Tmbim5 loss causes muscle atrophy without exacerbating mcu or slc8b1 knockout phenotypes

  1. Iga Wasilewska
  2. Dobrochna Adamek-Urbanska
  3. Sofiia Baranykova
  4. Lukasz Majewski
  5. Matilda Macias
  6. Aleksandra Szybinska
  7. Axel Methner

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Abstract

Mitochondria generate ATP by establishing a proton gradient across the inner membrane, which also drives calcium uptake via the mitochondrial calcium uniporter (MCU), while sodium-dependent NCLX (encoded by SLC8B1) facilitates efflux. The absence of severe phenotypes in MCU-knockout models suggests redundant calcium transport mechanisms. We investigated the role of TMBIM5, a potential bidirectional mitochondrial calcium transporter, by generating zebrafish lacking Tmbim5, Slc8b1, or both. tmbim5-knockout fish exhibited growth impairment, muscle atrophy (particularly in slow-twitch fibers), increased brain cell death, and reduced mitochondrial membrane potential without altering steady-state mitochondrial calcium levels. slc8b1-deficient fish demonstrated significantly attenuated sodium-dependent calcium efflux and increased larval mortality but were otherwise normal. Surprisingly, tmbim5/mcu and tmbim5/slc8b1 double knockouts were viable with normal Mendelian distribution. tmbim5/slc8b1 double knockouts displayed impaired mitochondrial calcium uptake capacity, diminished calcium efflux, and severely disrupted cristae architecture not observed in single knockouts. These findings suggest that Tmbim5 functions predominantly in mitochondrial calcium efflux rather than influx and indicate that robust compensatory mechanisms maintain mitochondrial calcium homeostasis despite disruption of these transport pathways.

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  1. Version published to 10.1101/2024.12.03.626543v2 on bioRxiv
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    Referee #3

    Evidence, reproducibility and clarity

    In this study, Wasilewska and colleagues generated tmbim5-/- zebrafish line and demonstrated that tmbim5 loss of function leads to decrease in zebrafish size and induces muscle atrophy. Authors used immunohistochemistry to suggest that tmbim5-/- zebrafish shows reduced glycogen levels in muscle and liver. However, most of the immunohistochemistry is not quantitated and only qualitative differences are shown. Next, the authors measured mitochondrial calcium levels in the brain of tmbim5-/- zebrafish but there was no behavioral phenotype in the fish. It would have be better to measure mitochondrial calcium levels in the muscles of tmbim5-/- zebrafish as phenotype is muscle atrophy. Further, it is reported that the mitochondrial membrane potential and glycogen levels were perturbed in tmbim5-/- zebrafish.

    Next, the authors generated a scl8b1-/- (a probable NCLX ortholog in zebrafish) zebrafish, which did not show any drastic phenotype. However, neither slc8b1 function nor the phenotype of scl8b1-/- zebrafish was well characterized. Further, authors created two double knockout zebrafish lines i.e. tmbim5-/-/mcu-/- and tmbim5-/-/slc8b1-/-. Interestingly, both these lines were viable and do not show any drastic phenotypes. The authors concluded that in these transgenic fishes compensatory and/or alternative mitochondrial Ca2+ mobilization pathways counterbalance the effects of silencing of these proteins.

    Although it is an interesting study, the conclusions are not well supported with the data. At several places only qualitative images are shown and quantitative data is missing. Similarly, Ca2+ imaging in muscles of tmbim5-/- zebrafish is not performed. Finally, no molecular mechanism or molecular details are provided. Though Tmbim5's potential role in EMRE degradation is discussed, it is not experimentally investigated. The quality of the manuscript would significantly enhance if authors perform the suggested experiments.

