A two-faced cysteine residue modulates skeletal muscle contraction. Focus on “S-nitrosylation and S-glutathionylation of Cys134 on troponin I have opposing competitive actions on Ca2+ sensitivity in rat fast-twitch muscle fibers

To test the hypothesis that the vasodilator complement that produces arteriolar vasodilation during muscle contraction depends on both stimulus and contraction frequency, we stimulated four to five skeletal muscle fibers in the anesthetized hamster cremaster preparation in situ and measured the change in diameter of arterioles at a site of overlap with the stimulated muscle fibers. Diameter was measured before, during, and after 2 min of skeletal muscle contraction stimulated over a range of stimulus frequencies [4, 20, and 40 Hz; 15 contractions/min (cpm), 250 ms train duration] and a range of contraction frequencies (6, 15, and 60 cpm; 20 Hz stimulus frequency, 250 ms train duration). Muscle fibers were stimulated in the absence and presence of an inhibitor of adenosine receptors [10−6 M xanthine amine congener (XAC)], an ATP-dependent potassium (K+) channel inhibitor (10−5 M glibenclamide), an inhibitor of a source of K+ by inhibition of voltage-dependent K+ channels [3 × 10−4 M 3,4-diaminopyridine (DAP)], and an inhibitor of nitric oxide synthase [10−6 M NG-nitro-l-arginine methyl ester (l-NAME) + 10−7 S-nitroso- N-acetylpenicillamine (a nitric oxide donor)]. l-NAME inhibited the dilations at all stimulus frequencies and contraction frequencies except 60 cpm. XAC inhibited the dilations at all contraction frequencies and stimulus frequencies except 40 Hz. Glibenclamide inhibited all dilations at all stimulus and contraction frequencies, and DAP did not inhibit dilations at any stimulus frequencies while attenuating dilation at a contraction frequency of 60 cpm only. Our data show that the complement of dilators responsible for the vasodilations induced by skeletal muscle contraction differed depending on the stimulus and contraction frequency; therefore, both are important determinants of the dilators involved in the processes of arteriolar vasodilation associated with active hyperemia.

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Diaphragmatic lymphatic vessel behavior during local skeletal muscle contraction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00701.2014 ◽

2015 ◽

Vol 308 (3) ◽

pp. H193-H205 ◽

Cited By ~ 13

Author(s):

Andrea Moriondo ◽

Eleonora Solari ◽

Cristiana Marcozzi ◽

Daniela Negrini

Keyword(s):

Skeletal Muscle ◽

Muscle Contraction ◽

Lymphatic Vessel ◽

Muscle Fibers ◽

Video Recording ◽

Three Dimensional ◽

Longitudinal Axis ◽

Vessel Diameter ◽

Skeletal Muscle Contraction ◽

Lymphatic Network

The mechanism through which the stresses developed in the diaphragmatic tissue during skeletal muscle contraction sustain local lymphatic function was studied in 10 deeply anesthetized, tracheotomized adult Wistar rats whose diaphragm was exposed after thoracotomy. To evaluate the direct effect of skeletal muscle contraction on the hydraulic intraluminal lymphatic pressures (Plymph) and lymphatic vessel geometry, the maximal contraction of diaphragmatic fibers adjacent to a lymphatic vessel was elicited by injection of 9.2 nl of 1 M KCl solution among diaphragmatic fibers while Plymph was recorded through micropuncture and vessel geometry via stereomicroscopy video recording. In lymphatics oriented perpendicularly to the longitudinal axis of muscle fibers and located at <300 μm from KCl injection, vessel diameter at maximal skeletal muscle contraction ( Dmc) decreased to 61.3 ± 1.4% of the precontraction value [resting diameter ( Drest)]; however, if injection was at >900 μm from the vessel, Dmc enlarged to 131.1 ± 2.3% of Drest. In vessels parallel to muscle fibers, Dmc increased to 122.8 ± 2.9% of Drest. During contraction, Plymph decreased as much as 22.5 ± 2.6 cmH2O in all submesothelial superficial vessels, whereas it increased by 10.7 ± 5.1 cmH2O in deeper vessels running perpendicular to contracting muscle fibers. Hence, the three-dimensional arrangement of the diaphragmatic lymphatic network seems to be finalized to efficiently exploit the stresses exerted by muscle fibers during the contracting inspiratory phase to promote lymph formation in superficial submesothelial lymphatics and its further propulsion in deeper intramuscular vessels.

