Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction

Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2005; 102(48):17519-24. DOI: 10.1073/pnas.0506846102
Source: PubMed


Repetitive stimulation potentiates contractile tension of fast-twitch 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 fast-twitch 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.

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Available from: Jian Huang, Feb 26, 2014
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    • "The force generated in muscular contraction increases after repetitive twitches. This response is called potentiation and depends on an increase of intracellular free Ca2+ concentration (Sandonà et al., 2005; Zhi et al., 2005) due to Ca2+ release from intracellular stores and Ca2+ inflow from the extracellular space in fast and slow twitch muscles, respectively. In the latter, Ca2+ uptake depends on the activation of purinergic ionotropic P2X4 receptors (Sandonà et al., 2005), which are highly expressed in this type of muscle. "
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    • "This is in contrast to smooth and skeletal MLCKs, which are also found in the heart but show dependence on Ca 2þ and calmodulin activity (Davis et al., 2001; Kamm and Stull, 2011). Interestingly, conventional loss of skeletal MLCK in mice was shown to have no effect on MLC2v phosphorylation and heart weight-to-body weight ratios (Zhi et al., 2005). In addition, conventional ablation of the long isoform of smooth muscle MLCK in mice resulted in no major effects on cardiac function (Ohlmann et al., 2005). "
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    • "All the RT-PCR primers were specifically designed for mouse occludin, claudin-1, ZO-1, myosin light chain kinase (MLCK), macrophage inflammatory protein 2 (MIP-2), lipopolysaccharide-induced CXC chemokine (LIX), TNF-α and β2-microglobulin (β2M) as a reference. Primer sequences for the RT-PCR were as follows: (1) mouse occludin [56], forward: 5′-ATG TCC GGC CGA TGC TCT C-3′, reverse: 5′-CTT TGG CTG CTC TTG GGT CTG TAT-3′; (2) mouse claudin-1 [57], forward: 5′-AGG AAA GGC CCT TCA GCA GAG CAA-3′, reverse: 5′-GTG CCC CCT CTT GACT CAT GCA AC-3′; (3) mouse ZO-1 [56], forward: 5′-ACC CGA AAC TGA TGC TGT GGA TAG-3′, reverse: 5′-AAA TGG CCG GGC AGA GAC TTG TGT A-3′; (4) mouse MLCK [58], forward: 5′-ACA TGC TAC TGA GTG GCC TCT CT-3′, reverse: 5′-GGC AGA CAG GAC ATT GTT TAA GG-3′; (5) mouse MIP-2 [59], forward: 5′-CGC CCA GAC AGA AGT CAT AG-3′, reverse: 5′-TCC TCC TTT CCA GGT CAG TTA-3′; (6) mouse LIX [59], forward: 5′-GGT CCA CAG TGC CCT ACG-3′, reverse: 5′-GCG AGT GCA TTC CGC TTA-3′; and (7) mouse TNF-α [60], forward: 5′-ATC ATC TTC TCA AAA TTC GAG TGA C-3′, reverse: 5′-CTA GTT GGT TGT CTT TGA GAT CCA T-3′. The cycle profile consisted of denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 20 seconds, 63°C for 30 seconds, and 72°C for 60 seconds. "
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