Mitochondrial fission process 1 (MTFP1) controls bioenergetic efficiency and prevents inflammatory cardiomyopathy and heart failure in mice

  1. Erminia Donnarumma
  2. Michael Kohlhaas
  3. Elodie Vimont
  4. Etienne Kornobis
  5. Thibault Chaze
  6. Quentin Giai Gianetto
  7. Mariette Matondo
  8. Maryse Moya-Nilges
  9. Christoph Maack
  10. Timothy Wai

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Abstract

Mitochondria are paramount to the metabolism and survival of cardiomyocytes. Here we show that Mitochondrial Fission Process 1 (MTFP1) is essential for cardiac structure and function. Constitutive knockout of cardiomyocyte MTFP1 in mice resulted in adult-onset dilated cardiomyopathy (DCM) characterized by sterile inflammation and cardiac fibrosis that progressed to heart failure and middle-aged death. Failing hearts from cardiomyocyte-restricted knockout mice displayed a general decline in mitochondrial gene expression and oxidative phosphorylation (OXPHOS) activity. Pre-DCM, we observed no defects in mitochondrial morphology, content, gene expression, OXPHOS assembly nor phosphorylation dependent respiration. However, knockout cardiac mitochondria displayed reduced membrane potential and increased non-phosphorylation dependent respiration, which could be rescued by pharmacological inhibition of the adenine nucleotide translocase ANT. Primary cardiomyocytes from pre-symptomatic knockout mice exhibited normal excitation-contraction coupling but increased sensitivity to programmed cell death (PCD), which was accompanied by an opening of the mitochondrial permeability transition pore (mPTP). Intriguingly, mouse embryonic fibroblasts deleted for Mtfp1 recapitulated PCD sensitivity and mPTP opening, both of which could be rescued by pharmacological or genetic inhibition of the mPTP regulator Cyclophilin D. Collectively, our data demonstrate that contrary to previous in vitro studies, the loss of the MTFP1 promotes mitochondrial uncoupling and increases cell death sensitivity, causally mediating pathogenic cardiac remodeling.

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    Referee #4

    Evidence, reproducibility and clarity

    Summary

    Fusion and fission of the mitochondrial network is one of the hottest topics in mitochondrial biology in the last years. The process is obviously necessary to allow cells to control the quality of small individual organelles, which are degraded by autophagy or mitophagy, if they are not working properly. Since a healthy mitochondrial network is essential for every cell in the body, the molecular players involved in these two processes are heavily investigated. In this paper, the authors investigate the role of MTFP1 in vitro and in vivo, a protein which has been studied and named because it seemed to be an important fission factor in cultured cells.

    Surprisingly and excitingly, the authors find that mitochondrial morphology and homeostasis is not affected by knocking out this protein in the heart of mice. On the contrary, it shows that this protein is a critical regulator of mitochondrial inner membrane coupling via the adenine-nucleotide-transporter (ANT). A loss of MTFP1 leads to a decline in the mitochondrial membrane potential, leading to cell death, which finally results in dilatative cardiomyopathy and causes early death of the animals. Therefore, this paper gives an important mitochondrial inner membrane protein a new role which may become very important to understand the opening of the large channel (MTPT-channel), which is responsible for some kinds of cell death in almost all cell types.

    Major comments:

    The conclusions are convincing, additional experiments on the molecular nature of the interaction between MTFP1 and ANT may be easily proposed by a reviewer; however, this will open a completely new line of research and should not be asked at this moment. Data and methods are presented in a perfect way, typical for the Wai lab. Statistical analysis has been performed meticulously, and there is nothing to add here. I have read the paper very carefully, but cannot find many points which should be changed.

    Minor Points:

    I must admit I hate the title, but the authors are in good company using the "genetic argument", as many others do. Mitochondrial fission process controls energetic efficiency - that is correct, but it does not prevent inflammatory cardiomyopathy and heart failure in mice. It is intact mitochondria which prevent inflammatory cardiomyopathy and heart failure, and as long as we do not know what exactly MTFP1 does, this title is misleading, although it may be considered attractive for readers. I would reformulate that and mention the new role of this protein in coupling of the mitochondrial inner membrane potential, but I leave this to the authors, of course.

