Volume 135, Issue 2, Pages 610-620.e2 (August 2008) Myosin Light Chain Kinase Is Central to Smooth Muscle Contraction and Required for Gastrointestinal Motility in Mice  Wei–Qi He, Ya–Jing Peng, Wen–Cheng Zhang, Ning Lv, Jing Tang, Chen Chen, Cheng–Hai Zhang, Song Gao, Hua–Qun Chen, Gang Zhi, Robert Feil, Kristine E. Kamm, James T. Stull, Xiang Gao, Min–Sheng Zhu  Gastroenterology  Volume 135, Issue 2, Pages 610-620.e2 (August 2008) DOI: 10.1053/j.gastro.2008.05.032 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Targeted disruption of Mlck gene in smooth muscle. (A) Schematic representation of Mlck smooth muscle–specific knockout strategy. The 8.9-kb genomic DNA fragment containing Mlck exons 23–25 was subcloned from 129/sv BAC using gap repair. The floxed Neo cassette was targeted upstream of exon 23, and excision of the floxed Neo cassette left behind a single loxP site (arrowheads in red) at the targeted locus. The single PGK-Neo cassette flanked by FRT sites (arrowheads in blue) and a downstream loxP site, was then introduced downstream of exon 25. The Neo cassette contains a BamHI site (blocks in red) that is favorable for Southern blot analysis. The floxed allele (Mlckflox) was formed after homologous recombination in ES cells. Mice containing the floxed allele were crossed with SM-CreERT2 (ki) mice that express a tamoxifen-activated Cre recombinase to generate Mlck+/flox; SM-CreERT2 and Mlckflox/flox; SM-CreERT2 mice. The ablation of exons 23–25 was induced by tamoxifen injection. The probe used for Southern blot analysis is shown as a solid blue bar, and the locations of the PCR primers a–d are indicated by green arrows. (B) Tail DNA isolated from homozygous (flox/flox) floxed, heterozygous (+/flox), and wild-type (+/+) mice was digested with BamHI and analyzed by Southern blot. The wild-type and floxed allele yield 18-kb and 9-kb fragments, respectively. (C) RT-PCR assay for MLCK mRNA. Various smooth muscle tissues were collected from MLCKSMKO mice induced with tamoxifen at different time points. The mRNA containing exons 23–25 was amplified by RT-PCR. The products in size of 907 bp and 576 bp reflect wild-type and mutated MLCK, respectively. (D) Western blots of MLCK in tamoxifen-treated tissues were collected at indicated days (d). Total actin stained with Coomassie Brilliant Blue G-250 was used as protein loading control. Tissues included the urinary bladder, internal anal sphincter, jejunum, and femoral artery. (E) Western blots of MLCK protein from floxed mice treated with tamoxifen (Tam+) or its vehicle (Tam-) for 16 days. The amount of loaded protein was normalized by total actin control. AO, aorta; LU, lung; ST, stomach; IL, ileum; ILM, ileum mucosa; JE, jejunum; CO, colon; BL, bladder. Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Expression of contractile and related regulatory proteins in jejunum from MLCKSMKO mice. Western blot of ILK, ROCK1, MYPT1, RLC, SM-MHC, and telokin (A and C) from MLCKSMKO (KO) and CTR jejunum. The samples were resolved by separate SDS-PAGE, and total actin stained with Coomassie brilliant blue was used as loading control. MYPT1 phosphorylations at residues 696 and 850 in response to ACh were measured by anti-MYPT1-P696 and anti-MYPT1-P850 antibody (B). Ileal total proteins (30 μg) in MLCKSMKO and CTR were resolved by SDS-PAGE and stained with Coomassie brilliant blue (D, left). To visualize more clearly the total myosin heavy chain and actin, we performed electrophoresis with different amounts of protein loading (100 μg) for comparisons of KO and CTR samples (D, right). Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Knockout of smooth muscle MLCK attenuates gastrointestinal motility. (A) Time course for food intake (left) and feces excretion (right) after tamoxifen treatment. Days were numbered with the start of tamoxifen injection. The value of each point represents the mean ± SEM of MLCKSMKO and the CTR mice (n = 3 each group). (B) Representative spontaneous contractions are shown for ileal segments from MLCKSMKO (left) and CTR (right) mice. (C) Quantification of the contraction amplitudes (left) and frequencies (right) are shown. (D) Tail blood pressure of MLCKSMKO and CTR mice was measured 15–16 days after beginning of tamoxifen injection (top). Micturition assay was performed at day 15. The total area of urine spots per hour (middle) and total number of urine spots per hour (bottom) were quantified. Bars represent means ± SEMs, n = 3–9, **P < .01 (t test). Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Abnormal gastrointestinal tract of MLCKSMKO mice. Appearance of gastrointestinal tract of an MLCKSMKO and a CTR mouse in situ (A) and out of the body (B). Note the dilated jejunum (arrows) and compacted feces in caecum and colon (arrowheads) in the MLCKSMKO mouse. Histologic examination of the small intestinal tract shows tissue changes with MLCK knockout in smooth muscle. Transverse sections of jejunum (C) and ileum (D) from MLCKSMKO and CTR mice were stained with hematoxylin and eosin. The lumen of the MLCKSMKO jejunum has a larger diameter with rare intestinal villus and a hypertrophic smooth muscle layer (indicated by an arrow). The structure of ileum from MLCKSMKO mice appears normal except for the hypertrophic smooth muscle layer. Scale bars for C and D: upper row, 500 μm; lower row, 50 μm. Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Contractions in intestinal smooth muscle and IAS. Representative recordings of ileum (A) and jejunum (B) and of IAS (C) from MLCKSMKO (upper row) and CTR (middle row) mice treated with 87 mmol/L KCl or 1 μmol/L ACh. Bars show duration of stimulation. Quantification of contraction responses to KCl and ACh (bottom row) for ileum (A), and jejunum (B) and IAS (C). Bars represent means ± SEMs, n = 4–10, **P < .01, *P < .05 (t test). Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Effects of atropine and Ca2+ depletion on smooth muscle contractility. KCl-induced contraction was not blocked by atropine (1 μmol/L) in ileal smooth muscle from MLCKSMKO (A) or CTR (B) mice. ACh (1 μmol/L) induced contractions in ileum from MLCKSMKO mice (C), and CTR mice (D) were inhibited by atropine. Depletion of Ca2+ in ileal strips by 1 mmol/L EGTA inhibited the contractile responses to repeated exposure to ACh (E). Quantitation of contractile responses of MLCKSMKO and CTR muscles to calcium depletion were normalized (F). Values are means ± SEMs (n = 3–4). Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Expression of exogenous MLCK partially restores contraction of MLCKSMKO smooth muscle. (A) A jejunum smooth muscle strip (6 mm) was infected with MLCK-expressing adenovirus (Adv-MLCK) and cultured up to 60 hours. The GFP expression driven by IRES as a tag of MLCK expression in a smooth muscle strip was examined by a dissecting fluorescence microscopy. (B) Exogenous MLCK was detected by Western blot in strips collected at 60 hours. The 2 panels shown were from the same gel but different lanes. Typical force recordings in response to 87 mmol/L KCl or 1 μmol/L ACh are shown for MLCK-deficient muscle infected with Adv-GFP control virus (C) or Adv-MLCK virus (D). Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 Time course of RLC phosphorylation in ileal smooth muscle from MLCKSMKO and CTR mice. RLC phosphorylation was measured in quick-frozen ileum smooth muscles from MLCKSMKO and control mice treated with 87 mmol/L KCl (A) or 1 μmol/L ACh (C) as shown by representative Western blots of glycerol/urea PAGE gels. Quantitation are shown of RLC phosphorylation with KCl (B) and ACh (D) treatments, respectively. Bars represent means ± SEMs, n = 4–10, **P < .01 (t test). Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 1 Genotyping analysis of MLCKSMKO mice. (A) PCR analysis of genomic DNA using primer sets specific for floxed mice as shown in Figure 1A. 5′, left arm; 3′, right arm. Primer a and b for left arm; primer c and d for right arm. (B) RT-PCR analysis for Cre and β-actin mRNA expression in different smooth muscle tissues of MLCKSMKO mice at the 16th day after tamoxifen injection. (C) Southern blot for Cre/loxP-mediated recombination in tissues from mice treated with (+) or without (−) tamoxifen for 16 days. Genomic DNA was digested with KpnI/NdeI and hybridized with α-P32-labeled probe (0.56 kb). The bands in sizes of 5.8 kb, 3.9 kb, and 2.6 kb represent floxed, wild-type, and deleted alleles, respectively. Tissues are Ta, tail; Lu, lung; Li, liver; Co, colon; and In, intestine (ileum). (D and E) Immunohistochemistry of Cre, MLCK, and SM22 in cross sections of CTR (lower panels) and MLCKSMKO (upper panels) ileum wall collected after tamoxifen treatment for 16 days. Nuclei were stained (blue) with Topro3 or PI (Invitrogen). Nuclear localization of Cre was most evident in hypertrophic circular layer of MLCKSMKO. Scale bars in panel D represent 40 μm; scale bars in panel E represent 80 μm. Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastrointestinal dysmotility of MLCKSMKO mice results in gut inflammation. At day 16 after tamoxifen induction, liquid contents in jejunum were cultured on LB agar plates at 37°C, and the bacteria colonies were counted. Knockout gut contents had more bacteria colonies than did CTR (7540 ± 7360 colonies/μL vs 40 ± 40 colonies/μL) as shown in panel A. To assess the proinflammatory response in gut, we measured intestinal TNF-α and IL-1β mRNA of knockout mice with RT-PCR (B). TNF-α expression increased, but IL-1β expression slightly decreased from the sixth day in MLCKSMKO mice by day 16. No obvious alteration in either cytokine was found in the CTR group (data not show). Hematology profile shows that the neutrophil and monocyte percentages were accordingly higher in knockout mice (C). Gastroenterology 2008 135, 610-620.e2DOI: (10.1053/j.gastro.2008.05.032) Copyright © 2008 AGA Institute Terms and Conditions