Noninvasive Assessments of Mitochondrial Capacity in People with Mitochondrial Myopathies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors describe the characteristics of skeletal muscle mitochondrial capacity at rest and post-exercise in patients with primary mitochondrial myopathy, comparing them with healthy controls. The study employs a solid and well-structured methodology. However, a significant limitation is the small sample size, which, although understandable given the rarity of these conditions, is particularly noticeable in the case study of a single patient who underwent a targeted aerobic training protocol (details of which should be more clearly defined). Additionally, the comparison between the 58% improvement in mitochondrial capacity observed in the single case study following aerobic training and the “young and older healthy controls” reported in other articles needs clarification (see below).

Below are some observations:

• Abstract, lines 19-20, and Conclusions, lines 301-302: The authors state that there is no difference in exercise oxygen desaturation between the MITO and AB cohorts. However, in the Results section (lines 88-89), they report a “greater (muscle oxygen desaturation during the exercise) in the MITO group than in the control group” with a p = 0.04. Please clarify this discrepancy.

• Keywords, line 28: Spelling error, "syndrome."

• Introduction, lines 35-36: Consider adding "eyes, ears, nerves" to the list of involved organs.

• Introduction, line 39: Consider adding “ophthalmoplegia, blindness, neuropathy, ataxia, stroke-like episodes, cardiomyopathy/arrhythmias, blood dyscrasias.”

• Introduction, line 46: Muscle biopsy is a relatively safe and minimally invasive procedure. I suggest removing this sentence.

• Introduction, lines 47-48: Brain MRI with spectroscopy is very useful for detecting lactate peaks, especially when brain signal abnormalities are identified. I suggest adding this detail.

• Introduction, line 51: I suggest rephrasing the concept as “could be helpful to provide evidence of mitochondrial dysfunction.”

• Introduction, line 65: I suggest removing the term “skeletal,” as other muscles (including smooth and cardiac muscles) are often involved in primary mitochondrial myopathies.

• Results, lines 75-77: I request the authors provide more detailed information on the diagnoses, specifically: summarized clinical features of the patients, whether nuclear DNA (gene panels linked to mitochondrial disorders) was analyzed, and identified variants with ACMG classification (especially for patient n.1). Please clarify “exome pending” for patient n.5 (was mtDNA sequencing also analyzed?), and clarify “mtDNA deletion” (is it a single macrodeletion, or were multiple deletions found? Which specimen was analyzed in patient n.7?).

• Results, line 96: Which participant performed the training session (diagnosis, age, gender, etc.)?

• Discussion, line 125: Spelling error, "heterogeneity."

• Discussion, line 178: The authors report an improvement of “57%,” whereas in other sections it was quantified as “58%.” Please clarify.

• Discussion, lines 178-179: Please further explain the comparison between the case study and “young controls and healthy older adults” who underwent “similar training programs.” Did other authors report a similar improvement when estimating wrist flexion mitochondrial capacity, or was this an overall figure for the whole body?

• Table 1: Consider changing “Leigh Disease” to “Leigh syndrome.”

• Table 1: Please clarify the diagnosis of “mito cytopathy” (clinical features?).

 

Comments on the Quality of English Language

The English used in the paper is clear, scientific, and allows for smooth reading, with only minimal adjustments needed.

Author Response

The authors describe the characteristics of skeletal muscle mitochondrial capacity at rest and post-exercise in patients with primary mitochondrial myopathy, comparing them with healthy controls. The study employs a solid and well-structured methodology. However, a significant limitation is the small sample size, which, although understandable given the rarity of these conditions, is particularly noticeable in the case study of a single patient who underwent a targeted aerobic training protocol (details of which should be more clearly defined). Additionally, the comparison between the 58% improvement in mitochondrial capacity observed in the single case study following aerobic training and the “young and older healthy controls” reported in other articles needs clarification (see below).

 

We appreciate the time and effort the reviewer made to evaluate our manuscript.  We agree that the sample size is small, and that larger sample sizes are difficult to obtain due to the rarity of the disorder.  We feel our sample size for the cross-sectional part of our study was adequate as the results for the six subjects was consistent.  All six subjects showed evidence of oxygen consumption with electrical stimulation, and recovery rate kinetics that were similar to control subjects.  The consistency of the results supports a general conclusion regarding the response to the testing in this study. 

 

The training study did use only one subject.  We considered it exploratory but also feel that the results are potentially very useful for future studies.  The change in symptoms during and following exercise training were large and support future studies on exercise training in this population.  If such improvements are seen in people with mitochondrial myopathies, then this would be useful to guide therapy for these people. 

 

The improvement of 58% is similar to what we have found in two studies of muscle specific training in first younger and then older adults (increases of ~40-60%).  References for previous studies has been added.

 

Below are some observations:

• Abstract, lines 19-20, and Conclusions, lines 301-302: The authors state that there is no difference in exercise oxygen desaturation between the MITO and AB cohorts. However, in the Results section (lines 88-89), they report a “greater (muscle oxygen desaturation during the exercise) in the MITO group than in the control group” with a p = 0.04. Please clarify this discrepancy.

This has been clarified.  The key observation is that the Mito group did not have reduced changes in oxygen levels. The values were actually greater but the important finding was that the value was not reduced compared to controls. 

• Keywords, line 28: Spelling error, "syndrome."

corrected

• Introduction, lines 35-36: Consider adding "eyes, ears, nerves" to the list of involved organs.

 

Added as suggested

 

• Introduction, line 39: Consider adding “ophthalmoplegia, blindness, neuropathy, ataxia, stroke-like episodes, cardiomyopathy/arrhythmias, blood dyscrasias.”

Added as suggested

 

• Introduction, line 46: Muscle biopsy is a relatively safe and minimally invasive procedure. I suggest removing this sentence.

 

The level of invasiveness of the muscle biopsy is a matter of opinion.  But we agree the statement can modified to be less negative.

 

• Introduction, lines 47-48: Brain MRI with spectroscopy is very useful for detecting lactate peaks, especially when brain signal abnormalities are identified. I suggest adding this detail.

 

We have including mentioning brain MRI to evaluate Mito myopaphy.

 

• Introduction, line 51: I suggest rephrasing the concept as “could be helpful to provide evidence of mitochondrial dysfunction.”

 

Edited as suggested

 

• Introduction, line 65: I suggest removing the term “skeletal,” as other muscles (including smooth and cardiac muscles) are often involved in primary mitochondrial myopathies.

 

We would prefer to leave in the word skeletal.  The topic being discussed is skeletal muscle, in particular the response of skeletal muscle to exercise.  We agree that cardiac and smooth muscles can be involved and are important to study, but that is beyond the scope of this study. 

 

• Results, lines 75-77: I request the authors provide more detailed information on the diagnoses, specifically: summarized clinical features of the patients, whether nuclear DNA (gene panels linked to mitochondrial disorders) was analyzed, and identified variants with ACMG classification (especially for patient n.1). Please clarify “exome pending” for patient n.5 (was mtDNA sequencing also analyzed?), and clarify “mtDNA deletion” (is it a single macrodeletion, or were multiple deletions found? Which specimen was analyzed in patient n.7?).

Unfortunately additional information is not available.  This section has been edited to remove ‘pending’ analysis.

 

• Results, line 96: Which participant performed the training session (diagnosis, age, gender, etc.)?

 

The participant performing the training is listed (#7)

 

• Discussion, line 125: Spelling error, "heterogeneity."

Corrected, thanks

• Discussion, line 178: The authors report an improvement of “57%,” whereas in other sections it was quantified as “58%.” Please clarify.

This has been clarified (58%), thanks

 

• Discussion, lines 178-179: Please further explain the comparison between the case study and “young controls and healthy older adults” who underwent “similar training programs.” Did other authors report a similar improvement when estimating wrist flexion mitochondrial capacity, or was this an overall figure for the whole body?

This has been clarified with references.  The previous protocols also used a one month wrist flexion exercise protocol. 

• Table 1: Consider changing “Leigh Disease” to “Leigh syndrome.”

Changed as suggested

• Table 1: Please clarify the diagnosis of “mito cytopathy” (clinical features?).

 At this time we do not have additional information other than the general description of mity cytopathy

 

Comments on the Quality of English Language

The English used in the paper is clear, scientific, and allows for smooth reading, with only minimal adjustments needed.

 

Thanks, minor changes have been made.

Reviewer 2 Report

Comments and Suggestions for Authors

Revision of the paper entitled: Noninvasive Assessments of Mitochondrial Capacity in People with Mitochondrial Myopathies

Kevin K. McCully, Hanah M. Bossie, and Fran D. Kendall

 

The authors hypothesized that mitochondria have been shown to play a key role in muscle oxygen consumption and that mitochondrial myopathies are often associated with reduced muscle oxygen consumption rates. They examined mitochondrial capacity in mitochondrial myopathies using infrared spectroscopy. Their results support the hypothesis that increased mitochondrial mass is associated with loss of mitochondrial functions. I think that the conclusions presented in this article are contradictory and therefore further experimental evidence should be presented and the number of patients and controls should be increased before concluding whether mitochondrial capacity does not change in MITO patients.

Author Response

The authors hypothesized that mitochondria have been shown to play a key role in muscle oxygen consumption and that mitochondrial myopathies are often associated with reduced muscle oxygen consumption rates. They examined mitochondrial capacity in mitochondrial myopathies using infrared spectroscopy. Their results support the hypothesis that increased mitochondrial mass is associated with loss of mitochondrial functions. I think that the conclusions presented in this article are contradictory and therefore further experimental evidence should be presented and the number of patients and controls should be increased before concluding whether mitochondrial capacity does not change in MITO patients.

 

We agree with the reviewer that testing more subjects would be useful.  We feel however, that our results are very consistent within the sample size that we tested.  Our main hypothesis was that muscle mVO2max would be reduced in the Mito patients.  However, our data did not support this conclusion.  In addition, our secondary outcomes such as the amount of reduction in oxygen levels with exercise, resting muscle metabolism supported our main conclusion.  So, we feel our results in this sample of Mito patients was consistent.  We have revised our figures to show individual data to highlight the consistency of our results.  And finally, our case study with a Mito patient performing endurance exercise showed similar increases in mVO2max as controls performing similar endurance training.  We feel worth presenting to the clinical and scientific community.  As with most studies, we hope our results will encourage future research. 

Reviewer 3 Report

Comments and Suggestions for Authors

McCully, K.K., et al assessed and investigated skeletal muscle mitochondrial capacity in people with mitochondrial myopathies (MITO) compared to able-bodied (AB) controls. The authors did not find any significant differences between the groups in mitochondrial capacity or muscle oxygen consumption. However, one MITO participant showed improved mitochondrial capacity and reduced fatigue after muscle-specific endurance training, suggesting that positive adaptations to training are possible for individuals with MITO.

The authors aim to clarify whether individuals with genetically confirmed skeletal muscle mitochondrial myopathies (MITO) have impaired mitochondrial capacity compared to healthy controls, using a novel NIRS-based measurement technique, also exploring the potential for endurance training to enhance mitochondrial function in an MITO patient, which eventually could offer valuable insights into both the pathology of MITO and the potential for therapeutic interventions.

High appreciation goes to the authors for providing appropriate research ethical statements. The designs for this study are justified. However other clinical tests could be done for further confirmation like the blood pannels and oxygen levels. Although the number of subjects in this study is low it may provide misleading information.

The basic approach to writing the manuscript is fine. The introduction, methods, and results were written shortly which needs to be improved with more information and references. More appreciation goes to the authors for the detailed result table. The discussion in the manuscript needs to explain the significance of this study to its readers and its importance.

Nonetheless, the article seemed to possess good value in the treatment of patients with MITO. Overall, the clarity of the text is understandable and needs some readjustments. The manuscript has minor typographical and grammatical errors.

In general, the manuscript can accomplish the caliber of quality for consideration for publication in the journal “Muscles”. The authors are advised to consider the comments below:

Comments:

1.      The number of participants was very low. And the ratio between male and female participants may create a big difference. It would be helpful to understand that the sex does not reflect in the data shown for this study either done before or in this study.

2.      The whole study was done in patients from different age groups. It has been shown/ proved before that Mitochondrial biogenesis declines with age due to alterations in mitochondrial dynamics and inhibition of mitophagy. It would be helpful if the authors could explain how the different age groups do not affect this study.

3.      The resolution of Figure 1 is very low. Please provide a high-resolution image. 

4.      Figure 2 – The markings are not visible in the figure where are 2A 2B or 2C? The graphs do not have statistical values. It would be helpful to provide one.

 

5.      Figure 3 – It is better to show this information in a table format. Although it is not clear from which patient the quotes are from?

Comments on the Quality of English Language

Overall, the clarity of the text is understandable and needs some readjustments. The manuscript has minor typographical and grammatical errors. 

Author Response

McCully, K.K., et al assessed and investigated skeletal muscle mitochondrial capacity in people with mitochondrial myopathies (MITO) compared to able-bodied (AB) controls. The authors did not find any significant differences between the groups in mitochondrial capacity or muscle oxygen consumption. However, one MITO participant showed improved mitochondrial capacity and reduced fatigue after muscle-specific endurance training, suggesting that positive adaptations to training are possible for individuals with MITO.

The authors aim to clarify whether individuals with genetically confirmed skeletal muscle mitochondrial myopathies (MITO) have impaired mitochondrial capacity compared to healthy controls, using a novel NIRS-based measurement technique, also exploring the potential for endurance training to enhance mitochondrial function in an MITO patient, which eventually could offer valuable insights into both the pathology of MITO and the potential for therapeutic interventions.

High appreciation goes to the authors for providing appropriate research ethical statements. The designs for this study are justified. However other clinical tests could be done for further confirmation like the blood pannels and oxygen levels. Although the number of subjects in this study is low it may provide misleading information.

