Skeletal muscle delimited myopathy and verapamil toxicity in SUR2 mutant mouse models of AIMS

Abstract ABCC9‐related intellectual disability and myopathy syndrome (AIMS) arises from loss‐of‐function (LoF) mutations in the ABCC9 gene, which encodes the SUR2 subunit of ATP‐sensitive potassium (KATP) channels. KATP channels are found throughout the cardiovascular system and skeletal muscle and couple cellular metabolism to excitability. AIMS individuals show fatigability, muscle spasms, and cardiac dysfunction. We found reduced exercise performance in mouse models of AIMS harboring premature stop codons in ABCC9. Given the roles of KATP channels in all muscles, we sought to determine how myopathy arises using tissue‐selective suppression of KATP and found that LoF in skeletal muscle, specifically, underlies myopathy. In isolated muscle, SUR2 LoF results in abnormal generation of unstimulated forces, potentially explaining painful spasms in AIMS. We sought to determine whether excessive Ca2+ influx through CaV1.1 channels was responsible for myopathology but found that the Ca2+ channel blocker verapamil unexpectedly resulted in premature death of AIMS mice and that rendering CaV1.1 channels nonpermeable by mutation failed to reverse pathology; results which caution against the use of calcium channel blockers in AIMS.

Specific comments: 1) Statistical comparisons in the bar graphs and figure legends are not well represented.
2) The authors should clarify the types of induced contractions (e.g., isometric). 3) No genotyping or validation of transgenic mice are given. No evidence of Cre-recombination. 4) Group labeling is confusing. Seeming use of inappropriate controls. 5) It is unclear what the numerical values associated R-R Interval variance represent. ECG tracings do not show a high degree of heart rate variability. 6) No or inappropriate scaling or axis labels in several figures, rendering data uninterpretable. 7) Inconsistent group labeling between manuscript text and figures. 8) Too much use of delta values.

Referee #3 (Comments on Novelty/Model System for Author):
The experiments and model systems were well thought out and the resulting data clarify mechanistic details of AIMS etiology and help guide potential therapeutic approaches for AIMS.
Referee #3 (Remarks for Author): The authors use both a global and a skeletal muscle-specific mouse model of ABCC9 truncation, which causes loss of function in the SUR2 subunit and therefore loss of function of ATP-sensitive potassium (KATP) channels. Using the mouse models, the authors demonstrate that loss of skeletal muscle KATP channel function underlies the myopathy in AIMS, rather than the cardiac channel loss. The authors also discover that ABCC9 truncation results in abnormal generation of unstimulated forces in skeletal muscle, reflecting aspects of human AIMS. They also find that excessive Ca2+ influx through CaV1.1 channels is not responsible for myopathology, and instead discovered that the Ca2+ channel blocker verapamil leads to premature death of AIMS mice, likely by bradyarrhythmia/heart block -suggesting against the use of calcium channel blockers to treat the symptoms of AIMS. The study is well-executed and the manuscript well-written with appropriate and clear figures. I have some minor points that should be addressable by adding clarification in the manuscript: 1) The authors mention they used mice of both sexes for most of the studies, but did not describe if there were sex differences on any of the phenotypes and/or if the sexes were match within groups, and pooled within groups. Please clarify in the manuscript.
2) Please describe any known sex differences in KATP and/or Cav1.1 function in mouse or human cardiac, skeletal muscle or other relevant tissue systems.
3) Are there differences in the role of Cav1.1 in mouse versus human cardiac or skeletal muscle tissue that could underlie potentially different responses to verapamil? 4) How did the authors arrive at the doses of verapamil used in the study? Is it possible that lower doses could be therapeutic without the toxicity? Were the control and STOP475 groups in these experiments matched for sex?
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Referee #1 (Comments on Novelty/Model System for Author):
The technical can be strengthened as per recommendation provided to authors. The novelty and medical impact are outlined below. The precision of the gene edited models is underscored.
