Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers.

Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (approximately 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.     

Myosin regulatory light chain expression in trout muscle

Muscle's contractile properties can vary along different trajectories, including between muscle fiber types, along the body (within a muscle fiber type), and between developmental stages. This study explores the role of the regulatory myosin light chain (MLC2) in modulating contractile properties in rainbow trout myotomal muscle. Rainbow trout show longitudinal variations in muscle activation and relaxation, with faster contractile properties in the anterior myotome. The expression of two muscle proteins, troponin T and parvalbumin, vary along the length of trout in concert with shifts in muscle activation and relaxation. However, there is no longitudinal variation in myosin heavy chain in trout. This study explores the role of MLC2 (or regulatory light chain), part of the myosin hexamer, in contributing to longitudinal variations in contractile properties of trout swimming muscle. We cloned and sequenced two isoforms of MLC2 from trout muscle and used real-time quantitative polymerase chain reaction to assess the relative expression of these two isoforms in red and white muscle from different body positions of two ages of rainbow trout: parr and smolt. Longitudinal variations in slow (sMLC2) but not fast (fMLC2) regulatory light chain isoforms were observed in young trout parr but not older trout smolts. The differences in sMLC2 expression correlated with shifts in muscle contractile properties in the parr. J. Exp. Zool. 309A:64-72, 2008. (c) 2007 Wiley-Liss, Inc.     

Myosin light chain patterns in histochemically typed single fibers of the rat skeletal muscle.

1. This study was conducted to investigate the relationship between histochemical fiber types and myosin light chain patterns in rat single muscle fibers. 2. The hybrid of fast and slow light chains was observed in type I and II fibers of the soleus and type II fibers of the red portion of lateral head of the gastrocnemius muscles. 3. We also observed 7 types of light chain composition. Of the 7 types, 5 types were explainable by assuming the coexistence of isomyosins with either fast or slow light chains. However, the other 2 types could not be accounted for without hypothesizing the presence of isomyosins with promiscuous light chain distribution.     

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.     

Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of isolated skeletal muscle fibers

This study examines the myosin isozyme heterogeneity (in terms of both alkali light chains and myosin heavy chains) among skeletal muscle fibers of the rabbit and correlates these isozyme differences with the differences in a contractile property, the velocity of unloaded shortening, of the fibers. The mean velocities of unloaded shortening (pCa 4.3; 12 degrees C) were as follows: psoas IIb fibers, 2.07 fiber lengths/s (n = 25); tibialis anterior (IIb) fibers, 1.63 fiber lengths/s (n = 18); vastus intermedius IIa fibers, 0.98 fiber lengths/s (n = 15); fibers (IIa) from chronically stimulated tibialis anterior, 0.86 fiber lengths/s (n = 16). Peptide maps of the myosins showed that the myosin heavy chains of the two groups of IIb fibers were indistinguishable from each other, but different from the heavy chains of the IIa fibers. However, the difference in maximal shortening velocity of the two groups of IIb fibers was correlated with a difference in the alkali light chain ratio deduced from the intensity ratio of myosin isoforms separated by gel electrophoresis under nondenaturing conditions. The vastus intermedius (IIa) and chronically stimulated tibialis anterior (IIa) fibers were indistinguishable in terms of either velocities of unloaded shortening or myosin isozyme contents. Soleus fibers contained only slow-twitch myosin. Thus, among fibers that contained a variety of myosin isozymes, differences in shortening velocities were correlated with the alkali light chain ratio, myosin heavy chain type, or a combination of both.     

Myosin turnover in cultured muscle fibers relaxed by tetrodotoxin

It is possible to maintain chick embryo muscle fibers in culture for several weeks. During this time the fibers mature and undergo spontaneous contractions. Contractions may be abolished by tetrodotoxin. When normal and tetrodotoxin (TTX) cultures are compared for myosin accumulation it is found that the relaxed or tetrodotoxin-treated cultures fail to accumulate myosin after about 4 days. Rates of myosin synthesis in normal and relaxed cultures are, however, identical. Failure to accumulate myosin in relaxed cultures is associated with a 30–40% increase in the rate of turnover of myosin heavy chains compared with normal, contracting cultures. It is suggested that one basis for hypotrophy is to be found in a post-translational mechanism for regulating myosin heavy chain (MHC) turnover.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.

