Talk:Iyer 2022 Abstract Bioblast

Comments by the Editor and response by the Authors

Gnaiger E 2022-05-26 / Response Iyer Shilpa 2022-5-28

1. mitoBHI

• mitoBHI was changed to mtBHI, according to abbreviation mt for mitochondria (see https://doi.org/10.26124/bec:2020-0001.v1, where you are coautor).

Yes, we agree with the change from mitoBHI to mtBHI.

• The 'four key aspects' are now listed in the abstract. In addition, it should be explained how the mtBHI was calculated from these four key aspects.

As per Dr. Rafael Moreno Sanchez’s suggestion, we are not including the detailed calculation in the abstract, but have described it in the main text of the manuscript.

2. Non-mitochondrial oxygen consumption

• non-mitochondrial oxygen consumption was changed to residual oxygen consumption Rox, since Rox may include mt-oxygen consumption that is not inhibited by rotenone and antimycin A (see https://doi.org/10.26124/bec:2020-0001.v1)

We are in agreement with the suggested editorial change already made in the abstract (v2).

3. Spare respiratory capacity

• Is it correct to assume that spare respiratory capacity was calculated as ET capacity minus ROUTINE respiration, E-R? How did you determine that E-R was a spare capacity, which implies that the cells did not make full use of their total capacity? The 'total capacity' of cell respiration may be compared to the OXPHOS capacity P of permeabilized cells (see https://doi.org/10.26124/bec:2020-0001.v1 for definition of P and E). A large E-P excess capacity is known for fibroblasts (Figure 6.12 in https://doi.org/10.26124/bec:2020-0002). Therefore, the actual spare capacity is P-R, and your estimation of spare capacity is overestimated by E-P. If it was not shown that E=P, then E-R is more appropriately termed "E-R reserve capacity". It is a reserve that cannot be fully activated by increasing ATP turnover but requires uncoupling or dyscoupling (https://doi.org/10.26124/bec:2020-0002).

In our study, we have used intact cells for measuring mitochondrial respiration. As per equation in Figure 1 provided by the manufacturer, spare respiratory capacity or (SRC) was calculated based as the difference between E=FCCP uncoupled respiration which is felt to reflect the maximal respiration (E) vs. basal respiration (R).

Since we used intact cells, it will be difficult to measure total capacity. In intact cells that utilize endogenous substrates and regulate their ADP availability; the uncoupled respiration rate is the best, though imperfect, estimate of maximal respiratory capacity. The physiologic significance of the “spare capacity” is open to interpretation. I agree E-R could be more appropriately termed as “E-R reserve capacity”. We have included the calculations below that was used for measuring different mitochondrial parameters. However, to minimize confusion, we are utilizing terminology used by most users of the Seahorse system.

4. ATP-linked respiration

• Is it correct to assume that ATP-linked respiration was calculated as R-L? If the cell would have a maximum shift of the ADP/ATP ratio towards ATP (T), then ATP-linked respiration would equal LEAK respiration L_T (Chance&Williams termminology: State 4), since phosphorylation is ADP limited. Therefore, 'ATP-linked respiration' should be changed to "ADP-linked respiration".

After replacing spare respiratory capacity by E-R: .. E-R, which was an indicator of the cell’s capacity to adapt to the defect.

• This simple interpretation cannot be applied in general: If the defect in dyscoupling, then E-R is diminished by a compensatory increase of R. If the phosphorylation system is strongly inhibited without injury of the ETS, then E-R is increased.

Please see the equation above in Figure 1, for calculation of ‘ATP-linked respiration’ is actually, as you know oxidative phosphorylation that produces ATP and of course represents the difference between state 3 and state 4 respiratory rates, as described by Chance & Williams. Since measurement was in intact cells and not permeabilized cells, I am hesitant to use “ADP-linked respiration” in our study.

We generally agree with the comment and the maximal respiration E indicates the maximal rate or respiration, which is decreased in cells with a defect in the electron transport chain. The fraction of this maximal capacity that is utilized in the intact cell represents the metabolic demand on the cell and the R needed to phosphorylate adequate ADP for cell survival and work. The fraction of the (decreased) maximal respiration in Leigh’s cells used would be expected to be greater than in normal cells.

5. The glycoBHI was based on four key aspects of glycolysis

• The 'four key aspects' are now listed in the abstract. In addition, it should be explained how the glycoBHI was calculated from these four key aspects.

As per Dr. Rafael Moreno Sanchez’s suggestion, we are not including the detailed information in the abstract.

6. Proton leak

• How did you measure the proton leak? If proton leak was measured as LEAK respiration L (which includes proton slip and cycling of cations other than protons) then the correct term LEAK respiration should be applied (https://doi.org/10.26124/bec:2020-0001.v1).

We measured proton leak based on calculation in Figure 1. We are in agreement that “proton leak” is LEAK respiration.

