Myosin Light Chain Kinase Is Central to Smooth Muscle Contraction and Required for Gastrointestinal Motility in Mice

Background & Aims: Smooth muscle is essential for maintaining homeostasis for many body functions and provides adaptive responses to stresses imposed by pathologic disorders. Identified cell signaling networks have defined many potential mechanisms for initiating smooth muscle contraction with or without myosin regulatory light chain (RLC) phosphorylation by myosin light chain kinase (MLCK). We generated tamoxifen-inducible and smooth muscle–specific MLCK knockout (KO) mice and provide direct loss-of-function evidence that shows the primary importance of MLCK in phasic smooth muscle contractions. Methods: We used the Cre-loxP system to establish Mlck floxed mice in which exons 23, 24, and 25 were flanked by 2 loxP sites. Smooth muscle–specific MLCK KO mice were generated by crossing Mlck floxed mice with SM-CreER^T2 (ki) mice followed by tamoxifen treatment. The phenotype was assessed by histologic, biochemical, molecular, cell biological, and physiologic analyses. Results: Targeted deletion of MLCK in adult mouse smooth muscle resulted in severe gut dysmotility characterized by weak peristalsis, dilation of the digestive tract, and reduction of feces excretion and food intake. There was also abnormal urinary bladder function and lower blood pressure. Isolated muscles showed a loss of RLC phosphorylation and force development induced by K⁺-depolarization. The kinase knockout also markedly reduced RLC phosphorylation and force development with acetylcholine which activates Ca²⁺-sensitizing signaling pathways. Conclusions: MLCK and its phosphorylation of RLC are required physiologically for smooth muscle contraction and are essential for normal gastrointestinal motility.

Abbreviations used in this paper: ACh, acetylcholine, Adv-MLCK, MLCK-expressing adenovirus, BAC, bacterial artificial chromosome, CTR, control, ES, embryonic stem, GFP, green fluorescent protein, IAS, internal anal sphincter, ILK, integrin-linked kinase, KO, knockout, MLCK, myosin light chain kinase, MYPT1, myosin phosphatase protein targeting subunit of the RLC phosphatase, RLC, regulatory light chain, ROCK1, Rho-associated coiled-coil–forming protein kinase 1, RT-PCR, reverse transcription–polymerase chain reaction, SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis