The mechanism underlying transient weakness in myotonia congenita
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Evaluation Summary:
Patients with myotonia congenita (or Becker disease) experience episodes of transient muscle weakness but the reasons underlying this phenomenon are unknown. This study provides the most definitive experimental evidence to date for the mechanistic basis of transient weakness in myotonia congenita and also suggests ranolazine may be beneficial for prophylactic management.
(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. Reviewers #1, #2, and #3 agreed to share their names with the authors.)
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Abstract
In addition to the hallmark muscle stiffness, patients with recessive myotonia congenita (Becker disease) experience debilitating bouts of transient weakness that remain poorly understood despite years of study. We performed intracellular recordings from muscle of both genetic and pharmacologic mouse models of Becker disease to identify the mechanism underlying transient weakness. Our recordings reveal transient depolarizations (plateau potentials) of the membrane potential to −25 to −35 mV in the genetic and pharmacologic models of Becker disease. Both Na + and Ca 2+ currents contribute to plateau potentials. Na + persistent inward current (NaPIC) through Na V 1.4 channels is the key trigger of plateau potentials and current through Ca V 1.1 Ca 2+ channels contributes to the duration of the plateau. Inhibiting NaPIC with ranolazine prevents the development of plateau potentials and eliminates transient weakness in vivo. These data suggest that targeting NaPIC may be an effective treatment to prevent transient weakness in myotonia congenita.
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Response to Reviewer #3 (Public Review):
Major Points:
- The major experimental limitation that prevented prior studies from establishing the mechanism for the transiently reduced excitability and weakness in MC was the concern that plateau depolarizations frequently occur as an artifact in studies of skeletal muscle membrane potential (e.g. secondary to leakage current from electrode impalement or failure to completely suppress contraction with motion-induced damage). The authors are to be commended for including many records of Vm (absolutely necessary for this publication) and for explicitly stating that a holding current was not applied to maintain Vrest. The confidence of these observation could be further increased by addressing these questions:
— Were recordings excluded from the analysis if the plateau potential was not followed …
Response to Reviewer #3 (Public Review):
Major Points:
- The major experimental limitation that prevented prior studies from establishing the mechanism for the transiently reduced excitability and weakness in MC was the concern that plateau depolarizations frequently occur as an artifact in studies of skeletal muscle membrane potential (e.g. secondary to leakage current from electrode impalement or failure to completely suppress contraction with motion-induced damage). The authors are to be commended for including many records of Vm (absolutely necessary for this publication) and for explicitly stating that a holding current was not applied to maintain Vrest. The confidence of these observation could be further increased by addressing these questions:
— Were recordings excluded from the analysis if the plateau potential was not followed by a subsequent return to Vrest? Was a criterion used to define successful return to the resting potential?
Recordings were excluded. The criterion used was a return of Vm to within 4 mV of the pre-stimulation resting potential.
— If fibers that failed to repolarize were excluded, was this a frequent or a rare event, and importantly, was the likelihood of failure different for control versus myotonic fibers?
We have now added quantitation of the percent of recordings excluded and the criteria for exclusion to the manuscript. Lack of repolarization to within 4 mV of the prior resting potential following plateau potentials never occurred in 9AC treated fibers (0/49 fibers) and occurred in 10/36 ClCadr fibers.
— The data clearly show a large variance for the duration of the plateau potential (e.g. horizontal extent of data in Fig. 3B), which is interesting and may provide additional insights on the balance of currents that contribute to this phenomenon. The authors also point out that the distribution was skewed toward briefer plateau periods for the 9-AC model than the adr mouse. It is suggested this difference may be a consequence of life-long reduced gCl in adr mice with some chronic compensation versus the acute block of ClC-1 in the 9-AC model. What about the possibility that the reduction of gCl is more severe in the adr fibers than in 9-AC treated animals? A residual Cl current could foreshorten the duration of the plateau potential. Another question with regard to the variable duration of the plateau potential is a "duration of 0". In other words, as shown in Fig 3C, how frequently was the absence of a PP encountered?
This is an excellent point regarding residual ClC-1 conductance following 9-AC, which is now included in a new section in the discussion.
In regards to the second point about fibers lacking plateau potentials, the frequency is included in the manuscript. 92% of 9AC treated fibers had plateau potentials, while only 30% of ClCadr fibers had plateau potentials. It is notable that plateau potentials are less frequent in ClCadr fibers, but when they occur, they are longer. This is now mentioned in the discussion.
— The possibility that activity-dependent accumulation of myoplasmic Ca may contribute to the PP is suggested (page 9 line 175), but this is not further commented upon in the Discussion. Namely, is the reduction of PP duration in ncDHPR fibers proposed to be a consequence of less inward charge movement or of less myoplasmic Ca accumulation (i.e. is it a balance of ionic currents or an intracellular signaling factor)? Moreover, with regard to an activity-dependent process that influences the likelihood and/or duration of the PP, the authors quantify the "mean firing rate" and the "mean membrane potential", both quantified during the preceding myotonic burst. Both of these factors may contribute to an activity-dependent process, but another factor has been omitted; namely the duration of the antecedent myotonic run. It would be interesting to test whether the duration of the myotonic burst had an influence on the PP.