(Remarks for Author): Genetically altered mice models were the mainstay of the study employed to gain insight into the myopathic component of the recently reported ABCC-9-related intellectual disability and myopathy syndrome (AIMS). The precision of the gene editing approaches used here, along with the remarkable muscle electrophysiological evaluation, enabled linkage of the SUR2 loss of function (compromising the KATP channel complex) with the skeletal muscle disease phenotype. This finding could guide the further understanding of disease pathobiology. In addition, the authors report lack of therapeutic efficacy and potential aggravated toxicity of the calcium channel blocker, verapamil, in the setting of AIMS, an observation of potential medical significance. We are grateful for the recognition of the novelty and medical impact of out studies and the suggestions for improvements.
Suggestions: 1. Authors should consider more direct evidence of muscle damage as currently provided muscle histology data seem insufficient to conclude of structural deterioration. We agree that this would be informative. In response, we are performing additional immunohistochemical and gene expression studies to attempt to identify molecular mechanisms of myopathy. The results of these experiments could be included in revision.
2. The pharmacological evaluation will be further strengthened by a more complete doseescalation study (to establish the LD50 value). Furthermore, authors may consider prospective electrocardiography acquisition (as terminal bradycardia reported here may not reflect the primary cause of death). Again, we agree that this would be informative, and plan to carry out a more detailed doseescalation study.
3. A negative cardiovascular hemodynamic effect of verapamil should also be considered. This is an interesting suggestion. We do not expect there to be any based on published norms, but to be certain, we plan to assess blood pressures in response to acute verapamil treatment in WT and SUR2-STOP mice. 4. As an alternative aimed to limit the excessive systemic verapamil toxicity, authors may consider the value of local calcium channel blocker administration and/or another calcium channel blocker, such as diltiazem used in skeletal myopathies. This is an astute suggestion, and we agree that further study of other potential calcium channel blockers will be informative. This would be a major undertaking and will constitute follow-up studies.
5. For broad readership, authors are encouraged to underscore this work in the context of KATP channelopathies reported across multiple organ systems. We will expand the discussion to include consideration of KATP channelopathies. The manuscript attempts to shed light on the specific involvement of skeletal muscle ablation of functioning K-ATPase activity on the pathology of AIMS. The superficial nature of the experiments reported here are largely uninformative and limit my enthusiasm for the work. We thank the reviewer for taking the time to evaluate the manuscript. To clarify, however, our studies relate to the ATP-sensitive potassium (KATP) channel, of which SUR2 is a key subunit -not the K-ATPase or Na,K,ATPase pump, as referred to in this review. This is not simply a nomenclature mistake: the reviewer notes -correctly -that "without functioning Na,K-ATPase activity the cells are likely to be wildly depolarized". This represents a fundamental misunderstanding of the manuscript, and is not a result of the way the manuscript is written, as the other referees clearly do understand it.
General comments: 1) It is difficult to make any interpretations regarding muscle performance in SUR2-STOP mice without CRISPR/Cas9 corrected controls. The possibility and influence of off-target effects by CRISPR/Cas9 gene editing have not been excluded. The suggestion that "CRISPR/Cas9 corrected controls" are needed to make interpretations is an unusual expectation for a genome-edited mouse model. While we appreciate the concern about off-target effects, we have (i) validated the on-target effect of the mutations on KATP channel activity in native cells, and (ii) mitigated the risk of potential off-target effects confounding these studies by extensive outbreeding, and by using littermate wildtype SUR2 control mice in all relevant experiments -the gold standard controls. Therefore, for any off-target mutation to confound the studies, any additional, unknown mutation would have to be in strong linkage disequilibrium with the intended ABCC9/SUR2 mutation. This is a formal possibility for this and all other studies of genetically modified animal models, but is highly unlikely. Our confidence in our conclusions is bolstered by identifying the key phenotypes (exercise intolerance and verapamil toxicity) in two distinct CRISPR mutant SUR2-STOP lines. The likelihood of both being confounded by additional mutations is significantly reduced.
2) It is not surprising the Myf5 driven channel ablation showed a similar phenotype given the assays reported here. The hang test is not demanding of the cardiovascular system, and the treadmill test cannot be interpreted as reported (see below). The authors must better design experiments to unequivocally determine the relative contribution of each tissue to the phenotype. The possibility of non-skeletal muscle delimited effects of SUR2/KATP loss-of-function on exercise tolerance has been proposed (Stoller et al., 2007). As we discuss, this was a reasonable proposal given the roles of vascular KATP channels in the dynamic perfusion of skeletal muscle. Our studies clearly demonstrate, for the first time, a consequence of KATP channel knockdown in skeletal muscle specifically.
3) Total treadmill distance was recorded but not reported. Rather, the authors use a dubious formula for workload to report running performance. The sum of kinetic and potential energy is total mechanical energy, which is generally conserved. The readout here appears to hinge on differences in the mass of the mouse and belt velocity (of which was under acceleration and is therefore not appropriate). No reports of mouse body weights were given. In any case, no conclusions can be made from this data. We chose this analysis to maintain consistency with previous analyses from multiple groups (PMID: 12271142, 25648265). However, given the concerns of the referee, we will now additionally report performance in terms of time sustained, and distance travelled, and include measurements of body weight.
4) The phenotyping methods chosen by the authors may not be sensitive enough to detect differences among genotypes. More objective (and less behavioral) methods are required to make any confident conclusions. The reader is left without any meaningful advances.
We are uncertain what more sensitive methods the referee is referring to but would be willing to perform additional phenotyping if justified. 5) What is the resting membrane potential of SUR2-STOP myofibers? Without functioning Na,K-ATPase activity the cells are likely to be wildly depolarized. How does this influence the observations made during the patch clamp experiments? Are there any compensatory mechanisms regulating ion flux?
As noted above, this study is not related to the Na,K-ATPase pump. The referee appears to have misunderstood which protein is in question. The comment regarding the consequence of Na,K-ATPase LoF is valid, but irrelevant. Loss of KATP has previously been shown to be without major effects on membrane potential in resting skeletal muscle, but does cause membrane potential depolarization in fatiguing stimuli, as we discuss.
6) The most important finding in the manuscript remains poorly explored. The authors uncover a dramatic effect of Verapamil on mortality in SUR2-STOP mice, which was treated like an afterthought. We agree that this is an important finding. It was not an afterthought, but should be considered alongside the preceding data showing that skeletal muscle specifically is a tissue for targeting with therapies.
Specific comments: 1) Statistical comparisons in the bar graphs and figure legends are not well represented. We would be happy to accommodate any specific suggestions.
2) The authors should clarify the types of induced contractions (e.g., isometric). We will clarify this in revision.
3) No genotyping or validation of transgenic mice are given. No evidence of Crerecombination. Dominant-negative gene expression was clearly demonstrated in patch-clamp studies, in which KATP conductances are abolished in SkM-DN mouse muscle. This is the most relevant measure of transgene expression, more important than Cre-recombination, or transcript level, as it demonstrates effects at the protein level. That said, we can incorporate evidence of CRE-recombination in revision. 4) Group labeling is confusing. Seeming use of inappropriate controls. We would be happy to make appropriate changes in labeling in response to any specific suggestions. The use of inappropriate controls is not clear to us.

5)
It is unclear what the numerical values associated R-R Interval variance represent. ECG tracings do not show a high degree of heart rate variability. We apologize for the absence of units of R-R interval variance, which will be corrected in revision. We disagree that the tracing does not show a variance in R-R interval, there is clearly an increased interval between the 3 rd and 4 th QRS complex, compared to the preceding intervals. 6) No or inappropriate scaling or axis labels in several figures, rendering data uninterpretable.
