Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
Although this study provides a comprehensive outlook on the ETC function in various tissues, the main caveat is that it's too technical and descriptive. The authors didn't invest much effort in putting their findings in the context of the biological function of the tissue analyzed, i.e., some tissues might be more glycolytic than others and have low ETC activity.
To better contextualize our results, we have added substantial amount of new information to the Discussion Section.
Also, it is unclear what slight changes in the activity of one or the other ETC complex mean in terms of mitochondrial ATP production.
Unfortunately, the method we used can only determine oxygen consumption rate through complex I (CI), CII, or CIV. It cannot tell us about ATP production. This method only measures maximal uncoupled respiration.
Likely, these small changes reported do not affect the mitochondrial respiration.
We are indeed looking at mitochondrial respiration. Some changes are more dramatic while others are much more modest. We are looking at the normal aging process across tissues (focusing on mitochondrial respiration) and not pathological states. As such, we expect many of the changes in mitochondrial respiration across tissues to be mild or relatively modest. After all, aging is slow and progressive. In fact, the variations we observed in mitochondrial respiration across tissues are consistent with the known heterogenous rate of aging across tissues.
With such a detailed dataset, the study falls short of deriving more functionally relevant conclusions about the heterogeneity of mitochondrial function in various tissues. In the current format, the readers get lost in the large amount of data presented in a technical manner.
We agree that the paper contains a large amount of information. In the revised manuscript, we did our best to contextualize our results by substantially expanding the Discussion Section.
Also, it is highly recommended that all the raw data and the values be made available as an Excel sheet (or other user-friendly formats) as a resource to the community.
We included all the data in two excel sheets (Figure 1 – data source 1; Figure 1 – data source 2). We presented them in such as way that it will be easy for other investigators to follow and re-use our dataset in their own studies for comparison.
Major concerns
(1) In this study, the authors used the method developed by Acin-Perez and colleagues (EMBO J, 2020) to analyze ETC complex activities in mitochondria derived from the snap-frozen tissue samples. However, the preservation of cellular/mitochondrial integrity in different types of tissues after being snap-frozen was not validated.
All the samples are actually maximally preserved due to being snap frozen. Freezing the samples disrupts the mitochondria to produce membrane fragments. Subsequent thawing, mincing, and homogenization in a non-detergent based buffer (mannose-sucrose) ensures that all tissue samples are maximally disrupted into fragments which contain ETC units in various combinations. This allows the assay to give an accurate representation of maximal respiratory capacity given the ETC units present in a tissue sample.
Since aging has been identified as the most important effector in this study, it is essential to validate how aging affects respiration in various fresh frozen tissues. Such analysis will ensure that the results presented are not due to the differential preservation of the mitochondrial respiration in the frozen tissue. In addition, such validations will further strengthen the conclusions and promote the broad usability of this "new" method.
The reason we adopted this method is because it has been rigorously validated in the original publication (PMID: 32432379) and a subsequent methods paper (PMID: 33320426). The authors in the original paper benchmarked their frozen tissue method with freshly isolated mitochondria from the same set of tissues. Their work showed highly comparable mitochondrial respiration from frozen tissues and isolated mitochondria. For this reason, we did not repeat those validation studies.
(2) In this study, the authors sampled the maximal activity of ETC complex I, II, and IV, but throughout the manuscript, they discussed the data in the context of mitochondrial function.
We apologize that we did not make it clearer in our manuscript. We corrected this in our revised manuscript (the Discussion Section). Our method we measure respiration starting at Complex I (CI; via NADH), starting at CII (via succinate), or starting at CIV (using TMPD and ascorbate). Regardless of whether electrons (donated by the substrate) enter the respiratory chain through CI, CII or CIV, oxygen (as the final electron acceptor) is only consumed at CIV. Therefor, the method measures mitochondrial respiration and function through CI, CII, or CIV. This high-resolution respirometry analysis method is different from the classic enzymatic method of assessing CI, CII, or CIV activity individually; the enzymatic method does not actually measure oxygen consumption due to electrons flowing through the respiratory complexes.
However, it is unclear how the changes in CI, CII, and CIV activity affect overall mitochondrial function (if at all) and how small changes seen in the maximal activity of one or more complexes affect the efficiency and efficacy of ATP production (OxPhos).
