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The presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists in skinned rat skeletal muscle fibres.

Duke AM, Steele DS - Cell Calcium (2008)

Bottom Line: When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05).However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished.These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.

View Article: PubMed Central - PubMed

Affiliation: Institute of Membrane and Systems Biology, University of Leeds, Woodhouse Lane, Leeds LS29JT, United Kingdom.

ABSTRACT
Single mechanically skinned extensor digitorum Longus (EDL) rat fibres were used as a model to study the influence of functional t-tubules on the properties of RyR1 in adult skeletal muscle. Fibres were superfused with solutions approximating to the intracellular milieu. Following skinning, the t-tubules re-seal and repolarise, allowing the sarcoplasmic reticulum (SR) Ca2+ release to be activated by field stimulation. However, in the present study, some fibres exhibited localised regions where depolarisation-induced SR Ca2+ release was absent, due to failure of the t-tubules to re-seal. When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05). This difference in RyR1 sensitivity was unaffected by sustained depolarisation of the t-tubule network. However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished. These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.

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Patchy Ca2+ release in mechanically skinned EDL fibres. (A) Composite transmission image of a mechanically skinned EDL muscle fibre produced by taking a series of x–y frames along its length. In this example, the sarcolemma was not removed along the entire fibre length allowing the “cuff” to be seen. The box (broken line) illustrates the maximum length of fibre observable in a single frame under 40× magnification. (B) Sequential x–y images (6.2 Hz) from a selected segment of a skinned fibre, stimulated repeatedly at 50 Hz for 400 ms. The graph shows changes in fluo-3 fluorescence, where each point was obtained by averaging the pixels within a single x–y frame. The individual frames obtained during the peak of each transient show that the tetanic responses are reproducible and that Ca2+ release is relatively uniform (above). A series of sequential x–y frames obtained during a single tetanic response is also shown (right) and a longitudinal line scan image obtained during a tetanic response (below). (C) Composite image showing peak tetanic [Ca2+] throughout the length of a mounted EDL fibre. Expanded sections reveal localised regions of Ca2+ release failure.
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fig1: Patchy Ca2+ release in mechanically skinned EDL fibres. (A) Composite transmission image of a mechanically skinned EDL muscle fibre produced by taking a series of x–y frames along its length. In this example, the sarcolemma was not removed along the entire fibre length allowing the “cuff” to be seen. The box (broken line) illustrates the maximum length of fibre observable in a single frame under 40× magnification. (B) Sequential x–y images (6.2 Hz) from a selected segment of a skinned fibre, stimulated repeatedly at 50 Hz for 400 ms. The graph shows changes in fluo-3 fluorescence, where each point was obtained by averaging the pixels within a single x–y frame. The individual frames obtained during the peak of each transient show that the tetanic responses are reproducible and that Ca2+ release is relatively uniform (above). A series of sequential x–y frames obtained during a single tetanic response is also shown (right) and a longitudinal line scan image obtained during a tetanic response (below). (C) Composite image showing peak tetanic [Ca2+] throughout the length of a mounted EDL fibre. Expanded sections reveal localised regions of Ca2+ release failure.

Mentions: While studies involving force measurements on mechanically skinned skeletal muscle fibres typically use sections 2–3 mm in length, confocal imaging systems allow only a fraction of the preparation to be viewed at one time. Fig. 1A shows a transmission image of a mechanically skinned rat EDL muscle fibre (∼2–3 mm long), tethered at each end using monofilament snares within stainless steel tubes. In this case, an image of the entire mounted fibre was produced by taking a series of x–y frames along its length, from which a composite image was constructed. The box (broken line) illustrates the maximum length of fibre that could be observed in a single frame under 40× magnification. Although this varies depending on optical arrangement and fibre orientation, this is typically ∼200–300 μm.


The presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists in skinned rat skeletal muscle fibres.

Duke AM, Steele DS - Cell Calcium (2008)

Patchy Ca2+ release in mechanically skinned EDL fibres. (A) Composite transmission image of a mechanically skinned EDL muscle fibre produced by taking a series of x–y frames along its length. In this example, the sarcolemma was not removed along the entire fibre length allowing the “cuff” to be seen. The box (broken line) illustrates the maximum length of fibre observable in a single frame under 40× magnification. (B) Sequential x–y images (6.2 Hz) from a selected segment of a skinned fibre, stimulated repeatedly at 50 Hz for 400 ms. The graph shows changes in fluo-3 fluorescence, where each point was obtained by averaging the pixels within a single x–y frame. The individual frames obtained during the peak of each transient show that the tetanic responses are reproducible and that Ca2+ release is relatively uniform (above). A series of sequential x–y frames obtained during a single tetanic response is also shown (right) and a longitudinal line scan image obtained during a tetanic response (below). (C) Composite image showing peak tetanic [Ca2+] throughout the length of a mounted EDL fibre. Expanded sections reveal localised regions of Ca2+ release failure.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571965&req=5

fig1: Patchy Ca2+ release in mechanically skinned EDL fibres. (A) Composite transmission image of a mechanically skinned EDL muscle fibre produced by taking a series of x–y frames along its length. In this example, the sarcolemma was not removed along the entire fibre length allowing the “cuff” to be seen. The box (broken line) illustrates the maximum length of fibre observable in a single frame under 40× magnification. (B) Sequential x–y images (6.2 Hz) from a selected segment of a skinned fibre, stimulated repeatedly at 50 Hz for 400 ms. The graph shows changes in fluo-3 fluorescence, where each point was obtained by averaging the pixels within a single x–y frame. The individual frames obtained during the peak of each transient show that the tetanic responses are reproducible and that Ca2+ release is relatively uniform (above). A series of sequential x–y frames obtained during a single tetanic response is also shown (right) and a longitudinal line scan image obtained during a tetanic response (below). (C) Composite image showing peak tetanic [Ca2+] throughout the length of a mounted EDL fibre. Expanded sections reveal localised regions of Ca2+ release failure.
Mentions: While studies involving force measurements on mechanically skinned skeletal muscle fibres typically use sections 2–3 mm in length, confocal imaging systems allow only a fraction of the preparation to be viewed at one time. Fig. 1A shows a transmission image of a mechanically skinned rat EDL muscle fibre (∼2–3 mm long), tethered at each end using monofilament snares within stainless steel tubes. In this case, an image of the entire mounted fibre was produced by taking a series of x–y frames along its length, from which a composite image was constructed. The box (broken line) illustrates the maximum length of fibre that could be observed in a single frame under 40× magnification. Although this varies depending on optical arrangement and fibre orientation, this is typically ∼200–300 μm.

Bottom Line: When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05).However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished.These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.

View Article: PubMed Central - PubMed

Affiliation: Institute of Membrane and Systems Biology, University of Leeds, Woodhouse Lane, Leeds LS29JT, United Kingdom.

ABSTRACT
Single mechanically skinned extensor digitorum Longus (EDL) rat fibres were used as a model to study the influence of functional t-tubules on the properties of RyR1 in adult skeletal muscle. Fibres were superfused with solutions approximating to the intracellular milieu. Following skinning, the t-tubules re-seal and repolarise, allowing the sarcoplasmic reticulum (SR) Ca2+ release to be activated by field stimulation. However, in the present study, some fibres exhibited localised regions where depolarisation-induced SR Ca2+ release was absent, due to failure of the t-tubules to re-seal. When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05). This difference in RyR1 sensitivity was unaffected by sustained depolarisation of the t-tubule network. However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished. These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.

Show MeSH
Related in: MedlinePlus