We will correct any absent labeling in revision, though are not sure of the specific figures referred to here. 7) Inconsistent group labeling between manuscript text and figures. We will proof-read for any labeling discrepancies, but again are not sure of specifics from this comment. 8) Too much use of delta values. Unfortunately, we are not sure what is referred to by "delta values"

Referee #3 (Comments on Novelty/Model System for Author):
The experiments and model systems were well thought out and the resulting data clarify mechanistic details of AIMS etiology and help guide potential therapeutic approaches for AIMS. (Remarks for Author): The authors use both a global and a skeletal muscle-specific mouse model of ABCC9 truncation, which causes loss of function in the SUR2 subunit and therefore loss of function of ATP-sensitive potassium (KATP) channels. Using the mouse models, the authors demonstrate that loss of skeletal muscle KATP channel function underlies the myopathy in AIMS, rather than the cardiac channel loss. The authors also discover that ABCC9 truncation results in abnormal generation of unstimulated forces in skeletal muscle, reflecting aspects of human AIMS. They also find that excessive Ca2+ influx through CaV1.1 channels is not responsible for myopathology, and instead discovered that the Ca2+ channel blocker verapamil leads to premature death of AIMS mice, likely by bradyarrhythmia/heart blocksuggesting against the use of calcium channel blockers to treat the symptoms of AIMS. The study is well-executed and the manuscript well-written with appropriate and clear figures. I have some minor points that should be addressable by adding clarification in the manuscript: We are grateful for the positive comments and welcome the suggestions.
1) The authors mention they used mice of both sexes for most of the studies, but did not describe if there were sex differences on any of the phenotypes and/or if the sexes were match within groups, and pooled within groups. Please clarify in the manuscript. All references to N numbers and sexes will be complete in the revised version. No significant differences were found between male and female mice of the same genotype in any experiment except in treadmill tests of dominant negative mice, where female Myf5-cre+ mice performed worse than males. For this reason, we focused on male mice for this experiment -this will be explained in the revised version.
2) Please describe any known sex differences in KATP and/or Cav1.1 function in mouse or human cardiac, skeletal muscle or other relevant tissue systems.
3) Are there differences in the role of Cav1.1 in mouse versus human cardiac or skeletal muscle tissue that could underlie potentially different responses to verapamil? We are unaware of any differences but will perform a detailed literature review and include any discussion in revision. 4) How did the authors arrive at the doses of verapamil used in the study? Is it possible that lower doses could be therapeutic without the toxicity? Were the control and STOP475 groups in these experiments matched for sex? These dosages were based on previous studies (in rats) showing tolerance and blood pressure lowering effects (and therefore in vivo efficacy) of the drug (PMID: 6184556). We show that the dose which causes death in the SUR2-STOP mice is without beneficial effects in the SkM-DN mice, which survive administration, and so do not expect lower doses to be beneficial. This can be tested in the dose escalation study suggested by reviewer 1 however and could be included in revision. Thank you for your appeal asking us to reconsider our decision on your manuscript. I have carefully read your letter and have reached out to referee #2 regarding the reference to the wrong channel in his/her report. Referee #2 replied: "Indeed, I incorrectly referenced the KATP channel in this particular comment. However, the concern remains: how does knockout of this channel affect resting membrane potential of the isolated myofibers, and are there any compensatory mechanisms. Regardless, the point still should be addressed by the authors. In viewing other comments that raise serious shortcomings of the manuscript, the channel becomes less important per se. For example, the highly behavioral-dependent phenotyping methods used remain a poor choice independent on the specific ion channel studied. Overall, the data presented in the manuscript are not convincing (due to inappropriate controls and questionable reporting methods); and the manuscript suffers from a serious lack of mechanistic understanding." To reach a balanced decision and as mentioned in a previous correspondence, I have also communicated your provisional pointby-point rebuttal letter to an external advisor. This adviser read your manuscript and the referees' reports, and suggested to allow resubmission of the manuscript after adequate revisions. Therefore, after internal discussion with my colleagues, we decided to reconsider our decision and invite revisions of the manuscript. I will involve an additional referee to evaluate the revised manuscript.