Please see the preceding response to the previous question. The method is measuring mitochondrial respiration through CI, CII or CIV. The limitation of this method is that it is maximal uncoupled respiration; namely, mitochondrial respiration is not coupled to ATP synthesis since the measurements are not performed on intact mitochondria. As such, we cannot say anything about the efficiency and efficacy of ATP production. This will be an interesting future studies to further investigating tissue level variations of mitochondrial OXPHOS.
The authors report huge variability between the activity of different complexes - in some tissues all three complexes (CI, CII, and CIV) and often in others, just one complex was affected. For example, as presented in Figure 4, there is no difference in CI activity in the hippocampus and cerebellum, but there is a slight change in CII and CIV activity. In contrast, in heart atria, there is a change in the activity of CI but not in CII and CIV. However, the authors still suggest that there is a significant difference in mitochondrial activity (e.g., "Old males showed a striking increase in mitochondrial activity via CI in the heart atria....reduced mitochondrial respiration in the brain cortex..." - Lines 5-7, Page 9). Until and unless a clear justification is provided, the authors should not make these broad claims on mitochondrial respiration based on small changes in the activity of one or more complexes (CI/CII/CIV). With such a data-heavy and descriptive study, it is confusing to track what is relevant and what is not for the functioning of mitochondria.
We have attempted to address these issues in the revised Discussion section.
(3) What do differences in the ETC complex CI, CII, and CIV activity in the same tissue mean? What role does the differential activity of these complexes (CI, CII, and CIV) play in mitochondrial function? What do changes in Oxphos mean for different tissues? Does that mean the tissue (cells involved) shift more towards glycolysis to derive their energy? In the best world, a few experiments related to the glycolytic state of the cells would have been ideal to solidify their finding further. The authors could have easily used ECAR measurements for some tissues to support their key conclusions.
We have attempted to address these issues in the revised Discussion section. The frozen tissue method does not involve intact mitochondria. As such, the method cannot measure ECAR, which requires the presence of intact mitochondria.
(4) The authors further analyzed parameters that significantly changed across their study (Figure 7, 98 data points analyzed). The main caveat of such analysis is that some tissue types would be represented three or even more times (due to changes in the activity of all three complexes - CI, CII, and CIV, and across different ages and sexes), and some just once. Such a method of analysis will skew the interpretation towards a few over-represented organ/tissue systems. Perhaps the authors should separately analyze tissue where all three complexes are affected from those with just one affected complex.
Figure 7 summarizes the differences between male vs female, and between young vs old. All the tissue-by-tissue comparisons (data separated by CI-linked respiration, CII-linked respiration, and CIV-linked respiration) can be found in earlier figures (Figure 1-6).
The focus of Figure 7 is to helps us better appreciate all the changes seen in the preceding Figure 1-6:
Panel A and B indicate all changes that are considered significant
Panel C indicates total tissues with at least one significantly affected respiration
Panel D indicates total magnitude of change (i.e., which tissue has the highest OCR) offering a non-relative view
Panel E indicates whole body separations
Panel F indicates whole body separations and age vs sex clustering
(5) The current protocol does not provide cell-type-specific resolution and will be unable to identify the cellular source of mitochondrial respiration. This becomes important, especially for those organ systems with tremendous cellular heterogeneity, such as the brain. The authors should discuss whether the observed changes result from an altered mitochondria respiratory capacity or if changes in proportions of cell types in the different conditions studied (young vs. aged) might also contribute to differential mitochondrial respiration.
We agree with the reviewer that this is a limitation of the method. We have addressed this issue in the revised Discussion section.
(6) Another critical concern of this study is that the same datasets were repeatedly analyzed and reanalyzed throughout the study with almost the same conclusion - namely, aging affects mitochondrial function, and sex-specific differences are limited to very few organs. Although this study has considerable potential, the authors missed the chance to add new insights into the distinct characteristics of mitochondrial activity in various tissue and organ systems. The author should invest significant efforts in putting their data in the context of mitochondrial function.
We have attempted to address these issues in the revised Discussion section.