7. Basal proton efflux rate

• "Basal respiration or basal metabolic rate (BMR) is the minimal rate of metabolism required to support basic body functions, essential for maintenance only. BMR (in humans) is measured at rest 12 to 14 hours after eating in a physically and mentally relaxed state at thermally neutral room temperature" (see Basal respiration). Was 'basal proton efflux rate' measured in the LEAK state or ROUTINE state of the cells? If the latter was the case, the term should be replaced by "ROUTINE proton efflux rates". Which buffering capacity was measured or assumed to convert the measured acidification rate (change of pH over time) to the proton efflux rate (change of proton concentration over time)?

We did not and do not imply that the basal proton efflux rate is related to the basal metabolic rate of the organism. As per manufacturer’s instructions, “a new metric ‘basal proton efflux rate’ or (basal PER) measured the extracellular acidification by accounting for buffering factor which has been predetermined and plate geometry. The basal PER measures the total cellular proton efflux rate”. In addition, by measuring and subtracting CO₂-dependent acidification, the glycolytic proton efflux rate (glycoPER) was calculated for intact live cells shown by the equation below:

• basalPER(total) = glycoPER + mito(PER)

Together, they demonstrate that accounting for mitochondrial CO₂ production, enables quantification of glycolytic rate in these extracellular flux assays.

8. Compensatory glycolysis

• Is it correct to assume that 'compensatory glycolysis' was calculated from the proton efflux rate after inhibition of respiration? Which corrections were applied to account for proton efflux not linked to glycolysis?

Yes, it is correct to assume that 'compensatory glycolysis' was calculated from the proton efflux rate after inhibition of respiration (see the illustration in Figure 2).

9. Mitochondrial proton efflux rate

• How was the mitochondrial proton efflux rate measured or calculated?

As shown in Figure 2, the contribution of the mitochondria to oxygen consumption and proton production was calculated as:

• mtPER = totalPER - (rotenone/antimycin) PER

• Gnaiger E 2022-05-28 The mt-proton pumps CI, CIII, and CIV are responsible for mt-proton efflux, which at steady-state is balanced by mt-proton influx (ATP synthase, proton leak, etc.). This contrasts with 'mitochondrial proton efflux rate' defined above. Should this ambiguous term be replaced by the explicit expression PER_ROUTINE - PER_ROX?

10. Basal (ROUTINE?) glycolysis, which was a measure of mitochondrial defect

• This simple interpretation cannot be applied in general: If the mitochondrial defect is mild dyscoupling, this can be compensated by a corresponding stimulation of R without impairment of the rate of aerobic ATP turnover, which then would not be reflected by a compensatory increase of ROUTINE glycolysis.

We agree. It was not a simple measure of basal glycolysis as an indicator of mitochondrial defect. The results from this study has attempted to provide a potential measure of multiple parameters contributing to overall mitochondrial dysfunction and attempted to link it with symptoms associated with disease severity.

GENERAL COMMENT

• Even if some of the terms discussed above are used by several authors in the literature, this provides no scientific justification to uncritically apply those terms, if they are ambiguous or even misleading - in the spirit of Gentle Science.

We have endeavored to clarify the terms used to describe measured components of oxidative and glycolytic function to be consistent with terms widely used by the multiple scientific groups using the Seahorse system to analyze intact cells.

» MitoPedia:_Terms_and_abbreviations#Harmonization

Comments by Reviewer 2

Moreno-Sanchez Rafael 2022-05-26

You made a thorough review of the abstract submitted by Iyer et al., with over 10 critical observations on terminology but also on concepts and definitions Nothing else to say, except for your remark (No. 1) "it should be explained how the mtBHI was calculated from these four key aspects". I do not think this request should be in the abstract; it would be expected in the main text of the full manuscript. "Therefore, 'ATP-linked respiration' should be changed to "ADP-linked respiration" " (No. 4) or OxPhos flux or net OxPhos flux. "it should be explained how the glycoBHI was calculated from these four key aspects." (No. 5). This information should not be in the abstract.

Harmonization of some terms in mitochondrial physiology

MitoPedia:_Terms_and_abbreviations#Harmonization

Version 2

2022-05-26

Leigh Syndrome (LS), is a severe neuro-metabolic disorder and has no current cure or adequate cellular models to understand the rapid fatality associated with the disease. Other symptoms are widespread tissue malfunction in brain stem and muscle in LS patients. We hypothesize that altered bioenergetic function caused by mitochondrial genome mutations in the electron transfer system (ETS) may lead to rapid fatality in LS. The extent to which pathogenic mtDNA variants regulate disease severity in LS is currently not well understood. To better understand this relationship, we computed the mitochondrial bioenergetics health index (mtBHI) and glycolytic bioenergetics health index (glycoBHI) for measuring overall mitochondrial dysfunction in LS patient fibroblast cells harboring varying percentages of pathogenic mutant mtDNA (T8993G, T9185C) exhibiting deficiency in ATP synthase or Complex I (T10158C, T12706C). The mitoBHI was based on four key aspects of mitochondrial respiration (spare respiratory capacity or SRC, ATP-linked respiration, non-mitochondrial oxygen consumption, and proton leak); while the glycoBHI was based on four key aspects of glycolysis (basal proton efflux rate, compensatory glycolysis, mitochondrial proton efflux rate, post 2-deoxy-D-glucose acidification).

