Mypt1^T694A/+ (KCl, n = 9; CCh, n = 7) and Mypt1^+/+ (KCl, n = 10; CCh, n = 12). All P > 0.05. E and F, quantification of dynamic alteration of sustained force by KCl or 30 μm CCh in bladder from mutant groups (Mypt1^T694A/− and Mypt1^T694A/+) compared to their corresponding control groups (Mypt1^+/– and Mypt1^+/+) at the indicated time points after stimulation. The values are represented as percentages of the peak force. Asterisks, Mypt1^T694A/− vs. Mypt1^+/−; daggers, Mypt1^T694A/+ vs. Mypt1^+/+. *P < 0.05, **P < 0.01, ***P < 0.001; ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001 (Student's t test); n values are identical to those of B and D. G, dose–response effects of CCh on sustained force development of bladders from Mypt1^T694A/−, Mypt1^T694A/+, Mypt1^+/– and Mypt1^+/+ mice. Values are expressed as percentages of the maximal force by 100 μm CCh at 5 min. Asterisks, Mypt1^T694A/− vs. Mypt1^+/−; n = 3–7, *P < 0.05, **P < 0.01.

Reduced RLC phosphorylation of MYPT1 T694A mutant muscle in the late phase of contraction

In vitro studies indicate that phosphorylation of MYPT1 at T694 inhibits MLCP dephosphorylation of phosphorylated RLC (Feng et al. 1999; Muranyi et al. 2005; Khromov et al. 2009). Therefore, we measured the extent of RLC phosphorylation at different times after initiating contractile responses in smooth muscle tissues containing mutant MYPT1 T694A. Upon KCl treatment, RLC phosphorylation of Mypt1^+/– control smooth muscle reached a maximal response at 15 s (40 ± 6%) and then declined gradually; Mypt1^T694A/− smooth muscle had a comparable RLC phosphorylation at 15 s (38 ± 7%) and then declined more rapidly (16 ± 2 vs. 9 ± 2% at 180 s) (Fig. 7A and B). Similar results were obtained by comparing RLC phosphorylation responses in bladder smooth muscles from Mypt1^T694A/+ and Mypt1^+/+ mice (60 s: 5 ± 0.5 vs. 13 ± 2%; 180 s: 3 ± 1 vs. 8 ± 2%, Fig. 7C and D). To test if the reduced force of T694A muscle was caused by the reduced RLC phosphorylation, we applied calyculin A, an inhibitor of PP1 and PP2A phosphatases (Ishihara et al. 1989; Henson et al. 2003). After incubation with calyculin A, control bladder smooth muscle generated a force response that was 43.7 ± 4% of the maximal force induced by KCl (Fig. 7E and F), while muscles from Mypt1^T694A/− mice generated force to a similar extent after calyculin A treatment, indicating the reduction of force could be reversed by PP1c inhibition. These results show that the T694A mutation led to a reduction of RLC phosphorylation during force maintenance, probably by reducing the inhibition of MLCP activity normally associated with phosphorylation of T694.

Figure 7.

MYPT1 T694A mutation decreases RLC phosphorylation in bladder tissue

A and C, after stimulation with 124 mm KCl, bladder muscles were quickly frozen for sample preparation. Phosphorylated RLC (p-RLC) was measured by Western blot analysis of glycerol/urea PAGE gels. RLC, non-phosphorylated; p-RLC, monophosphorylated. B, quantification of A shows a decreased RLC phosphorylation in mutant muscle after 3 min compared to control muscle; n = 7–9, *P < 0.05 (Student's t test). The p-RLC level was expressed as the percentage of the total RLC. D, quantification of C shows lower level of phosphorylation of RLC in Mypt1^T694A/+ smooth muscle at 1 min and 3 min after KCl treatment compared to Mypt1^+/+ but comparable at 10 s and 20 s; n = 6–13, *P < 0.05, **P < 0.01 (Student's t test). E, representative force tracing of bladder strips evoked by 1 μm calyculin A (CLA). F, quantification of calyculin A-induced contraction. Values are expressed as percentages of the maximal force by 124 mm KCl; n = 3–4, P > 0.05.