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(Remarks for Author): Genetically altered mice models were the mainstay of the study employed to gain insight into the myopathic component of the recently reported ABCC-9-related intellectual disability and myopathy syndrome (AIMS). The precision of the gene editing approaches used here, along with the remarkable muscle electrophysiological evaluation, enabled linkage of the SUR2 loss of function (compromising the KATP channel complex) with the skeletal muscle disease phenotype. This finding could guide the further understanding of disease pathobiology. In addition, the authors report lack of therapeutic efficacy and potential aggravated toxicity of the calcium channel blocker, verapamil, in the setting of AIMS, an observation of potential medical significance. We are grateful for the recognition of the novelty and medical impact of our studies and the suggestions for improvements.
Suggestions: 1. Authors should consider more direct evidence of muscle damage as currently provided muscle histology data seem insufficient to conclude of structural deterioration. We thank the reviewer for this suggestion and have added additional histopathological data. These data show that muscle from skeletal muscle dominant-negative (SkM-DN) mice also exhibits necrotic fibers, as observed in HE stained sections, and as further illustrated by macrophage infiltration. Such necrotic fibers were not observed from WT muscle samples. SkM-DN muscle also exhibited increased fibrosis, as determined by picrosirius red staining. Finally, consistent with increased muscle injury response in sedentary mice, we observed increased numbers of Pax7 positive satellite cells in SkM-DN muscle. These new findings have been included in the new Fig 3. 2. The pharmacological evaluation will be further strengthened by a more complete doseescalation study (to establish the LD50 value). Furthermore, authors may consider prospective electrocardiography acquisition (as terminal bradycardia reported here may not reflect the primary cause of death). We agree that it would be informative to explore this further. Our data so far at least indicate that the LD50 for verapamil in SUR2-STOP 475 mice is in between the doses tested 12th Mar 2023 2nd Authors' Response to Reviewers (i.e. between 0.3 g/l and 0.9 g/l in drinking water), which narrows this to a relatively small range. Our findings suggest that detailed further study of the effects of verapamil alongside other calcium channel blockers is warranted and should form the basis of follow up studies (see below). Notably, our ECG analysis did not show any initial obvious bradycardia or arrythmia in the SUR2-STOP mice (Fig. 5D), although the footpad ECG method is probably not sensitive enough to identify subtle abnormalities. Thus, we would like to carry out additional follow up studies (including comparison of verapamil with other calcium channel blockers) using more sophisticated telemetry recording and analysis, although these are not currently available to us. 3. A negative cardiovascular hemodynamic effect of verapamil should also be considered. This is an interesting suggestion. As we have previously reported (Smeland et al., Nat Comms, 2019), SUR2-STOP mice exhibit higher than normal blood pressures -most likely due to the loss of KATP channel in vascular smooth muscle. Therefore, of course, the naïve prediction would be that verapamil should decrease and potentially normalize blood pressures, via calcium channel blockade in smooth muscle. We cannot rule out unexpected hemodynamic effects, but in a limited experiment (see Fig i below), invasive blood pressure measurement via carotid artery cannulation in isoflurane-anesthetized mice that survived verapamil revealed no drastic hemodynamic effects (SUR2-STOP without verapamil, n = 3; SUR2-STOP with verapamil, n = 2). 4. As an alternative aimed to limit the excessive systemic verapamil toxicity, authors may consider the value of local calcium channel blocker administration and/or another calcium channel blocker, such as diltiazem used in skeletal myopathies. This is an astute suggestion, and we agree that further study of other potential calcium channel blockers will be informative. This would be a major undertaking and will constitute follow-up studies. 5. For broad readership, authors are encouraged to underscore this work in the context of KATP channelopathies reported across multiple organ systems. We have expanded the discussion to include consideration of KATP channelopathies, as below: "Interestingly, exercise intolerance, muscle fatigue, and histopathology are also observed in the rare KATP channelopathy, Cantú Syndrome, which arises from gain-of-function mutations in either ABCC9 (SUR2) or KCNJ8 (Kir6.1)45-47. Whether this is due to intrinsic defects in skeletal muscle physiology or is due to altered cardiovascular function and/or systemic perfusion remains to be established. Individuals with LoF mutations in KCNJ11 (Kir6.2) suffer from congenital hyperinsulinism (CHI). Although muscle fatigue is not typically reported, an extended consanguineous family, exhibiting a syndrome of CHI and rhabdomyolysis has been reported in association with the Kir6.2[R34H] mutation48. In this case, the CHI was severe, and potentially the loss of Kir6.2-dependent KATP was profound: previous analysis of mutation of this arginine residue renders the channels completely insensitive to the essential activator PIP249. Why myopathy is not more widely observed in CHI is not clear but could be explained by incomplete loss-of-function or possibly some underappreciated contribution of Kir6.1 subunits to skeletal muscle channels."