These are two excellent points.
Regarding the first point, a brief discussion of how Ca flux through Cav1.1 channels might prolong plateau potentials has now been added to the discussion. We have deferred study of the role of intracellular Ca in sustaining plateau potentials to future studies.
In response to the second point regarding the role of duration of myotonia in triggering plateau potentials, we examined whether the duration of prior myotonia correlates with development of plateau potentials. We have added a new figure (Fig 4) showing the repeated occurrence of plateau potentials within individual fibers in response to repeated stimulation. The triggering of plateau potentials does not appear to correlate with duration of preceding myotonia.
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Reviewer #3 (Public Review):
Myotonia congenita is a heritable disorder of muscle fiber excitability in which a severe reduction of the resting chloride conductance (gCl, CLCN1 mutations) produces susceptibility to involuntary after-contractions and transient weakness. Fifty years ago, Bryant, Adrian and colleagues showed that loss of > 50% of gCl is sufficient to cause myotonic bursts of after-discharges. Much less is known about the mechanistic basis for the transient weakness (several seconds, up to 1 minute) that occurs with initial contractions after rest. This study elegantly confirms what has long been suspected; that sustained depolarization of the resting potential is the basis for the transient weakness. The experimental approach employed several new techniques to achieve this demonstration. First, the use of repeated in situ …
Reviewer #3 (Public Review):
Myotonia congenita is a heritable disorder of muscle fiber excitability in which a severe reduction of the resting chloride conductance (gCl, CLCN1 mutations) produces susceptibility to involuntary after-contractions and transient weakness. Fifty years ago, Bryant, Adrian and colleagues showed that loss of > 50% of gCl is sufficient to cause myotonic bursts of after-discharges. Much less is known about the mechanistic basis for the transient weakness (several seconds, up to 1 minute) that occurs with initial contractions after rest. This study elegantly confirms what has long been suspected; that sustained depolarization of the resting potential is the basis for the transient weakness. The experimental approach employed several new techniques to achieve this demonstration. First, the use of repeated in situ contraction tests every 4 sec (Fig. 1) clearly shows the coincidence of myotonia and transient weakness, both of which exhibit warm-up. This animal model for the transient weakness in a low gCl state was essential for the success of this study. Secondly, the remarkably stable measurements of membrane potential (Vm), without the need to apply a holding current to achieve the normal resting potential (Figure 2) is necessary to convincingly demonstrate the plateau depolarizations are a consequence of the myotonic condition, and not a stimulation artifact. Moreover, a severe reduction of fiber excitability was directly demonstrated by application of brief current pulses during the plateau depolarization (Figure 2E). Third, the authors have used the ncDHPR mouse (non-conducting CaV1.1) to show the Ca current has some role in prolonging the duration of the plateau. This is an important contribution because the sluggish, low-amplitude Ca current in skeletal muscle has not previously been implicated in the pathogenesis of myotonia. Finally, the authors built upon their recent work showing ranolazine suppresses myotonia in low gCl muscle to also show this drug abolishes the plateau potential. Taken together, this excellent study provides the most definitive experimental evidence to date for the mechanistic basis of transient weakness in myotonia congenita and also suggests ranolazine may be beneficial for prophylactic management.
Major Points:
- The major experimental limitation that prevented prior studies from establishing the mechanism for the transiently reduced excitability and weakness in MC was the concern that plateau depolarizations frequently occur as an artifact in studies of skeletal muscle membrane potential (e.g. secondary to leakage current from electrode impalement or failure to completely suppress contraction with motion-induced damage). The authors are to be commended for including many records of Vm (absolutely necessary for this publication) and for explicitly stating that a holding current was not applied to maintain Vrest. The confidence of these observation could be further increased by addressing these questions:
— Were recordings excluded from the analysis if the plateau potential was not followed by a subsequent return to Vrest? Was a criterion used to define successful return to the resting potential?
— If fibers that failed to repolarize were excluded, was this a frequent or a rare event, and importantly, was the likelihood of failure different for control versus myotonic fibers?
The data clearly show a large variance for the duration of the plateau potential (e.g. horizontal extent of data in Figure 3B), which is interesting and may provide additional insights on the balance of currents that contribute to this phenomenon. The authors also point out that the distribution was skewed toward briefer plateau periods for the 9-AC model than the adr mouse. It is suggested this difference may be a consequence of life-long reduced gCl in adr mice with some chronic compensation versus the acute block of ClC-1 in the 9-AC model. What about the possibility that the reduction of gCl is more severe in the adr fibers than in 9-AC treated animals? A residual Cl current could foreshorten the duration of the plateau potential. Another question with regard to the variable duration of the plateau potential is a "duration of 0". In other words, as shown in Fig 3C, how frequently was the absence of a PP encountered?