MYPT1 T852A mutation did not reduce contractile or RLC phosphorylation responses

To investigate the role of MYPT1 T852 phosphorylation in regulating MLCP activity physiologically, we analysed the contractile properties of bladder smooth muscle of E18.5 or P0 mice with MYPT1 T852A mutations. In response to KCl, the bladder smooth muscle from Mypt1^T852A/− and Mypt1^+/− mice showed similar maximal forces (Mypt1^T852A/−: 3.08 ± 0.43 g mm⁻² vs. Mypt1^+/−: 3.07 ± 0.31 g mm⁻², P > 0.05) and similar reductions in sustained contractile forces (all P > 0.05) (Fig. 8A and E). Similar results were obtained with CCh treatment (Fig. 8C and F). We also determined the dose–response effects of CCh on sustained force, in which the force at 5 min after CCh treatment was measured relative to the maximal force response induced by 100 μm CCh. Upon stimulation with various doses of CCh, for Mypt1^T852A/− mice mean force values were less in embryonic bladder smooth muscle than in Mypt1^+/– control muscle, but no statistical significance was observed (all P > 0.05) (Fig. 8G). Measurements with muscles from Mypt1^T852A/T852A and Mypt1^+/+ mice treated with KCl showed that the former tissues had less force than the latter tissues at various times, although the differences were modest (Fig. 8E); for example, 5 min after KCl treatment, the sustained force had declined to 10.2 ± 1.0% of maximal force in muscles from Mypt1^T852A/T852A mice, which was significantly lower than the value (14.1 ± 0.9%; P < 0.01) from Mypt1^+/+ control mice (Fig. 8E). Similar results were obtained from Mypt1^T852A/T852A mice of P0 (Fig. 8H). Upon stimulation with CCh (E18.5) or BCh (P0), the average sustained force of Mypt1^T852A/T852A bladder smooth muscle was less than that of Mypt1^+/+ control, but no statistical significance was observed (all P > 0.05) (Fig. 8F and I). Thus, the MYPT1 T852A mutation had modest effects on the sustained contraction of embryonic bladder smooth muscles.

Figure 8.

MYPT1 T852A mutation slightly decreases sustained phase in bladder

A and C, representative force traces evoked by depolarization or 30 μm CCh in Mypt1^T852A/− and Mypt1^+/− bladder smooth muscles from E18.5 mice. B and D, maximal force evoked by depolarization or 30 μm CCh in Mypt1^T852A/− and Mypt1^+/− bladder smooth muscle (n = 8–10). E and F, quantification of the sustained force in Mypt1^T852A/−, Mypt1^+/−, Mypt1^+/+ and Mypt1^T852A/T852A smooth muscle by KCl or 30 μm CCh. Consistently, bladder smooth muscle slightly reduced sustained contraction evoked by KCl (daggers, Mypt1^T852A/T852A vs. Mypt1^+/+; ^††P < 0.01, ^†P < 0.05). Values are as of the peak force. G, dose–response effect of CCh on contraction of bladders from different genotypes of mice at E18.5. Values of the sustained force evoked by CCh at 5 min are expressed as percentages of the maximal force at 100 μm CCh. H and I, quantification of the sustained phase evoked by KCl or 200 μm BCh in bladders from Mypt1^T852A/T852A and Mypt1^+/+ mice (P0). Data were from six measurements and each from different bladders. Mypt1^T852A/T852A vs. Mypt1^+/+, ^†P < 0.05 (Student's t test). J, quantification of phosphorylation of RLC for bladder muscles from Mypt1^T852A/− and Mypt1^+/− mice (E18.5) in response to 124 mm KCl at 15 s and thereafter. The p-RLC level was expressed as the percentage of the total RLC; n = 4–7, P > 0.05.

As MYPT1 T852 phosphorylation inhibits MLCP activity in vitro, we measured RLC phosphorylation during contraction. Consistent with results obtained on contractile force responses, RLC phosphorylation of Mypt1^T852A/− bladder smooth muscle was comparable to that of Mypt1^+/− (all P > 0.05 at 15 s, 1 min and 3 min after stimulation) (Fig. 8J). This indicates that MYPT1 T852 phosphorylation does not apparently influence MLCP activity towards RLC dephosphorylation in vivo.