Referee #2 (Comments on Novelty/Model System for Author):
Many of the physiological readouts can be influenced by behavior. Inappropriate controls for CRISPR/Cas9 edited mice. Dubious mathematical formulation. Lack of detailed mechanistic understanding. (Remarks for Author): The manuscript attempts to shed light on the specific involvement of skeletal muscle ablation of functioning K-ATPase activity on the pathology of AIMS. The superficial nature of the experiments reported here are largely uninformative and limit my enthusiasm for the work. We thank the reviewer for taking the time to evaluate the manuscript. To clarify, however, our studies relate to the ATP-sensitive potassium (KATP) channel, of which SUR2 is a key subunit -not the K-ATPase or Na,K,ATPase pump, as referred to in this review. This appears to represent a fundamental misunderstanding of the manuscript, but not one that results from the way the manuscript is written, as the other referees clearly do understand this.
General comments: 1) It is difficult to make any interpretations regarding muscle performance in SUR2-STOP mice without CRISPR/Cas9 corrected controls. The possibility and influence of off-target effects by CRISPR/Cas9 gene editing have not been excluded.
The suggestion that CRISPR/Cas9 corrected controls are needed to make interpretations is an unusual expectation for a genome-edited mouse model. While we appreciate the concern about off-target effects, we have (i) validated the on-target effect of the mutations on KATP channel activity in native cells, and (ii) mitigated the risk of potential off-target effects confounding these studies by extensive outbreeding, and by using littermate wildtype SUR2 control mice in all relevant experiments -the gold standard controls. Therefore, for any off-target mutation to confound the studies, an additional unknown mutation would have to be in strong linkage disequilibrium with the intended ABCC9/SUR2 mutation. This is a formal possibility for this and all other studies of genetically modified animal models, but is highly unlikely. Our confidence in our conclusions is bolstered by identifying the key phenotypes (exercise intolerance and verapamil toxicity) in two distinct CRISPR mutant SUR2-STOP lines. The likelihood of both being confounded by additional mutations is significantly reduced.
2) It is not surprising the Myf5 driven channel ablation showed a similar phenotype given the assays reported here. The hang test is not demanding of the cardiovascular system, and the treadmill test cannot be interpreted as reported (see below). The authors must better design experiments to unequivocally determine the relative contribution of each tissue to the phenotype. The possibility of non-skeletal muscle delimited effects of SUR2/KATP loss-of-function on exercise tolerance has been proposed (Stoller et al., 2007). As we discuss, this was a reasonable proposal given the roles of vascular KATP channels in the dynamic perfusion of skeletal muscle. Our studies clearly demonstrate, for the first time, a consequence of KATP channel knockdown in skeletal muscle specifically.
3) Total treadmill distance was recorded but not reported. Rather, the authors use a dubious formula for workload to report running performance. The sum of kinetic and potential energy is total mechanical energy, which is generally conserved. The readout here appears to hinge on differences in the mass of the mouse and belt velocity (of which was under acceleration and is therefore not appropriate). No reports of mouse body weights were given. In any case, no conclusions can be made from this data. We chose this analysis to maintain consistency with previous analyses from multiple groups (PMID: 12271142, 25648265). However, given the concerns of the referee, we have substituted figures showing distance travelled on the treadmill in place of the workload figures (new Fig. 1D and Fig, 2D). The treadmill protocol underwent step-wise increases in velocity (increased by 3m/min every 3 minutes). The differences observed definitively did not arise due to differences in the mass of the mice. We have now included data on mouse weights in the relevant figure legends; there was no significant difference in weight between WT and SUR2-STOP mice, nor for any group used in the dominant-negative studies.