Reviewer #2 (Public Review):
Summary:
The authors utilize a new technique to measure mitochondrial respiration from frozen tissue extracts, which goes around the historical problem of purifying mitochondria prior to analysis, a process that requires a fair amount of time and cannot be easily scaled up.
Strengths:
A comprehensive analysis of mitochondrial respiration across tissues, sexes, and two different ages provides foundational knowledge needed in the field.
Weaknesses:
While many of the findings are mostly descriptive, this paper provides a large amount of data for the community and can be used as a reference for further studies. As the authors suggest, this is a new atlas of mitochondrial function in mouse. The inclusion of a middle aged time point and a slightly older young point (3-6 months) would be beneficial to the study.
We agreed with the reviewer that inclusion of additional time points (e.g., 3-6 months) would further strengthen the study. However, the cost, labor, and time associated with another set of samples (660 tissue samples from male and female mice and 1980 respirometry assays) are too high for our lab with limited budget and manpower. Regrettably, we will not be able to carry out the extra work as requested by the reviewer.
Reviewer #3 (Public Review):
The aim of the study was to map, a) whether different tissues exhibit different metabolic profiles (this is known already), what differences are found between female and male mice and how the profiles changes with age. In particular, the study recorded the activity of respirasomes, i.e. the concerted activity of mitochondrial respiratory complex chains consisting of CI+CIII2+CIV, CII+CIII2+CIV or CIV alone.
The strength is certainly the atlas of oxidative metabolism in the whole mouse body, the inclusion of the two different sexes and the comparison between young and old mice. The measurement was performed on frozen tissue, which is possible as already shown (Acin-Perez et al, EMBO J, 2020).
Weakness:
The assay reveals the maximum capacity of enzyme activity, which is an artificial situation and may differ from in vivo respiration, as the authors themselves discuss. The material used was a very crude preparation of cells containing mitochondria and other cytosolic compounds and organelles. Thus, the conditions are not well defined and the respiratory chain activity was certainly uncoupled from ATP synthesis. Preparation of more pure mitochondria and testing for coupling would allow evaluation of additional parameters: P/O ratios, feedback mechanism, basal respiration, and ATP-coupled respiration, which reflect in vivo conditions much better. The discussion is rather descriptive and cautious and could lead to some speculations about what could cause the differences in respiration and also what consequences these could have, or what certain changes imply.
Nevertheless, this study is an important step towards this kind of analysis.
We have attempted to address some of these issues in the revised Discussion Section. The frozen tissue method can only measure maximal uncoupled respiration. Because we are not measuring mitochondrial respiration using intact mitochondria, several of the functional parameters the reviewer alluded to (e.g., P/O ratios, feedback mechanism, basal respiration, and ATP-coupled respiration) simply cannot be obtained with the current set of samples. Nevertheless, we agree that all the additional data (if obtained) would be very informative.
Reviewer #1 (Recommendations For The Authors):
(1) For most of the comparative analysis, the authors normalized OCR/min to MitoTracker Deep RedFM (MTDR) fluorescence intensity. Why was the data normalized to the total protein content not used for comparative analysis? Is there a correlation between MTDR fluorescence and the protein content across different tissues?
Given that we used the crude extract method, total protein content does not equal total mitochondrial protein content. This is why the MTDR method was used, as this represents a high throughput method of assessing mitochondrial mass in this volume of samples. In general, the total protein concentration is used to ensure the respiration intensity was approximately the same across all samples loaded into the Seahorse machine.
(2) To test the mitochondrial isolation yield, the authors should run immunoblot against canonical mitochondrial proteins in both homogenates and mitochondrial-containing supernatants and show that the protocol followed effectively enriched mitochondria in the supernatant fraction. This would also strengthen the notion that the "µg protein" value used to normalize the total mitochondrial content comes from isolated mitochondria and not other extra-mitochondrial proteins.
Because we are using crude tissue lysate (from frozen tissue), the total ug protein content does not come from isolated mitochondria; for this reason, it was not used and this is why MTDR was. Total mitochondrial protein content is subject to change depending on tissue for non-mitochondrial reasons. This method does not use isolated mitochondria; we only use tissue lysates enriched for mitochondrial proteins. This method has been rigorously validated in the original study (PMID: 32432379) and a subsequent methods paper (PMID: 33320426). In those studies, the authors had performed requisite quality checks the reviewer has asked for (e.g., immunoblot against canonical mitochondrial proteins in both homogenates and mitochondrial-containing supernatants to show effective enrichment of mitochondrial proteins). For this reason, we did not repeat this.
