The role of action potential changes in depolarization-induced failure of excitation contraction coupling in mouse skeletal muscle
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Curated by eLife
Evaluation Summary:
This addresses an important area of excitation contraction coupling failure of potential clinical translational importance. They report that progressive depolarization of the resting potential upon excitation contraction coupling results in a persistence of action potential generation in the face of a failure of Ca2+ release.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K + is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a narrow range of resting potentials. Simultaneous imaging of Ca 2+ transients and recording of action potentials (APs) demonstrated failure to generate Ca 2+ transients when APs peaked at potentials more negative than –30mV. An AP property that closely correlated with failure of the Ca 2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca 2+ transients and APs with electrodes separated by 1.6mm revealed AP conduction fails when APs peak below –21mV. We hypothesize propagation of APs and generation of Ca 2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca 2+ release from the sarcoplasmic reticulum is governed by AP integral. The reason distinct AP properties may govern distinct steps of ECC is the kinetics of the ion channels involved. Na channels, which govern propagation, have rapid kinetics and are insensitive to AP width (and thus AP integral) whereas Ca 2+ release is governed by gating charge movement of Cav1.1 channels, which have slower kinetics such that Ca 2+ release is sensitive to AP integral. The quantitative relationships established between resting potential, AP properties, AP conduction and Ca 2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential.
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Author Response
Reviewer #2 (Public Review):
Strengths
The strongest aspect of this study is about the relationship between resting potential, action potential and calcium transient. This is the first study demonstrating such relationships and this was quite overdue.
Weaknesses
There is a misunderstanding about the all or none concept. The authors argued that some researchers had proposed that the potassium effects on force was an all or none effect, which is not true. It is true that the range of potassium concentrations and resting potentials over which the decreases in twitch forces is very narrow, it does not implicate an all or none concept because the decreases are steep but still gradual. The all or non concept for action potential does not implicate that the action potential shape is constant in all physiological …
Author Response
Reviewer #2 (Public Review):
Strengths
The strongest aspect of this study is about the relationship between resting potential, action potential and calcium transient. This is the first study demonstrating such relationships and this was quite overdue.
Weaknesses
There is a misunderstanding about the all or none concept. The authors argued that some researchers had proposed that the potassium effects on force was an all or none effect, which is not true. It is true that the range of potassium concentrations and resting potentials over which the decreases in twitch forces is very narrow, it does not implicate an all or none concept because the decreases are steep but still gradual. The all or non concept for action potential does not implicate that the action potential shape is constant in all physiological conditions; instead for a given physiological condition the action potential shape is constant but changes between physiological condition.
We have extensively rewritten the manuscript to remove the all or none concept.
There is a major lack of information about the calcium indicator that has been used for this study. Consequently, it is difficult to validate the relationship between peak calcium and resting potential or overshoot relationship. It is not clear whether the indicator get saturated with calcium and there is a major issue about a short twitch contraction in msec and a calcium transient lasting minutes.
We have added more information on the Ca2+ indicator dye to the manuscript. The reviewer is correct about potential issues of interpretation and a section on limitations has been added to the discussion. A point of clarification, the signal from GCAMP6f lasts 1 s, not minutes.
Finally the experimental temperature was 25°C when during exercise muscle temperature often exceeds 37°C. So, it will not be possible to use any of the relationships provided in this study to understand the role of potassium in fatigue because the potassium effects on all three parameters are very temperature sensitive.
This is an excellent point. We hope, however, that the reviewer will agree that the given the significant number of new experiments performed, repeating all of the experiments at 37 is beyond the scope of the current work. We plan to do studies at a more physiologic temperature in the future.
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Evaluation Summary:
This addresses an important area of excitation contraction coupling failure of potential clinical translational importance. They report that progressive depolarization of the resting potential upon excitation contraction coupling results in a persistence of action potential generation in the face of a failure of Ca2+ release.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors explore the effects of progressive depolarization of the resting potential upon excitation contraction coupling seeking to attribute its failure to either failure of action potential generation or of its subsequent coupling to Ca2+ release. The authors report a persistence of action potential generation in the face of a failure of Ca2+ release. The observation is empirically a helpful one in itself, but the further interpretation in the paper must address a number of issues require resolution bearing upon (a) whether there is a possible tubular action potential conduction failure despite persistent surface membrane excitation and (b) the relative inactivation properties of Na channel function and of excitation contraction coupling.
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Reviewer #2 (Public Review):
Summary
The objective of this study is to further understand the mechanism by which increases in extracellular [K+] affect action potential, calcium transient and twitch force. They show that the decreases in action potential overshoot and twitch force occur over a very narrow range of resting potential as others have demonstrated. What is new and interesting are the relationship between resting potential, action potential and peak calcium during twitch contractions. They demonstrated the gradual decrease in calcium peak over a narrow range of resting potential and a linear relationship between action potential area and peak calcium.
Strengths
The strongest aspect of this study is about the relationship between resting potential, action potential and calcium transient. This is the first study demonstrating …
Reviewer #2 (Public Review):
Summary
The objective of this study is to further understand the mechanism by which increases in extracellular [K+] affect action potential, calcium transient and twitch force. They show that the decreases in action potential overshoot and twitch force occur over a very narrow range of resting potential as others have demonstrated. What is new and interesting are the relationship between resting potential, action potential and peak calcium during twitch contractions. They demonstrated the gradual decrease in calcium peak over a narrow range of resting potential and a linear relationship between action potential area and peak calcium.
Strengths
The strongest aspect of this study is about the relationship between resting potential, action potential and calcium transient. This is the first study demonstrating such relationships and this was quite overdue.