 

We do not have additional clinical test results to add to our paper.  We have listed this as a potential limitation to our study.  We have added that NIRS testing of skeletal muscle has increased in number in recent year. Since the year 2000 there have been 107 published papers using the mVO2max measurement.  This includes many studies of clinical populations.  Since the year 2000, there have been 1,208 published papers using NIRS to measure oxygen levels in skeletal muscle.  So we feel the NIRS measurements in this study have been shown to have research and clinical utility.  This information has been added to the discussion.

 

The basic approach to writing the manuscript is fine. The introduction, methods, and results were written shortly which needs to be improved with more information and references. More appreciation goes to the authors for the detailed result table. The discussion in the manuscript needs to explain the significance of this study to its readers and its importance.

 

We have added to the discussion to address the potential significance of the study.  We appreciate this comment as it is very important. 

 

Nonetheless, the article seemed to possess good value in the treatment of patients with MITO. Overall, the clarity of the text is understandable and needs some readjustments. The manuscript has minor typographical and grammatical errors.

 

We have attempted to remove errors in the manuscript.

 

In general, the manuscript can accomplish the caliber of quality for consideration for publication in the journal “Muscles”. The authors are advised to consider the comments below:

Comments: 

1. The number of participants was very low. And the ratio between male and female participants may create a big difference. It would be helpful to understand that the sex does not reflect in the data shown for this study either done before or in this study.

 

We have listed our sample size as a potential limitation to the study.  In our previous studies, we have not found any sex related differences in our NIRS measurements.  We have added sex as a biological variable in the discussion of our revised paper.

 

2. The whole study was done in patients from different age groups. It has been shown/ proved before that Mitochondrial biogenesis declines with agedue to alterations in mitochondrial dynamics and inhibition of mitophagy. It would be helpful if the authors could explain how the different age groups do not affect this study.

We agree that age can influence mitochondrial function.  Although we feel with the age range in this study, age is not a major explanatory variable.  The age ranges in our MITO and controls groups were similar, which we feel reduces the potential impact of age on our results.  Discussion of age as a potential variable has been added to the discussion.

 

3. The resolution of Figure 1 is very low. Please provide a high-resolution image. 

The resolution of the figures has been improved.

4. Figure 2 – The markings are not visible in the figure where are 2A 2B or 2C? The graphs do not have statistical values. It would be helpful to provide one.

The figures have been modified as suggested.

 

5. Figure 3 – It is better to show this information in a table format. Although it is not clear from which patient the quotes are from?

 

The figure has been put in table form, and the subject linked to the results of the cross sectional study.

 

Comments on the Quality of English Language

Overall, the clarity of the text is understandable and needs some readjustments. The manuscript has minor typographical and grammatical errors. 

 

Hopefully the minor concerns have been addressed.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have now been permitted to revise, but in my opinion the manuscript still does not meet the minimum standards required for publication. In particular, the conclusions presented in this article are contradictory and therefore further experimental evidence should be presented and the number of patients and controls increased before concluding whether mitochondrial capacity does not change in MITO patients.

Author Response

The authors have now been permitted to revise, but in my opinion the manuscript still does not meet the minimum standards required for publication. In particular, the conclusions presented in this article are contradictory and therefore further experimental evidence should be presented and the number of patients and controls increased before concluding whether mitochondrial capacity does not change in MITO patients.

We are sorry the reviewer does not accept our conclusions.  We feel our test results are internally consistent.  To summarize: the rate of recovery of muscle metabolism (mVO2max) was not lower/slower in MITO patients.  Supporting this, resting oxygen consumption was not different, the MITO patients lowered their oxygen levels as much or more than the controls with exercise, and had initial rates of muscle metabolism that were similar to controls.  All of these secondary measures suggest that the MITO patients were consuming oxygen in a similar fashion to controls.  All of these measures are not consistent with the traditional hypothesis of lowered oxygen utilization as a result of the MITO diseases.  Thus, the conclusion from this study is that forearm muscle oxidative metabolism is not impaired.  

We did test a small group of MITO patients.  MITO disease is relatively rare and it was hard to recruit large numbers of patients.  However, the results of our tests were very consistent between patients.  That is they all showed the same general response.  Testing more patients would be unlikely to change the outcome of the study (in our opinion).  We feel that testing more patients than necessary to test our hypothesis is also something to avoid.

One point of concern is that this group of MITO patients does not represent all MITO patients.  We feel our results are very important because our results suggest that not all MITO patients demonstrate deficients in oxidative metabolism in their muscles.  This is important for future research and treatment of MITO patients.  MITO defects can occur in the brain and other parts of the body, but not necessarily in skeletal muscle.  In addition, the care study of endurance training is a demonstration that oxidative metabolism may improve with endurance training in MITO patients.  

We have made a few edits to the manuscript to highlight our results are from the forearm muscles and our conclusions are appropriate for the forearm muscles.  We hope that helps make the paper clearer.