(3) MitoTracker loads into mitochondria in a membrane potential-dependent manner. The authors should rule out the possibility that samples from different ages and sexes might have different mitochondrial membrane potentials and exhibit a differential MitoTracker loading capacity. This becomes relevant for data normalization based on MTDR (MTDR/µg protein) since it was assumed that loading capacity is the same for mitochondria across different tissue and age groups.
MitoTracker Deep Red is not membrane potential dependent and can be effectively used to quantify mitochondrial mass even when mitochondrial membrane potential is lost. This is highlighted in the original study (PMID: 32432379).
(4) Page 11, line 3 typo - across, not cross.
Response: We have fixed the typo.
Reviewer #2 (Recommendations For The Authors):
If possible, I would include a middle aged time point between 12 and 14 months of age.
We agreed with reviewer that inclusion of additional time points (e.g., 3-6 months) would further strengthen the study. However, the cost, labor, and time associated with another set of samples (660 tissue samples from male and female mice and 1980 respirometry assays) are too high for our lab with limited budget and manpower. Regrettably, we will not be able to carry out the extra work as requested by the reviewer.
Reviewer #3 (Recommendations For The Authors):
Overall, the work is well done and the data are well processed making them easy to understand. Some minor adjustments would improve the manuscript further:
- Significance OCR in Figure 2, maybe add error bars?
We have added the error bars and statistical significance to revised Figure 2.
- Tissue comparison A-C, right panel: graphs are cropped
We are not sure what the reviewer meant here. We have double checked all our revised figures to make sure nothing is accidentally cropped.
- Heart ventricle: Old males and females have higher CI- and CII-dependent respiration than young males and females? Only CIV respiration is lower?
Comparing old to young male or female heart ventricle respiration via CI or CII shows an increase in maximal capacity with age. CIV-linked respiration is in the upward direction as well, although not significant, when comparing old to young. When comparing the respiration values among themselves within a mouse, i.e. old male CI- or CII-linked respiration compared to old male CIV- linked respiration, we can see that the old male CIV-linked respiration is very similar. When comparing the same in the old female mouse, there appears to be something special about electrons entering through CI as compared to CII or CIV, as CI-linked respiration appears to be elevated compared to both CII and CIV. Although we do not know if this is significantly different, the trend in the data is clear. We do not know the exact reason as to why this occurred in the heart ventricles. To differing degrees, the connected nature of CI-, CII-, and CIV-linked respirations seems to be in a generally similar style in most skeletal muscles as well, and the old male heart atria. Again, the root of this discrepancy is unknown and potentially indicates an interesting physiologic trait of certain types of muscle and merits further exploration.
- What is plotted in Fig.3: The mean of all OCR of all tissues? A,B,C: Plot with break in x-axis to expand the violin, add mean/median values as numbers to the graph (same for Fig4)
The left most side of Figure 3 A, B, and C shows the average OCR/MTDR value across all tissues in a group. Each tissue assayed is represented in the violin plot as an open circle.
- Fig. 3D: add YM/YF to graph for better understanding, same in following figures
This is in the scale bar next to all heat maps presented in the figures. We also added to the revised figure as well to improve clarity.
- Additional figures: x-axis title (time) is missing in OCR graphs
Time has been added to the x axis of all additional figures for clarity.
- Also a more general question is: where the concentrations of substrates and inhibitors optimized before starting the series of experiments?
All the details of assay optimization was carried out in the original study (PMID: 32432379) and the subsequent methods paper (PMID: 33320426). Because we had to survey 33 different tissues, we tested and optimized the “optimal” protein concentrations we need to use; the primary goal of this was to balance enough respiration signal without too much respiration signal across all tissue types as to keep all the diverse tissues analyzed under the Seahorse machine’s capabilities of detection. Through our optimization of mostly the very high respiring tissues like heart and kidney, we were also able to prove that all substrates and inhibitors were in saturating concentrations since we could get respiration to go higher if more sample was added and that all signal could be lost in these samples with the same amount of inhibitors.