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S-nitrosylation and S-glutathionylation of Cys134 on troponin I have opposing competitive actions on Ca2+ sensitivity in rat fast-twitch muscle fibers

AJP Cell Physiology ◽

10.1152/ajpcell.00334.2016 ◽

2017 ◽

Vol 312 (3) ◽

pp. C316-C327 ◽

Cited By ~ 25

Author(s):

T. L. Dutka ◽

J. P. Mollica ◽

C. R. Lamboley ◽

V. C. Weerakkody ◽

D. W. Greening ◽

...

Keyword(s):

Troponin I ◽

Muscle Fibers ◽

Contractile Apparatus ◽

Slow Twitch ◽

Mechanistic Basis ◽

Opposing Effects ◽

Slow Twitch Fibers ◽

Competitive Actions ◽

Fast Twitch ◽

Biotin Switch

Nitric oxide is generated in skeletal muscle with activity and decreases Ca2+ sensitivity of the contractile apparatus, putatively by S-nitrosylation of an unidentified protein. We investigated the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca2+ sensitivity in mammalian fast-twitch (FT) fibers mediated by S-glutathionylation of Cys134 on fast troponin I (TnIf). Force-[Ca2+] characteristics of the contractile apparatus in mechanically skinned fibers were assessed by direct activation with heavily Ca2+-buffered solutions. Treatment with S-nitrosylating agents, S-nitrosoglutathione (GSNO) or S-nitroso- N-acetyl-penicillamine (SNAP), decreased pCa50 ( = −log10 [Ca2+] at half-maximal activation) by ~−0.07 pCa units in rat and human FT fibers without affecting maximum force, but had no effect on rat and human slow-twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca2+ sensitivity decrease was 1) fully reversed with dithiothreitol or reduced glutathione, 2) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and 3) irreversibly blocked by low concentration of the alkylating agent, N-ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused S-nitrosylation of TnIf. S-glutathionylation pretreatment blocked the effects of S-nitrosylation on Ca2+ sensitivity, and vice-versa. S-nitrosylation pretreatment prevented NEM from irreversibly blocking S-glutathionylation of TnIf and its effects on Ca2+ sensitivity, and likewise S-glutathionylation pretreatment prevented NEM block of S-nitrosylation. Following substitution of TnIf into rat slow-twitch fibers, S-nitrosylation treatment caused decreased Ca2+ sensitivity. These findings demonstrate that S-nitrosylation and S-glutathionylation exert opposing effects on Ca2+ sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnIf.

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Deep muscle-proteomic analysis of freeze-dried human muscle biopsies reveals fiber type-specific adaptations to exercise training

Nature Communications ◽

10.1038/s41467-020-20556-8 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

A. S. Deshmukh ◽

D. E. Steenberg ◽

M. Hostrup ◽

J. B. Birk ◽

J. K. Larsen ◽

...

Keyword(s):

Skeletal Muscle ◽

Exercise Training ◽

Fiber Type ◽

Muscle Fibers ◽

Human Muscle ◽

Fiber Types ◽

Freeze Dried ◽

Fast Twitch Muscle ◽

Fast Twitch ◽

Muscle Biopsies

AbstractSkeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

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Lymph flow pattern in pleural diaphragmatic lymphatics during intrinsic and extrinsic isotonic contraction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00640.2015 ◽

2016 ◽

Vol 310 (1) ◽

pp. H60-H70 ◽

Cited By ~ 10

Author(s):

Andrea Moriondo ◽

Eleonora Solari ◽

Cristiana Marcozzi ◽

Daniela Negrini

Keyword(s):

Skeletal Muscle ◽

Shear Stress ◽

Muscle Contraction ◽

Ex Vivo ◽

Muscle Fibers ◽

Lymphatic Vessels ◽

Smooth Muscle Contraction ◽

Skeletal Muscle Fibers ◽

Skeletal Muscle Contraction

Peripheral rat diaphragmatic lymphatic vessels, endowed with intrinsic spontaneous contractility, were in vivo filled with fluorescent dextrans and microspheres and subsequently studied ex vivo in excised diaphragmatic samples. Changes in diameter and lymph velocity were detected, in a vessel segment, during spontaneous lymphatic smooth muscle contraction and upon activation, through electrical whole-field stimulation, of diaphragmatic skeletal muscle fibers. During intrinsic contraction lymph flowed both forward and backward, with a net forward propulsion of 14.1 ± 2.9 μm at an average net forward speed of 18.0 ± 3.6 μm/s. Each skeletal muscle contraction sustained a net forward-lymph displacement of 441.9 ± 159.2 μm at an average velocity of 339.9 ± 122.7 μm/s, values significantly higher than those documented during spontaneous contraction. The flow velocity profile was parabolic during both spontaneous and skeletal muscle contraction, and the shear stress calculated at the vessel wall at the highest instantaneous velocity never exceeded 0.25 dyne/cm2. Therefore, we propose that the synchronous contraction of diaphragmatic skeletal muscle fibers recruited at every inspiratory act dramatically enhances diaphragmatic lymph propulsion, whereas the spontaneous lymphatic contractility might, at least in the diaphragm, be essential in organizing the pattern of flow redistribution within the diaphragmatic lymphatic circuit. Moreover, the very low shear stress values observed in diaphragmatic lymphatics suggest that, in contrast with other contractile lymphatic networks, a likely interplay between intrinsic and extrinsic mechanisms be based on a mechanical and/or electrical connection rather than on nitric oxide release.