4) The phenotyping methods chosen by the authors may not be sensitive enough to detect differences among genotypes. More objective (and less behavioral) methods are required to make any confident conclusions. The reader is left without any meaningful advances.
We are uncertain what more sensitive methods the referee is referring to as no specific details have been provided. 5) What is the resting membrane potential of SUR2-STOP myofibers? Without functioning Na,K-ATPase activity the cells are likely to be wildly depolarized. How does this influence the observations made during the patch clamp experiments? Are there any compensatory mechanisms regulating ion flux?
As noted above, this study is not related to the Na,K-ATPase pump. The referee appears to have misunderstood which protein is in question, and thus the comment regarding the consequence of Na,K-ATPase LoF is valid, but not relevant. Kir6.2-KO or pharmacological inhibition of KATP has previously been shown to be without major effects on membrane potential in resting skeletal muscle but does cause membrane potential depolarization in fatiguing stimuli (Cifelli et al., 2008, DOI: 10.1113/expphysiol.2008Zhu et al., 2014Zhu et al., , doi: 10.1085063). We appreciate the prompt to clarify this and have amendment the text as below: "KATP channels likely contribute little to determination of the resting membrane potential in resting muscle, but in fatiguing stimuli the activation of functional KATP channels protects isolated myofibers from excessive membrane depolarization, cytosolic Ca2+ overload, and abnormal development of unstimulated tension21-23,31" 6) The most important finding in the manuscript remains poorly explored. The authors uncover a dramatic effect of Verapamil on mortality in SUR2-STOP mice, which was treated like an afterthought. We agree that this is an important finding. It was not an afterthought, but should be considered alongside the preceding data showing that skeletal muscle specifically is a tissue for targeting with therapies.
Specific comments: 1) Statistical comparisons in the bar graphs and figure legends are not well represented.
We have modified the figures to include p values as per the journal standards. Description of N numbers and p values are now also included in the figure legends, and full description of test statistics have been included with source data files.
2) The authors should clarify the types of induced contractions (e.g., isometric). Thank you for the suggestion, we have added this.
3) No genotyping or validation of transgenic mice are given. No evidence of Crerecombination. Dominant-negative gene expression was clearly demonstrated in patch-clamp studies, which show that KATP conductances are abolished in SkM-DN mouse muscle. This is the most relevant measure of transgene expression, more important than Cre-recombination, or transcript level, as it demonstrates the intended functional effects at the protein level. 4) Group labeling is confusing. Seeming use of inappropriate controls. We are unsure of the specific issues but have endeavored to label all groups as clearly as possible.
5) It is unclear what the numerical values associated R-R Interval variance represent. ECG tracings do not show a high degree of heart rate variability. We apologize for the absence of units of R-R interval variance, which has been corrected in revision. We disagree that the tracing does not show a variance in R-R interval, there is clearly an increased interval between the 3 rd and 4 th QRS complex, compared to the preceding intervals. 6) No or inappropriate scaling or axis labels in several figures, rendering data uninterpretable. We have added scale bars to histology images. 7) Inconsistent group labeling between manuscript text and figures. We have additionally proof-read for labeling discrepancies, but again are not sure of specifics from this comment. The authors use both a global and a skeletal muscle-specific mouse model of ABCC9 truncation, which causes loss of function in the SUR2 subunit and therefore loss of function of ATP-sensitive potassium (KATP) channels. Using the mouse models, the authors demonstrate that loss of skeletal muscle KATP channel function underlies the myopathy in AIMS, rather than the cardiac channel loss. The authors also discover that ABCC9 truncation results in abnormal generation of unstimulated forces in skeletal muscle, reflecting aspects of human AIMS. They also find that excessive Ca2+ influx through CaV1.1 channels is not responsible for myopathology, and instead discovered that the Ca2+ channel blocker verapamil leads to premature death of AIMS mice, likely by bradyarrhythmia/heart blocksuggesting against the use of calcium channel blockers to treat the symptoms of AIMS. The study is well-executed and the manuscript well-written with appropriate and clear figures. I have some minor points that should be addressable by adding clarification in the manuscript: We are grateful for the positive comments and welcome the suggestions.