Contractile response to ROCK inhibitor in MYPT1 mutant smooth muscle

MYPT1 phosphorylation is reported to be associated with calcium sensitization through ROCK signalling pathways (Somlyo & Somlyo, 2003; Wang et al. 2009; Kitazawa, 2010; Hudson et al. 2012). To test whether MYPT1 T694 and T852 phosphorylation is functionally dependent on ROCK signalling, we measured the contractile responses of mutant bladder smooth muscles with ROCK inhibition. In smooth muscles from control mice and mice containing MYPT1 T694A mutations, pre-addition of 10 μm Y27632 did not inhibit the maximal contractile responses to KCl (all P > 0.05) (Fig. 9A and B). However, sustained contractile responses were significantly inhibited in tissues from control mice (Mypt1^+/+: 3.8 ± 0.9 vs. 16.0 ± 2.3%, n = 6; Mypt1^+/−: 12.5 ± 0.7 vs. 32.8 ± 1.9%, n = 9, all P < 0.001) (Fig. 9A and C). In Mypt1^T694A/− and Mypt1^T694A/+ smooth muscles, pre-addition of 10 μm Y27632 also significantly inhibited sustained force responses to KCl at an extent comparable to the results obtained with corresponding control muscles (Mypt1^T694A/−: 7.8 ± 2.6 vs. 20.1 ± 2.8, n = 3; Mypt1^T694A/+: 3.4 ±  ± 1.0 vs. 10.4 ± 2.1, n = 4, P < 0.05) (Fig. 9C). Note that the residual sustained tensions of Mypt1^+/− and Mypt1^T694A/− smooth muscle after Y27632 treatment were significantly higher than those of Mypt1^+/+ and Mypt1^T694A/+. Similarly, we also examined the effect of ROCK inhibitor H1152 on the CCh-evoked contractile response. The sustained tension of Mypt1^T694A/− smooth muscle was significantly inhibited in the presence of 0.1 μm H1152 (P < 0.05), in a similar manner to that of Mypt1^+/− control (Fig. 9D–F). These results indicate that force maintenance by T694 phosphorylation was independent of ROCK signalling, consistent with MYPT1 T694 phosphorylation measurements (Fig. 1E and I).

Figure 9.

The MYPT1 T694A mutation does not confer resistance to force reduction by the ROCK inhibitor

A and D, representative force traces evoked by KCl (A) or CCh (D) in the presence of ROCK inhibitor Y27632 (A) or H1152 (D) or vehicle in Mypt1^T694A/− and Mypt1^+/− bladder smooth muscles. B, C, E and F, quantitative summary of the effects of Y27632 (10 μm) (B and C) and H1152 (0.1 μm) (E and F) on peak (B and E) and sustained (5 min) (C and F) force evoked by KCl (B and C) or 30 μm CCh (E and F) in bladders from T694A mutant mice and control mice (E18.5). Force values are expressed relative to the peak force recorded in the presence of KCl (B and C) or CCh (E and F) (line at 100%). *P < 0.05, **P < 0.01, ***P < 0.001 (Student's paired t test).

We also measured the effects of ROCK inhibitor on smooth muscle responses with tissues from mice containing the MYPT1 T852A mutation. Similar to the T694A mutation, the bladder smooth muscles isolated from Mypt1^T852A/−, Mypt1^T852A/T852A, Mypt1^+/− and Mypt1^+/+ mice were sensitive to ROCK inhibitor in sustained but not maximal force responses (Fig. 10A–C). A more selective inhibitor of ROCK, H1152, showed a similar effect (Fig. 10D–I). These results suggest that T852 phosphorylation is not necessary for the force inhibitory effects of the ROCK inhibitor. To investigate why inhibition of ROCK was able to relax these mutant smooth muscles, we examined the effect of ROCK inhibition on LIM kinases (LIMK)/cofilin/F-actin cascade. Upon pretreatment with vehicle, KCl modestly induced cofilin phosphorylation within 20 s and 5 min in Mypt1^+/+ bladder smooth muscle. Upon pretreatment with 10 μm Y27632, cofilin phosphorylation was significantly inhibited at 5 min (P < 0.05) (Fig. 11A and B). Interestingly, in Mypt1^T694A/− and control muscle, treatment with Y27632 also significantly inhibited cofilin phosphorylation at 5 min (Fig. 11C). Thus, the inactivation of cofilin by phosphorylation may contribute to force development by enhanced actin dynamics. The inhibition of ROCK appears to inhibit cofilin phosphorylation and thus inhibits force development.

Figure 10.