    Major Comments:

    1. As a potential mechanism, Tmbim5's potential role in EMRE degradation is discussed but it is not experimentally investigated. It is very easy to test this hypothesis. If this is the case, it would be a very good contribution to the field.
    2. On Page 16, authors state that slc8b1 does not constitutes the major mitochondrial Ca2+ efflux transport system. Authors should do calcium imaging experiments just like they did with tmbim5 and mcu double knockouts (data presented in Figure 4C) to make any comments on functioning of slc8b1 in mitochondrial Ca2+ transport. This is important because slc8b1 is only a predictive ortholog of human NCLX and it is not experimentally examined yet.
    3. The data presented in Fig. 4C is very important but it is not fully explained and discussed in the results. Please discuss all the data sets presented in Fig4C in detail. As such, it is very difficult to follow and interpret the data.
    4. In tmbim5-/- zebrafish, what happens to mitochondrial Ca2+ signaling in muscle as phenotype is muscle atrophy only?
    5. Please validate the observation of decreased glycogen levels in tmbim5-/- fish by one more way. Only immunohistochemistry that too without quantitation is not convincing (Fig. 2E-H).

    Minor Comments:

    1. Authors state that tmbim5 loss of function leads to metabolic changes but the only data provided is decrease in glycogen levels. It would be helpful for the authors to focus comments specifically on the data presented in the manuscript to avoid potential over-interpretation.
    2. While discussing Fig4., authors mention that Tmbim5 may act as a MCU independent Ca2+ uptake mechanism and therefore they crossed tmbim5 mutants with mcu KO fish. But from the data presented in Fig.3 and as concluded by the authors themselves tmbim5 mutants do not show changes in the mitochondrial Ca2+ levels. Authors may clarify this point.
    3. Does tmbim5 contributes to mitochondrial Ca2+ uptake in presence or along with MCU. Further analysis of Fig4C may shed some light on this. Authors should test significance between tmbim5-/- and WT as well as between tmbim5-/- and tmbim5+/+ in mcu-/- background.
    4. Please check the labeling on traces in Fig3D.
    5. Please include quantitation of data presented in EV2E-F.
    6. Please include quantitation of immunohistochemistry data presented in 2E-H.

    Referee cross-commenting

    Several comments are common between the reviewers highlighting that those experiments are critical. Secondly, I agree with the concerns raised by other two reviewers.

    Significance

    In this study, authors report couple of new transgenic zebrafish lines. However, further characterization of slc8b1-/- is required. This study reinforces the existing idea that there are very robust compensatory mechanisms that maintain mitochondrial Ca2+ homeostasis. While the work provides useful insights, it could benefit from a broader scope to provide substantial advancement to existing knowledge.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: The work of Wasilewska et al. focusses on the MCU independent basal Ca2+ uptake mechanisms and the effects of MCU, NCLX, and TMBIM5 KO on Zebrafish Ca2+ homeostasis, mortality, anatomy and metabolism. The authors found evidence that tmbim5 potentially has a bidirectional mode of operation and is able to extrude Ca2+ from the matrix as well as transfer Ca2+ into mitochondria. Further, a reduced membrane potential in tmbim5-/- fish and altered metabolism was found. While the conclusion drawn are well argumented, a few points have to be addressed.

    Major Points:

    1. While all mitochondrial genes seem collectively reduced compared to control, it would be interesting to assess the mitochondrial mass and/or mitochondrial turnover rate in regard to e.g. mitophagy. The reduced membrane potential could lead to PINK1 accumulation on the outer mitochondrial membrane to mediate mitophagy leading overall to reduced mitochondrial count and mass.
    2. The characterization of slc8b1-KO fish needs some improvement to facilitate a better understanding of the molecular interactions of slc8b1 and tmbim5. This would also greatly improve the understanding of the phenotypical characterization and behavioral response to CGP.
    3. Functional Ca2+ measurements of the activity of slc8b1 gene product have to be done to ensure a KO phenotype. Especially in light of the surprising results presented in Figure 6A showing an effect of CGP on slc8b1-KO fish but not on tmbim5-KO fish I advise mitochondrial isolation to conduct mitochondrial basal and extrusion Ca2+experiments of slc8b1-KO fish, tmbim5-KO fish, and double KO-fish.

    Minor Points:

    The authors claim that mRNA levels of mitochondrial proteins involved in Ca2+ transport in tmbim5-/- are unaffected (Figure EV3). While the T-tests show no significant alteration, what happens if a 2-way ANOVA shows a more general effect revealed between WT and TMBIM5-/-?