1) The authors mention they used mice of both sexes for most of the studies, but did not describe if there were sex differences on any of the phenotypes and/or if the sexes were match within groups, and pooled within groups. Please clarify in the manuscript. We appreciate the reviewer highlighting this. We did not observe any significant difference between male and female mice of the same genotype in any experiment (p > 0.05 according to Mann Whitney U tests comparing male v female of the same genotype), except for the treadmill test, where female Myf5+ mice appeared to fatigue more quickly than their male counterparts. Therefore, for the dominant-negative treadmill study we used only male mice for all groups -as now explained more fully in the methods. In all other experiments, where no sex dependent effects were apparent, we combined male and female mice (as now fully described in the figure legends) to maximize statistical power and conserve mice required for studies.
2) Please describe any known sex differences in KATP and/or Cav1.1 function in mouse or human cardiac, skeletal muscle or other relevant tissue systems.
For KATP channels, increased SUR2A transcript levels and Kir6.2 and SUR2A protein levels have been reported in female hearts compared with males; and sex differences in KATP may impact myocardial infarct sizes (Ranki et al. 2001, DOI: 10.1016/s0735-1097 01428-0;Johnson et al., 2006, DOI: 10.1152/ajpheart.01291.2005. Importantly, we observed verapamil induced deaths in both male and female mice (7 of 8 male and 4 of 6 female SUR2-STOP 475 mice died within 28 days of 0.9 g/l verapamil). To our knowledge, sex differences in skeletal muscle KATP channels have not been studied. Subtle sex-dependent differences are possible and warrant further study, but again we saw no significant difference in exercise tolerance between WT or SUR2-STOP male and female mice (according to Mann Whitney U tests comparing male v female of the same genotype). For Cav1.1, a recent study showed there was no difference in L-type currents, Ca2+ release, or skeletal muscle EC coupling between male and female mice (Beqollari et al., 2020, DOI: 10.1016/j.bbrc.2019. 3) Are there differences in the role of Cav1.1 in mouse versus human cardiac or skeletal muscle tissue that could underlie potentially different responses to verapamil? We are unaware of significant differences between mouse and human Ca V 1.1. The IC50 values for inhibition of cardiac Cav channels is in the lower µM range for racemic verapamil in human and rodent model systems and the major amino acids responsible for verapamil binding to L-type Ca2+ channels are highly conserved between mice and humans (Striessnig et al., 1998 DOI: 10.1016/s0165-6147(98)01171-7 ; Fig. 3b). 4) How did the authors arrive at the doses of verapamil used in the study? Is it possible that lower doses could be therapeutic without the toxicity? Were the control and STOP475 groups in these experiments matched for sex? These doses were based on previous studies (in rats) that showed tolerance and blood pressure lowering effects (and therefore in vivo efficacy) of the drug (PMID: 6184556). We show that the dose which causes death in the SUR2-STOP mice is without beneficial effects in the SkM-DN mice (all ofwhich survive administration), and so we do not expect lower doses to be beneficial. We observed verapamil-induced deaths in both male and female mice (7 of 8 male and 4 of 6 female SUR2-STOP475 mice died within 28 days of 0.9 g/l verapamil). Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. This revised version was sent back to referees #1 and #3, and we additionally asked the independent expert who had advised us on your initial submission to review the revised version. We have now received the report from this expert (now referee #4) and referee #3. Referee #1 has not gotten back to us yet, but referee #4 also evaluated your answers to this referee. As you will see, both referees #3 and #4 are now supportive of publication, and we will therefore be able to accept your manuscript once the following editorial points will be addressed: 1/ Main manuscript text: -Please address the queries from our data editors in the data edited manuscript file. Please remove the yellow highlights, and only keep in track changes mode any new modification.
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