The MYPT1 T852A mutation does not confer resistance to force reduction by the ROCK inhibitor

A, D and G, representative force traces evoked by KCl (A) or 10 μm CCh (D and G) in the presence of ROCK inhibitor Y27632 (A), H1152 (D and G) or vehicle in T852A mutant and control bladder smooth muscles from E18.5 (A) or adult mice (D and G). B, E, C, F, H and I, quantitative summary of the effects of Y27632 (10 μm) (B and C) and H1152 (0.1 μm) (E, F, H and I) on peak (B, E and H) and sustained (5 min) (C, F and I) tension evoked by KCl (B, C, E and F) or CCh (H and I) in bladders from T852A mutant mice and control mice. Force values are expressed relative to the peak force recorded in the presence of KCl (B, C, E and F) or CCh (H and I) (line at 100%). *P < 0.05, **P < 0.01, ***P < 0.001 (Student's paired t test).

Figure 11.

Depolarization-induced cofilin phosphorylation was attenuated by ROCK inhibitor Y-27632

A, bladder strips from P0 mice were preincubated for 15 min with 10 μm Y27632 followed by 124 mm KCl containing Y27632 as indicated. Cofilin phosphorylation was analysed by Western blot assay, and total cofilin was used for loading control. B, quantification of the data and analysed with Student's paired t test; n = 5, *P < 0.05 (compared with vehicle at the same time). C, Western blot analysis shows preincubation with Y27632 attenuates cofilin phosphorylation both in T694A mutant and control bladder strips at 5 min after 124 mm KCl stimulation.

Discussion

As the main regulatory subunit of MLCP, MYPT1 appears to regulate phosphatase activity biochemically through multiple mechanisms (e.g. serving as a binding subunit, phosphorylation at various sites affecting phosphatase activity and scaffolding with different proteins), and functions in multiple important biological processes (Matsumura & Hartshorne, 2008; Grassie et al. 2011). Global deletion of MYPT1 causes embryonic lethality as reported previously (Okamoto et al. 2005), suggesting a critical role of MYPT1-regulated MLCP during early developmental processes. If the MYPT1 knockout is restricted to smooth muscle cells, developmental defects are not found (He et al. 2013; Tsai et al. 2014). Herein, we blocked MYPT1 phosphorylation at T694 and T852 sites by introduction of alanine, and did not observe abnormal developmental defects except omphalocoele, a hernia around the base of the umbilical cord. Ablation of MYPT1 phosphorylation appears not to affect most developmental processes. Interestingly, either T694A or T852A mutations led to omphalocoele, but their penetrance was different. This difference may be attributable to the different phosphorylation properties between T694 and T852 (the former is constitutive and the latter is inducible). ROCK knockout mice also exhibit a similar omphalocoele (Shimizu et al. 2005; Thumkeo et al. 2005) so a ROCK/MYPT1 signalling cascade may regulate closure of the ventral body wall at the umbilical ring. The genetic causes of omphalocoele in humans are largely unknown although gene mutations as well as environmental and epigenetic contributions are appreciated (Dauve & McLin, 2013). No one single gene is probably responsible for omphalocoele in humans so the potential involvement of MYPT1 as well as ROCK should be explored.

In response to physiological and pharmacological stimuli, smooth muscles initially develop a rapid maximal force response through activation of Ca²⁺/calmodulin-dependent MLCK (Kamm & Stull, 1985, 2001; He et al. 2008, 2011; Zhang et al. 2010). Following this maximal force response, [Ca²⁺]_i decreases but RLC phosphorylation and force may be maintained to some extent by reduced MLCK activity combined with Ca²⁺ sensitization mechanisms involving MYPT1 phosphorylation by ROCK and CPI-17 phosphorylation by PKC. For embryonic mouse bladder smooth muscle, we confirmed that MYPT1 T852 and CPI-17 are phosphorylated by K⁺ depolarization or muscarinic receptor stimulation, which could be reduced by ROCK and PKC inhibitors, respectively. The constitutive MYPT1 T694 phosphorylation was not inhibited by ROCK or PKC inhibitors, similar to other reports for bladder smooth muscle (Wang et al. 2009, 2012). Additionally, treatment of bladder tissues with calyculin A did not further increase MYPT1 T694 phosphorylation. Because calyculin A treatment results in 100% phosphorylation of MYPT1 T694 (as well as T852) in adult bladder smooth muscle (Tsai et al. 2014), MYPT1 T694 may be similarly phosphorylated in embryonic bladder smooth muscle.