    Significance

    This is a well-designed and carefully executed piece of work. The experimental design is thoughtfully elaborated, and the topic is worthy of investigation. The strengths of this study lie in translating our knowledge of TMBIN5 from single cells to organism and organ function. Moreover, the work provides important new information that will help the scientific community working on mitochondrial regulation AND muscle diseases to understand how ions coordinately regulate mitochondrial function.

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    Referee #1

    Evidence, reproducibility and clarity

    Although the experimental approach is promising (see below), the results do not significantly expand our current understanding. This is partly due to the challenges of interpreting negative results, which are nonetheless worth reporting. Some of the conclusions and interpretations of the results could benefit from further clarification and contextualization to enhance their impact:

    • Figure 1D: The distribution of fiber size in wt vs. Tmbim5-ko fish shows a notable difference limited to one size range. Can the authors clarify this observation? Could this indicate a switch in fiber type? Is there a correlation between this finding and the differential PAS staining?
    • Figure 3: one of the advantages of the zebrafish model is its transparency, allowing for fluorescence imaging. Unfortunately, this proves to be impossible in the case of cepia2mt. The data provided by the authors show that the fluorescence of this probe does not vary following physiological stimuli. The only change is that induced by CCCP (Fig 3C-D), which according to the authors causes a discharge of mitochondrial calcium. However, the use of CCCP with GFP-based probes should be avoided, as the acidification caused by CCCP treatment leads to quenching of the fluorophore, resulting in a fluorescence decrease which is independent of Ca2+ levels. Although the experimental approach aims to detect dynamic changes in mitochondrial Ca2+ levels, the presented results in Figure 3 do not provide conclusive evidence to support this capability. While significant experimental effort is evident, these findings may require further validation or additional data to strengthen their impact. Alternatively, the authors could remove this Figure 3 and relevant text from the manuscript.
    • Figure 6A: In my opinion, this dataset is impossible to understand. To my knowledge, the precise molecular target of CGP-37157 remains elusive. While CGP is often considered an NCLX inhibitor, this classification lacks definitive experimental support. Although CGP is known to inhibit mitochondrial Na+-dependent Ca2+ extrusion, direct binding of CGP to NCLX has yet to be conclusively demonstrated. With this in mind, the authors show that pharmacological intervention with CGP elicits a distinct phenotype in the fish model. While this effect appears to persist in SLC8B1-KO fish, it is absent in Tmbim5-KO fish, suggesting Tmbim5 as a potential molecular target for CGP. However, this interpretation is inconsistent with the following observations: i) CGP remains effective in Tmbim5/Slc8b1 double-KO fish and ii) Tmbim5-KO fish exhibit no discernible phenotype. A comprehensive explanation that reconciles these findings is sought.
    • Figure 6B: according to the authors, the phenotype induced by CGP treatment is specific because a different substance with a completely different effect, CCCP, causes the same phenotype in both wt and Tmbim5-KO fish. Also in this case, the rationale and reasoning behind this experiment in not very evident. As I see it, CCCP blocks zebrafish motility because it is a metabolic poison, and its effect does not depend on any transporter.

    Significance

    The manuscript submitted by Wasilewska et al investigates the functional relationship between different mitochondrial calcium transporters using zebrafish as a model. The topic is of great interest. In the last 15 years, many mitochondrial calcium transporters have been identified. In some cases, their mechanism is not fully understood, such as in the case of TMBIM5, recently described by some as an H/Ca exchanger, or as a Ca channel by others. Furthermore, the functional relationship between different transporters has so far been studied in a partial and superficial way. I believe that this work is therefore of great interest because it aims to contribute to a fundamental problem that is still poorly studied. The idea of using zebrafish is interesting, as it is an organism that is easy to manipulate and phenotype, and because it is transparent, making it possible to use specific biosensors to characterize mitochondrial calcium dynamics, at least in principle. The paper therefore deserves attention.

  6. Version published to 10.1101/2024.12.03.626543v1 on bioRxiv