Weaknesses
There is a misunderstanding about the all or none concept. The authors argued that some researchers had proposed that the potassium effects on force was an all or none effect, which is not true. It is true that the range of potassium concentrations and resting potentials over which the decreases in twitch forces is very narrow, it does not implicate an all or none concept because the decreases are steep but still gradual. The all or non concept for action potential does not implicate that the action potential shape is constant in all physiological conditions; instead for a given physiological condition the action potential shape is constant but changes between physiological condition.
There is a major lack of information about the calcium indicator that has been used for this study. Consequently, it is difficult to validate the relationship between peak calcium and resting potential or overshoot relationship. It is not clear whether the indicator get saturated with calcium and there is a major issue about a short twitch contraction in msec and a calcium transient lasting minutes.
Finally the experimental temperature was 25oC when during exercise muscle temperature often exceeds 37oC. So, it will not be possible to use any of the relationships provided in this study to understand the role of potassium in fatigue because the potassium effects on all three parameters are very temperature sensitive.
Impact of the study
Although the data about the effects on twitch force is nothing new, the data for the relationship between membrane potential and calcium peak is the first of his kind and extremely important for our understanding of the potassium effect on contraction and its role during fatigue.
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Reviewer #3 (Public Review):
This paper by Wang et al., presents an elegant approach to study the role of depolarization of the resting membrane potential for excitation-contraction-coupling in skeletal muscle with relevance for basic physiology and muscle disease. The study aims to provide a more detailed understanding of how depolarization affects skeletal muscle fiber excitability and action potential waveform, and how this in turn reduces Ca2+ release from the sarcoplasmic reticulum eventually leading to the loss of force that is associated with depolarization.
Methodologically, two intracellular electrodes are inserted into EDL muscle fibers from mice. The electrodes are used to trigger and record single fiber action potentials in conjunction with recordings of intracellular Ca2+ transients reported by genetically encoded Ca2+ …
Reviewer #3 (Public Review):
This paper by Wang et al., presents an elegant approach to study the role of depolarization of the resting membrane potential for excitation-contraction-coupling in skeletal muscle with relevance for basic physiology and muscle disease. The study aims to provide a more detailed understanding of how depolarization affects skeletal muscle fiber excitability and action potential waveform, and how this in turn reduces Ca2+ release from the sarcoplasmic reticulum eventually leading to the loss of force that is associated with depolarization.
Methodologically, two intracellular electrodes are inserted into EDL muscle fibers from mice. The electrodes are used to trigger and record single fiber action potentials in conjunction with recordings of intracellular Ca2+ transients reported by genetically encoded Ca2+ sensing GCAMP6f. Muscle movement is reduced using an inhibitor of the contractile filaments.
The study clearly shows that depolarizing muscle with elevation of extracellular K+ from 3.5 to 10 mM causes increased twitch force. When further elevating extracellular K+, the twitch force declines and at 14 - 16 mM K+ the twitch force is largely abolished. The increase in force at moderately elevated K+ (10 mM) remains unclear especially because it is associated with clear reduction in action potential peak. At high extracellular K+ (12-16 mM) the action potentials become further compromised. In a separate series of experiments, muscle fibers are excited to trigger action potentials every 5 seconds for 7 mins either at 3.5 mM K+ throughout or initially at 3.5 mM K+ but during the 7 minutes a sudden elevation of extracellular K+ to 16 mM is imposed. This series of experiments is productive because it gives both action potential wave form characteristics and Ca2+ transients from the same muscle fibers as the resting membrane potential changes throughout the experiment. The approach therefore provides co-temporal measurements of action potentials and Ca2+ transients that enable correlations to be made between action potential waveform characteristics and the amplitude of the Ca2+ transients.
A fundamental and important finding of the study is that Ca2+ release is highly dependent on the action potential shape showing that changes in action potential waveform such as reduced amplitude and altered width of the action potential are decisive of Ca2+ release. The authors analyze this in more details and arrives at the conclusion that the integral of action potential waveform more depolarized than -30 mV is the best predictor of Ca2+ release.
The data is discussed in relation to loss of force in depolarized muscles, and the authors challenge an existing notion of loss of force in depolarized muscle being caused by all-or-none excitation failure. This notion stems from the close correlation between force and propagating compound muscle action potentials in isolated muscle at elevated extracellular K+ that has been reported in other studies. The present study shows that changes in action potential waveform with reduced waveform integral more depolarized above - 30 mV could reduce Ca2+ release and force without representing a complete loss of excitation. This is then taken to a discussion on how to best define an action potential.
Some considerations regarding the methodology deserve to be mentioned. The electrodes that are inserted in the muscle fibers appear to be at very close proximity and hence the recordings would not be propagating action potentials but rather action potentials or local responses around the recording electrode. Similarly, the Ca2+ transients are also recorded in this same small part of the muscle fiber. On this basis it remains unclear whether the compromised action potentials at extracellular K+ above 10 mM would propagate along the entire muscle fiber to trigger contractions or merely be local responses at the site of the electrodes. Another methodological aspect to mention is that the sampling in the Ca2+ imaging appears slow relative to the rapid upstroke and spike of the Ca2+ transient. The authors acknowledge and discuss these limitations but still draw conclusions based on the data of the recorded amplitude of the Ca2+ transient. Additional experiments that measures propagating action potentials and Ca2+ imaging/fluorescence systems with higher sampling rate and better suited for detecting peaks of Ca2+ transient will be needed to fully validate some of the rather strong conclusions by the authors.
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