Pharmacological perturbations affecting MYPT1 T852 and CPI-17 phosphorylation associated with diminution of force responses have formed a fundamental model of signalling mechanisms for force maintenance in a variety of smooth muscles (Kitazawa et al. 2003; Niiro et al. 2003; Dimopoulos et al. 2007; Wang et al. 2009). However, using a genetic approach to knockin non-phosphorylatable alanine for T852 or T694, we found that the MYPT1 T852A mutation had no significant effect on maximal force development and little effect on force maintenance in isolated bladder tissues. Information about MYPT1 T852 phosphorylation as well as its functional roles are conflicting (Feng et al. 1999; Muranyi et al. 2005; Khromov et al. 2009; Khasnis et al. 2014). For example, Feng et al. (1999) suggested that T852 is not an inhibitory site for MLCP activity; Khromov et al. (2009) demonstrated in vitro that T852 phosphorylation displayed 30-fold less activity than T694; and Muranyi et al. (2005) concluded that T852 phosphorylation regulated MLCP activity equivalently to T694. Several reports propose that the regulatory mechanism of T852 is direct inhibition of PP1c activity through T852 phosphorylation (Feng et al. 1999; Khromov et al. 2009), while others suggest that T852 phosphorylation triggers the dissociation of PP1 and MYPT1 complex or MYPT1 fragment (714–1004) from myosin filaments (Velasco et al. 2002). Most recently, Khasnis et al. (2014) developed a novel procedure to express MLCP reconstituted in mammalian cells that retained characteristics of the native enzyme. MYPT1 T694 phosphorylation inhibited MLCP activity whereas T852 phosphorylation did not. These results are consistent with our results showing no differences in RLC phosphorylation and relaxation in control vs. tissues containing the MYPT1 T852A mutation.

A primary argument for a role of MYPT1 T852 phosphorylation in contributing to enhanced RLC phosphorylation in intact tissues depends mostly on interpretations of results obtained with pharmacological inhibitors of ROCK. However, MYPT1 is not the only protein phosphorylated by ROCK, and other potential physiological substrates and processes affecting smooth muscle contraction have been identified. For example, ROCK appears to be involved in agonist activation of Ca²⁺ entry distinct from voltage or store operated channels (Ghisdal et al. 2003). ROCK also affects actin dynamics in multiple signalling modules, including in particular phosphorylation of LIMK, which phosphorylates and inactivates cofilin's ability to sever F-actin (Amano et al. 2000; Walsh & Cole, 2013). The resulting increase in active polymerization is associated with enhanced force development through membrane adhesions linking the actomyosin motor complex with the extracellular matrix (Gunst & Zhang, 2008). Treatment of smooth muscle tissue with a ROCK inhibitor inhibited F-actin polymerization and contraction (Moreno-Dominguez et al. 2013). Thus, even selective pharmacological inhibition of ROCK will inhibit phosphorylation of multiple proteins affecting smooth muscle contraction and not be selective for inhibiting MYPT1 T852 phosphorylation.

Physiological activation, in contrast to pharmacological stimulation, of smooth muscle contraction elicits different phosphorylation responses for MYPT1 and CPI-17. Neurostimulation resulted in increased phosphorylation of CPI-17, but not MYPT1, in gastric fundus or urinary bladder smooth muscles (Tsai et al. 2012; Bhetwal et al. 2013). However, pharmacological stimulation of muscarinic receptors increased MYPT1 as well as CPI-17 phosphorylation in both types of smooth muscles. The inducible increase of T852 phosphorylation observed in these two smooth muscle tissues may be due to pharmacologically intense activation of signalling modules not observed physiologically. In contrast, the myogenic response in arterial resistance vessels was associated with MYPT1 T852 phosphorylation and increased actin polymerization, but no CPI-17 phosphorylation (Moreno-Dominguez et al. 2013). These responses were all diminished with ROCK inhibition, implicating a ROCK-dependent contribution to the myogenic response by MYPT1 T852 and/or cofilin phosphorylation. Recent studies confirmed the importance of ROCK in cofilin phosphorylation and enhanced actin polymerization in the myogenic response (Moreno-Dominguez et al. 2014).

