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.

Download full-text


Available from: Jian Huang, Feb 26, 2014
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Normal myotubes and adult innervated skeletal myofibers express the glycoprotein pannexin1 (Panx1). Six of them form a "gap junction hemichannel-like" structure that connects the cytoplasm with the extracellular space; here they will be called Panx1 channels. These are poorly selective channels permeable to ions, small metabolic substrate, and signaling molecules. So far little is known about the role of Panx1 channels in muscles but skeletal muscles of Panx1(-/-) mice do not show an evident phenotype. Innervated adult fast and slow skeletal myofibers show Panx1 reactivity in close proximity to dihydropyridine receptors in the sarcolemma of T-tubules. These Panx1 channels are activated by electrical stimulation and extracellular ATP. Panx1 channels play a relevant role in potentiation of muscle contraction because they allow release of ATP and uptake of glucose, two molecules required for this response. In support of this notion, the absence of Panx1 abrogates the potentiation of muscle contraction elicited by repetitive electrical stimulation, which is reversed by exogenously applied ATP. Phosphorylation of Panx1 Thr and Ser residues might be involved in Panx1 channel activation since it is enhanced during potentiation of muscle contraction. Under denervation, Panx1 levels are upregulated and this partially explains the reduction in electrochemical gradient, however its absence does not prevent denervation-induced atrophy but prevents the higher oxidative state. Panx1 also forms functional channels at the cell surface of myotubes and their functional state has been associated with intracellular Ca(2+) signals and regulation of myotube plasticity evoked by electrical stimulation. We proposed that Panx1 channels participate as ATP channels and help to keep a normal oxidative state in skeletal muscles.
    Frontiers in Physiology 04/2014; 5:139. DOI:10.3389/fphys.2014.00139 · 3.53 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Thin (actin) filament accessory proteins are thought to be the regulatory force for muscle contraction in cardiac muscle; however, compelling new evidence suggests that thick (myosin) filament regulatory proteins are emerging as having independent and important roles in regulating cardiac muscle contraction. Key to these new findings is a growing body of evidence that point to an influential and, more recently, direct role for ventricular myosin light chain-2 (MLC2v) phosphorylation in regulating cardiac muscle contraction, function, and disease. This includes the discovery and characterization of a cardiac-specific myosin light chain kinase capable of phosphorylating MLC2v as well as a myosin phosphatase that dephosphorylates MLC2v in the heart, which provides added mechanistic insights on MLC2v regulation within cardiac muscle. Here, we review evidence for an emerging and critical role for MLC2v phosphorylation in regulating cardiac myosin cycling kinetics, function, and disease, based on recent studies performed in genetic mouse models and humans. We further provide new perspectives on future avenues for targeting these pathways as therapies in alleviating cardiac disease.
    Trends in cardiovascular medicine 08/2013; 24(4). DOI:10.1016/j.tcm.2013.07.004 · 2.91 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: Exclusive enteral nutrition (EEN) is a well-established approach to the management of Crohn's disease. Aim. To determine effects of EEN upon inflammation and gut barrier function in a colitis mouse model. Methods: Interleukin-10-deficient mice (IL-10(-/-)) were inoculated with Helicobacter trogontum and then treated with EEN, metronidazole, hydrocortisone, or EEN and metronidazole combination. Blood and tissue were collected at 2 and 4 weeks with histology, mucosal integrity, tight junction integrity, inflammation, and H. trogontum load evaluated. Results: H. trogontum induced colitis in IL-10(-/-) mice with histological changes in the cecum and colon. Elevated mucosal IL-8 mRNA in infected mice was associated with intestinal barrier dysfunction indicated by decreased transepithelial electrical resistance and mRNA of tight junction proteins and increased short-circuit current, myosin light chain kinase mRNA, paracellular permeability, and tumor necrosis factor- α and myeloperoxidase plasma levels (P < 0.01 for all comparisons). EEN and metronidazole, but not hydrocortisone, treatments restored barrier function, maintained gut barrier integrity, and reversed inflammatory changes along with reduction of H. trogontum load (versus infected controls P < 0.05). Conclusion: H. trogontum infection in IL-10(-/-) mice induced typhlocolitis with intestinal barrier dysfunction. EEN and metronidazole, but not hydrocortisone, modulate barrier dysfunction and reversal of inflammatory changes.
    08/2013; 2013(234):909613. DOI:10.1155/2013/909613
Show more