The possibility that activity-dependent accumulation of myoplasmic Ca may contribute to the PP is suggested (page 9 line 175), but this is not further commented upon in the Discussion. Namely, is the reduction of PP duration in ncDHPR fibers proposed to be a consequence of less inward charge movement or of less myoplasmic Ca accumulation (i.e. is it a balance of ionic currents or an intracellular signaling factor)? Moreover, with regard to an activity-dependent process that influences the likelihood and/or duration of the PP, the authors quantify the "mean firing rate" and the "mean membrane potential", both quantified during the preceding myotonic burst. Both of these factors may contribute to an activity-dependent process, but another factor has been omitted; namely the duration of the antecedent myotonic run. It would be interesting to test whether the duration of the myotonic burst had an influence on the PP.
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Reviewer #2 (Public Review):
The manuscript by Myers et al provides new insight into the mechanism of transient muscle in myotonia congenita, a question that has escaped understanding since its first description over >40 years ago. The authors use a complementary set of approaches (including measurements of in situ muscle force production, membrane voltage and ion currents) to determine the membrane conductances that underlie transient weakness in muscle from both genetic (Clc1-/- adr mice) and pharmacologic (9-AC-treated WT mice) models of myotonia congenita. The authors utilize a combination of a non-conducting Cav1.1 mouse and treatment with ranolazine to dissect the relative contribution of Cav1.1 and persistent Nav1.4 conductances, respectively, to sustained plateau membrane depolarizations observed following myotonic runs, which …
Reviewer #2 (Public Review):
The manuscript by Myers et al provides new insight into the mechanism of transient muscle in myotonia congenita, a question that has escaped understanding since its first description over >40 years ago. The authors use a complementary set of approaches (including measurements of in situ muscle force production, membrane voltage and ion currents) to determine the membrane conductances that underlie transient weakness in muscle from both genetic (Clc1-/- adr mice) and pharmacologic (9-AC-treated WT mice) models of myotonia congenita. The authors utilize a combination of a non-conducting Cav1.1 mouse and treatment with ranolazine to dissect the relative contribution of Cav1.1 and persistent Nav1.4 conductances, respectively, to sustained plateau membrane depolarizations observed following myotonic runs, which are proposed to underlie the transient weakness observed following myotonic runs.
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Reviewer #1 (Public Review):
Patients with myotonia congenita caused by loss-of-function mutations in ClC-1 experience muscle stiffness (due to hyperexcitability) as well as transient muscle weakness. This study examines the mechanisms underlying the transient muscle weakness seen myotonia congenita. The authors show that a ClC-1 null mouse exhibits the transient weakness after muscle stimulation observed in humans. Current clamp recordings of muscle fibers from ClC-1-null mice showed indicated myotonia after electrical stimulation that often terminated in a plateau potential for varying periods, during which the muscle was unexcitable, before repolarization to the resting membrane potential. The myotonia and plateau potentials could be recapitulated in wild type muscle fibers with acute pharmacological inhibition of ClC-1. Experiments …
Reviewer #1 (Public Review):
Patients with myotonia congenita caused by loss-of-function mutations in ClC-1 experience muscle stiffness (due to hyperexcitability) as well as transient muscle weakness. This study examines the mechanisms underlying the transient muscle weakness seen myotonia congenita. The authors show that a ClC-1 null mouse exhibits the transient weakness after muscle stimulation observed in humans. Current clamp recordings of muscle fibers from ClC-1-null mice showed indicated myotonia after electrical stimulation that often terminated in a plateau potential for varying periods, during which the muscle was unexcitable, before repolarization to the resting membrane potential. The myotonia and plateau potentials could be recapitulated in wild type muscle fibers with acute pharmacological inhibition of ClC-1. Experiments in fibers from a non-conducting Cav1.1 knockin mouse indicated Ca2+ influx is important for sustaining, but not initiating, plateau potentials. Ranolazine blocked both the myotonia and development of a plateau potential in isolated muscle fibers, as well as the in vivo transient muscle weakness observed in ClC-1-null mice, implicating Na+ persistent inward currents through Nav1.4 (NAPIC) as the molecular mechanism.
Overall, the experiments presented in this work are well-executed and the results convincing. While the role of NAPIC in the development of myotonia in ClC mice has been previously reported this work provides the new insight that it is also responsible for the development of plateau potentials that underlie muscle weakness in myotonia congenita.
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Evaluation Summary:
Patients with myotonia congenita (or Becker disease) experience episodes of transient muscle weakness but the reasons underlying this phenomenon are unknown. This study provides the most definitive experimental evidence to date for the mechanistic basis of transient weakness in myotonia congenita and also suggests ranolazine may be beneficial for prophylactic management.
(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. Reviewers #1, #2, and #3 agreed to share their names with the authors.)
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