We also found that most MYPT1 T694 was phosphorylated constitutively, and was not reduced by ROCK or PKC inhibitors that did affect MYPT1 T852 or CPI-17 phosphorylation. Such a constitutive phosphorylation is obviously not catalysed by ROCK1/2 and PKC. Whether it is catalysed by ZIPK, ILK or p21-activated kinase remains unknown (MacDonald et al. 2001; Muranyi et al. 2002; Takizawa et al. 2002). Our previous observations from the selective knockout of MYPT1 in smooth muscle tissue (He et al. 2013; Qiao et al. 2014; Tsai et al. 2014) suggest existence of a constitutive Ca²⁺ sensitization under resting conditions. Biochemical analysis shows that the reduced PP1cδ activity in MYPT1-deficient tissues appears similar to the attenuated MLCP activity in wild-type tissues resulting from the constitutive phosphorylation of MYPT1 T694 (Tsai et al. 2014). In this report, we show that the MYPT1 T694A mutation significantly inhibited sustained force as well as RLC phosphorylation. Thus, we suggest that smooth muscles have a constitutive Ca²⁺ sensitization mechanism contributing to force maintenance via MYPT1 T694 phosphorylation. In contrast to constitutive phosphorylation, pharmacological agonist-induced Ca²⁺ sensitization may primarily comprise the CPI-17 phosphorylation response (Kitazawa et al. 2000; Sakai et al. 2007; Kitazawa, 2010) and other protein phosphorylations by ROCK.

There are some caveats in considering these results on functional effects related to MYPT1 T694A or T852A mutations. Due to development of omphalocoele, results were limited to E18.5 or P0 tissues. There might be some minor distinctions in contractile performance between E18.5/newborn and adult tissues (Ekman et al. 2005). We thus are cautious in interpretations relative to adult tissues. Secondly, these results are focused on bladder smooth muscle and additional investigations are needed to extend observations to other kinds of phasic smooth muscles in addition to tonic smooth muscles.

In conclusion, constitutive phosphorylation of MYPT1 at T694 appears to mediate sensitized force maintenance through inhibition of MLCP activity, whereas agonist-induced T852 phosphorylation has no apparent role in regulating contractile responses. Our observations provide novel mechanistic insights into the specific role of MYPT1 phosphorylation on physiological and pharmacological Ca²⁺ sensitization.

Glossary

Abbreviations

α-SMA

α-smooth muscle actin

BCh

bethanechol

CCh

carbachol

CPI-17

protein kinase C-potentiated protein phosphatase 1 inhibitor protein of 17 kDa

DTT

dithiothreitol

ES cells

embryonic stem cells

GPCR

G-protein coupled receptor

ILK

integrin-linked kinase

LIMK

LIM kinases

LZ

leucine zipper

MLCK

myosin light chain kinase

MLCP

myosin light chain phosphatase

M-RIP

myosin phosphatase-rho interacting protein

MYPT1

myosin phosphatase targeting subunit-1

Par-4

prostate apoptosis response-4

PKC

protein kinase C

PP1c

a catalytic type 1 phosphatase subunit

RLC

myosin regulatory light chain

ROCK

RhoA-associated protein kinase

SM-MHC

smooth muscle myosin heavy chain

TCA

trichloroacetic acid

ZIPK

zipper interacting protein kinase

Additional information

Competing interests

The authors declare that no conflict of interest exists.

Author contributions

M.S.Z., C.P.C., K.E.K. and J.T.S. conceived the experimental approaches; M.S.Z., C.P.C., W.Q.H., C.H.Z., K.E.K and J.T.S. designed the experiments; C.P.C., X.C., Y.N.Q., P.W., W.Q.H., C.H.Z., W.Z., Y.Q.G., J.S. and Y.W. performed the experiments; C.P.C., P.W., X.C., Y.N.Q., Y.Q.G., C.C., W.Z., T.T., J.S., N.G. and M.S.Z. analysed and interpreted the data; C.P.C., M.S.Z., K.E.K. and J.T.S. wrote or revised the manuscript. All authors approved the final version of the manuscript.

Funding

This work was supported by the National Key Scientific Research Program of China (2014CB964701 to M.S.Z.), National Natural Science Funding of China (31272311 and 31330034 to M.S.Z.), National Institutes of Health Grants (R01 HL112778 and P01 HL110869 to J.T.S.), Moss Heart Fund (J.T.S.), and the Fouad A. and Val Imm Bashour Distinguished Chair in Physiology (J.T.S.).