Our results indicated that (1) high heteroplasmy was detected in disease lines affecting ATP synthase and low heteroplasmy was detected in disease lines affecting NADH dehydrogenase; (2) levels of defective enzyme activities of the ETS correlated with the percentage of pathogenic mtDNA; (3) mitochondrial respiration was disrupted in diseased lines with variable SRC; (d) mitochondrial ATP synthesis rate was decreased while glycolytic ATP synthesis rate was elevated in diseased cell lines.

Based on the overall analysis of the five diseased patient-specific fibroblasts, the glycoBHI emerged as a sensitive indicator of mitochondrial defects because the cells had switched ‘on’ the glycolytic pathway. GlycoBHI was significantly increased in all cell lines compared to control BJ-FB and was indeed sensitive to mitochondrial dysfunction. We also computed the ‘composite BHI ratio’ (OXPHOS/Glycolysis) by dividing mtBHI/glycoBHI values because the cell lines were utilizing both OXPHOS (although highly defective) and glycolysis pathways to maintain the energy requirements in the individual cell line. Two important parameters associated with the composite BHI ratio were basal glycolysis (PER), which was a measure of mitochondrial defect, and SRC, which was an indicator of the cell’s capacity to adapt to the defect.

Overall, these results suggest that as long as the precise mechanism of LS has not been elucidated, a multi-pronged approach that takes into consideration the specific pathogenic mtDNA variant, along with a composite BHI ratio, can aid in better diagnosis and understanding the factors influencing disease severity and rapid fatality in LS.

Future experiments will determine whether mitochondrial morphology depend on mtDNA mutation load and whether they influence bioenergetics within a cell. Our ongoing studies are focused on evaluating mutation burden in human induced pluripotent stem cells (hiPSCs) reprogrammed from these patient fibroblast cells, followed by bioenergetic analyses in differentiated neurons and muscle cells derived from hiPSCs. Results from these studies will address the knowledge gaps that exist in the understanding of relationships among mtDNA mutations, morphology, function, and cell fate that may ultimately contribute to devastating mitochondrial disorders.

Version 1

2022-05-15

Leigh Syndrome (LS), is a severe neuro-metabolic disorder and has no current cure or adequate cellular models to understand the rapid fatality associated with the disease. Other symptoms are widespread tissue malfunction in brain stem and muscle in LS patients. We hypothesize that altered bioenergertic function caused by mitochondrial genome mutations in the electron transport chain (ETC) may lead to rapid fatality in LS. The extent to which pathogenic mtDNA variants regulate disease severity in LS is currently not well understood. To better understand this relationship, we computed a glycolytic bioenergetics health index (BHI) for measuring mitochondrial dysfunction in LS patient fibroblast cells harboring varying percentages of pathogenic mutant mtDNA (T8993G, T9185C) exhibiting deficiency in complex V or complex I (T10158C, T12706C). Our results indicated that (a) high heteroplasmy was detected in was detected in disease lines affecting ATP Synthase and low heteroplasmy was detected in disease lines affecting NADH Dehydrogenase; (b) levels of defective enzyme activities of the ETC correlated with the percentage of pathogenic mtDNA; (c) mitochondrial respiration was disrupted in diseased lines with variable spare respiratory capacity (SRC) (d) mitochondrial ATP synthesis rate is decreased while glycolytic ATP synthesis rate is elevated in diseased cell lines. Based on the overall analysis of the five diseased patient-specific fibroblasts, the ‘glycoBHI’ emerged as a sensitive indicator of mitochondrial defects as the cells had switched ‘on’ the glycolytic pathway. GlycoBHI was significantly increased in all the cell lines compared to control BJ-FB and was indeed sensitive to mitochondrial dysfunction. We also computed the ‘composite BHI ratio’ oxphos/glycolysis because the cell lines were utilizing both oxphos (although highly defective) and glycolysis pathways to maintain the energy requirements in the individual cell line. Two important parameters associated with the composite BHI ratio were basal glycolysis (PER), which was a measure of mitochondrial defect, and SRC, which was an indicator of the cell’s capacity to adapt to the defect. Overall, these results suggest that as long as the precise mechanism of LS has not been elucidated, a multi-pronged approach that takes into consideration the specific pathogenic mtDNA variant, along with a composite BHI ratio, can aid in better diagnosis and understanding the factors influencing disease severity and rapid fatality in LS. Future experiments will determine whether mitochondrial morphology depend on mitochondrial DNA mutation load and whether they influence bioenergetics within a cell. Our ongoing studies are focused on evaluating mutation burden in human induced pluripotent stem cells (hiPSCs), followed by bioenergetic analyses in differentiated neurons and muscle cells from LS-hiPSCs. Results from these studies will address the knowledge gaps that exist in the understanding of relationships among mtDNA mutations, morphology, function and cell fate that may ultimately contribute to devastating mitochondrial disorders.