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Little progress has been made toward the use of embryonic stem (ES) cells to study and isolate skeletal muscle progenitors. This is due to the paucity of paraxial mesoderm formation during embryoid body (EB) in vitro differentiation and to the lack of reliable identification and isolation criteria for skeletal muscle precursors. Here we show that expression of the transcription factor Pax3 during embryoid body differentiation enhances both paraxial mesoderm formation and the myogenic potential of the cells within this population. Transplantation of Pax3-induced cells results in teratomas, however, indicating the presence of residual undifferentiated cells. By sorting for the PDGF-alpha receptor, a marker of paraxial mesoderm, and for the absence of Flk-1, a marker of lateral plate mesoderm, we derive a cell population from differentiating ES cell cultures that has substantial muscle regeneration potential. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function without the formation of teratomas. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy.

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Repetitive stimulation potentiates contractile tension of fasttwitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca 2؉ ͞calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fasttwitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca 2؉ ͞calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.calcium ͉ calmodulin ͉ twitch S keletal muscle contraction depends on a voltage-driven conformational change in the L-type Ca 2ϩ channel in the transverse tubule that triggers Ca 2ϩ release from the sarcoplasmic reticulum through the intracellular ryanodine receptor (1, 2). The Ca 2ϩ binds to troponin in thin filaments, thereby allowing myosin cross bridges to bind actin and generate muscle tension (3). However, muscle contractions involve more complex mechanisms that affect performance. Since Ranke noted in 1865 (4) that, with stimuli uniform in strength the later twitch contractions were stronger than the first, there has been considerable interest in identifying the mechanisms involved in isometric twitch potentiation during trains of stimuli at low frequency (staircase) or after a tetanus (posttetanic potentiation). Considerations have been given to changes in compliance of the series elastic elements, to activation of more fibers within a muscle, to increased Ca 2ϩ release within a single fiber to activate fully the contractile proteins, and to changes in excitation-contraction coupling processes (5-8).Ca 2ϩ released during muscle contraction can also activate the dedicated protein kinase Ca 2ϩ ͞calmodulin-dependent skeletal muscle myosin light chain kinase (skMLCK) to initiate myosin regulatory light chain (RLC) phosphorylat...

Background & Aims-Smooth muscle is essential for maintaining homeostasis for many body functions and provides adaptive responses to stresses imposed by pathological 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 generate 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.