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Local and remote arteriolar dilations initiated by skeletal muscle contraction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.5.h2285 ◽

2000 ◽

Vol 279 (5) ◽

pp. H2285-H2294 ◽

Cited By ~ 45

Author(s):

Coral L. Murrant ◽

Ingrid H. Sarelius

Keyword(s):

Skeletal Muscle ◽

Muscle Contraction ◽

Muscle Fibers ◽

Muscle Metabolism ◽

Cremaster Muscle ◽

Remote Site ◽

Upstream Site ◽

Skeletal Muscle Contraction ◽

Perivascular Nerves ◽

The Relationship

To investigate the relationship between skeletal muscle metabolism and arteriolar dilations in the region local to contracting muscle fibers as well as dilations at remote arteriolar regions upstream, we used a microelectrode on cremaster muscle of anesthetized hamsters to stimulate four to five muscle fibers lying approximately perpendicular to and overlapping a transverse arteriole. Before, during, and after muscle contraction, we measured the diameter of the arteriole at the site of muscle fiber overlap (local) and at a remote site ∼1,000 μm upstream. Two minutes of 2-, 4-, or 8-Hz stimulation (5–10 V, 0.4-ms duration) produced a significant dilation locally (8.2 ± 2.0-, 22.5 ± 2.4-, and 30.9 ± 2.1-μm increase, respectively) and at the remote site (4.2 ± 0.8, 11.0 ± 1.1, and 18.9 ± 2.7 μm, respectively). Muscle contraction at 4 Hz initiated a remote dilation that was unaffected by 15-min micropipette application of either 2 μM tetrodotoxin, 0.07% halothane, or 40 μM 18-β-glycyrrhetinic acid between the local and upstream site. Therefore, at the arteriolar level, muscle contraction initiates a robust remote dilation that does not appear to be transmitted via perivascular nerves or gap junctions.

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Phosphorylation of the regulatory light chains of myosin affects Ca2+sensitivity of skeletal muscle contraction

Journal of Applied Physiology ◽

10.1152/japplphysiol.00858.2001 ◽

2002 ◽

Vol 92 (4) ◽

pp. 1661-1670 ◽

Cited By ~ 67

Author(s):

Danuta Szczesna ◽

Jiaju Zhao ◽

Michelle Jones ◽

Gang Zhi ◽

James Stull ◽

...

Keyword(s):

Skeletal Muscle ◽

Steady State ◽

Muscle Contraction ◽

Muscle Fibers ◽

Light Chains ◽

Regulatory Light Chains ◽

Force Development ◽

Skeletal Muscle Contraction ◽

State Force ◽

Kinetics Of

The role of phosphorylation of the myosin regulatory light chains (RLC) is well established in smooth muscle contraction, but in striated (skeletal and cardiac) muscle its role is still controversial. We have studied the effects of RLC phosphorylation in reconstituted myosin and in skinned skeletal muscle fibers where Ca2+sensitivity and the kinetics of steady-state force development were measured. Skeletal muscle myosin reconstituted with phosphorylated RLC produced a much higher Ca2+sensitivity of thin filament-regulated ATPase activity than nonphosphorylated RLC (change in −log of the Ca2+concentration producing half-maximal activation = ∼0.25). The same was true for the Ca2+sensitivity of force in skinned skeletal muscle fibers, which increased on reconstitution of the fibers with the phosphorylated RLC. In addition, we have shown that the level of endogenous RLC phosphorylation is a crucial determinant of the Ca2+sensitivity of force development. Studies of the effects of RLC phosphorylation on the kinetics of force activation with the caged Ca2+, DM-nitrophen, showed a slight increase in the rates of force development with low statistical significance. However, an increase from 69 to 84% of the initial steady-state force was observed when nonphosphorylated RLC-reconstituted fibers were subsequently phosphorylated with exogenous myosin light chain kinase. In conclusion, our results suggest that, although Ca2+binding to the troponin-tropomyosin complex is the primary regulator of skeletal muscle contraction, RLC play an important modulatory role in this process.

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