The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged for labeled cgRLC in a low [Mg2+] rigor solution at 30 degrees C. Troponin and troponin C removed in this procedure were replaced. RLC exchange had little effect on active force production. X-ray diffraction showed normal structure in rigor after RLC exchange, but loss of axial and helical order in relaxation. In isolated myofibrils labeled cgRLC was confined to the regions of the sarcomere containing myosin heads. The ATR dipoles showed a preference for orientations perpendicular to the fiber axis, combined with limited nanosecond rotational motion, in all conditions studied. The perpendicular orientation preference was more marked in rigor than in either relaxation or active contraction. Stretching relaxed fibers to sarcomere length 4 microns to eliminate overlap between actin- and myosin-containing filaments had little effect on the orientation preference. There was no change in orientation preference when fibers were put into rigor at sarcomere length 4.0 microns. Qualitatively similar results were obtained with ATR-labeled rabbit skeletal RLC.     

Orientation of the N-terminal lobe of the myosin regulatory light chain in skeletal muscle fibers

The orientation of the N-terminal lobe of the myosin regulatory light chain (RLC) in demembranated fibers of rabbit psoas muscle was determined by polarized fluorescence. The native RLC was replaced by a smooth muscle RLC with a bifunctional rhodamine probe attached to its A, B, C, or D helix. Fiber fluorescence data were interpreted using the crystal structure of the head domain of chicken skeletal myosin in the nucleotide-free state. The peak angle between the lever axis of the myosin head and the fiber or actin filament axis was 100—110° in relaxation, isometric contraction, and rigor. In each state the hook helix was at an angle of ～40° to the lever/filament plane. The in situ orientation of the RLC D and E helices, and by implication of its N- and C-lobes, was similar in smooth and skeletal RLC isoforms. The angle between these two RLC lobes in rigor fibers was different from that in the crystal structure. These results extend previous crystallographic evidence for bending between the two lobes of the RLC to actin-attached myosin heads in muscle fibers, and suggest that such bending may have functional significance in contraction and regulation of vertebrate striated muscle.     

Myosin types in human skeletal muscle fibers

By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

Myosin types in human skeletal muscle fibers

Summary By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

Myosin regulatory light chain diphosphorylation slows relaxation of arterial smooth muscle

The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC(20)) at Ser(19) by Ca(2+)/calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca(2+)-independent phosphorylation at both Ser(19) and Thr(18) by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr(18) on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser(19) and Thr(18) was achieved by treatment with adenosine 5'-O-(3-thiotriphosphate) in the presence of Ca(2+), which induced stoichiometric thiophosphorylation at Ser(19), followed by microcystin (phosphatase inhibitor) in the absence of Ca(2+), which induced phosphorylation at Thr(18). Phosphorylation at Thr(18) had no effect on steady-state force induced by Ser(19) thiophosphorylation. However, phosphorylation of Ser(19) or both Ser(19) and Thr(18) to comparable stoichiometries (0.5 mol of P(i)/mol of LC(20)) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: t(½) values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser(19)) and diphosphorylated LC(20), respectively. We conclude that phosphorylation at Thr(18) decreases the rates of LC(20) dephosphorylation and smooth muscle relaxation compared with LC(20) phosphorylated exclusively at Ser(19). These effects of LC(20) diphosphorylation, combined with increased Ser(19) phosphorylation (Ca(2+)-independent), may underlie the hypercontractility that is observed in response to certain physiological contractile stimuli, and under pathological conditions such as cerebral and coronary arterial vasospasm, intimal hyperplasia, and hypertension.     

Myosin light chain phosphorylation and contractile performance of human skeletal muscle

Twitch tension and maximal unloaded velocity of human knee extensor muscles were studied under conditions of low phosphate content of the phosphorylatable light chains (P-light chains) of myosin and elevated phosphate content, following a 10-s maximal voluntary isometric contraction (MVC). After the MVC, twitch tension was significantly potentiated, with greater potentiation observed at a shorter muscle length (p less than 0.05). The MVC was associated with at least a twofold increase in phosphate content of the fast (LC2F) and two slow (LC2S and LC2S') P-light chains, but this increase was unrelated to muscle length. No significant differences in knee extension velocity were observed between conditions where P-light chains had low or elevated phosphate content. Positive but nonsignificant correlations were noted between the extent of twitch potentiation and phosphate content of individual P-light chains as well as the percentage of type II muscle fibres in vastus lateralis muscle. No significant relationships were determined for myosin light chain kinase activity and either P-light chain phosphorylation or type II fibre percentage. These data suggest that, unlike other mammalian fast muscles, P-light chain phosphorylation of mixed human muscles is not strongly associated with altered contractile performance.     

Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin

The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were significantly faster in phasic (0.57 +/- 0.06 s(-1)) than tonic (0.29 +/- 0.03 s(-1)) smooth muscles. Thiophosphorylation of regulatory light chains increased and strain decreased the release rate approximately twofold. The calculated (k-ADP/k+ADP) dissociation constant, Kd of unstrained, unphosphorylated cross-bridges for ADP was 0.6 microM for rabbit bladder and 0.3 microM for femoral artery. The rates of ADP release from rigor bridges and reported values of Pi release (corresponding to the steady-state adenosine triphosphatase (ATPase) rate of actomyosin (AM)) from cross-bridges during a maintained isometric contraction are similar, indicating that the ADP-release step or an isomerization preceding it may be limiting the adenosine triphosphatase rate. We conclude that the strain- and dephosphorylation-dependent high affinity for and slow ADP release from smooth muscle myosin prolongs the fraction of the duty cycle occupied by strongly bound actomyosin.ADP state(s) and contributes to the high economy of force.     

Novel sensors of the regulatory switch on the regulatory light chain of smooth muscle Myosin

Smooth muscle myosin can be switched on by phosphorylation of Ser-19 of the regulatory light chain. Our previous photocross-linking results suggested that an element of the structural mechanism for the regulatory switch was a phosphorylation-induced motion of the regulatory light chain N terminus (Wahlstrom, J. L., Randall, M. A., Jr., Lawson, J. D., Lyons, D. E., Siems, W. F., Crouch, G. J., Barr, R., Facemyer, K. C., and Cremo, C. R. (2003) J. Biol. Chem. 278, 5123-5131). Here we used three different approaches to test this notion, which are reactivity of cysteine thiols, pyrene and acrylodan spectral analysis, and pyrene fluorescence quenching. All methods detected significant differences between the unphosphorylated and phosphorylated regulatory light chain N termini in heavy meromyosin, a double-headed subfragment with an intact regulatory switch. These differences were not observed for subfragment-1, a single-headed, unregulated subfragment. In the presence of either ATP or ADP, phosphorylation increased the solvent exposure and decreased the polarity of the environment about position 23 of the regulatory light chain of heavy meromyosin. These phosphorylation-induced structural changes were not as evident in the absence of nucleotides. Nucleotide binding to unphosphorylated heavy meromyosin caused a decrease in exposure and an increase in polarity of the N terminus, whereas the effects of nucleotide on phosphorylated heavy meromyosin were the opposite. We showed a direct correlation between the kinetics of nucleotide binding/turnover and the conformational change reported by acrylodan at position 23 of the regulatory light chain. Acrylodan-A23C also reports the heads up (extended) to flexed (folded) transition in unphosphorylated heavy meromyosin. This is the first demonstration of direct coupling of nucleotide binding to conformational changes in the N terminus of the regulatory light chain.     

Myosin regulatory domain orientation in skeletal muscle fibers: application of novel electron paramagnetic resonance spectral decomposition and molecular modeling methods

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers using a modified method that results in near-stoichiometric levels of RLC and fully functional muscle. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (+/-15 degrees ) with the spin-label z axis parallel to actin, and a second population with a large distribution (+/-60 degrees ). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. The comparison with the electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10 degrees of the electron microscopy model.     

Myosin light chain 2 modulates calcium-sensitive cross-bridge transitions in vertebrate skeletal muscle.

We investigated the mechanism of the Ca2+ sensitivity of cross-bridge transitions that limit the rate of force development in vertebrate skeletal muscle. The rate of force development increases with Ca2+ concentration in the physiological range. We show here that at low concentrations of Ca2+ the rate of force development increases after partial extraction of the 20-kD light chain 2 subunit of myosin, whereas reconstitution with light chain 2 fully restores native sensitivity to Ca2+ in skinned single skeletal fibers. Furthermore, elevated free Mg2+ concentration reduces Ca2+ sensitivity, an effect that is reversed by extraction of the light chain but not by disruption of thin-filament activation by partial removal of troponin C, the Ca2+ binding protein of the thin filament. Our findings indicate that the Ca2+ sensitivity of the rate of force development in vertebrate skeletal muscle is mediated in part by the light chain 2 subunit of the myosin cross-bridge.