    P. 2, line 45: The loss of MTFP1 promotes ... (erase "the")

    P. 12, line 321: There is clearly no indication of mitochondrial elongation, but I do see clearly in these pictures a separation between the organelles in the mutant mice in contrast to wild type, where mitochondria touch each other (Fig. 3c to d). If this is consistent, it should be mentioned. P. 12, line 324: I am not a true expert in fusion and fission, so wouldn't be a blot showing all the OPA1 isoforms necessary here?

    P. 13, line341: The same argument is repeated in two sentences following each other. I suggest to write here "Our data collectively indicate that MTFP1, unlike DRP1, is not an essential fission protein, contrary to its namesake, either in vitro or in vivo.".

    P. 13, line 349: "We sought to investigate..."

    Significance

    Understanding mitochondrial dynamics (fusion and fission) and bioenergetics (which some people considered to be fully known since the 1950s) is of utmost importance for biology and biomedicine. Since this paper gives a prominent protein, which the field believes is a fission factor, a completely new role, it is a paper of high interest. As the authors state, using these mice the protein may help to understand the molecular function of the mitochondrial membrane permeability transition pore (MPTP), which is still enigmatic, but important for so many ways of cell death. The paper is therefore state of the art and at the frontline of cell biology, and the large mitochondrial community will be very interested to read the paper.

    I have been working on mitochondria for 35 years, starting with bioenergetics, switching then to mitochondrial biogenesis regulated by transcription of nuclear genes as well as the mitochondrial genome, followed by studying the consequences of mtDNA mutations, and now considering how mitochondrial dysfunction may be involved in the normal aging process. Therefore, I feel myself competent to critically judge the quality of this paper. I am not a molecular biologist, therefore, the molecular details of protein-protein interaction do not lie in the focus of my interest; on the contrary, I feel that sometimes too much emphasis is laid on such molecular details, while the big question - in this case, how mitochondrial membrane potential is regulated - is not addressed at all.

    Referee Cross-commenting

    I guess we all suffer from reviewers of our own papers asking for more mechanistic insight. This paper unexpectedly shows a new role for MTFP1 - which is important for the mito community - and opens the door to more mechanistic studies how it uncouples the mitos and leads to cell death via ANT and MPTP - which is imprtant for a very broad community.

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    Referee #3

    Evidence, reproducibility and clarity

    Donnarumma et al. characterize cardiac-specific KO of Mitochondrial fission process 1 (MTFP1), a mysterious mitochondrial protein thought to be involved in mitochondrial inner membrane fission. They initially demonstrate that the survival, cardiac function and respiration is diminished in the KO mouse and seek to find a mechanism. In MEF cells, surprisingly, they do not report any changes in fission, though mitochondrial morphology is altered. The authors then identify loss of MTFP1 as being damaging through exacerbation of cell death, possibly due to enhanced activation of the mitochondrial permeability transition. This is a beautiful and thorough paper. The data presented is of high quality and the conclusions are well supported by the figures. There was little to criticize in the manuscript!

    1. Although total mtDNA levels were no different, was there mtDNA release into the cytoplasm in Mtfp1 cKO? This is one possible mechanism to consider regarding the interferon response, as this would be a potent trigger for the innate immune response, as pointed out in the discussion and in PMC4409480.
    2. The authors show mitochondrial morphology in the pre-symptomatic period. What happens during DCM? Does this effect become exacerbated in the KO compared to WT?
    3. Given the cellular phenotypes seen in the ppif/Mtfp1 DKO cells, does this translate into a survival benefit in these animals? (If this data is easily available would recommend showing it, even if negative; but if entirely new crosses and 20-30 weeks of follow-up are required then it's fine to not address this question here).
    4. Methods (line 1281): It appears that only male mice were imaged from 10-34 weeks? Why only show one sex, especially as the authors note a difference in survival between males and females? Also, it is unclear why the data on female HF is relegated to the Supplement. This should be in the main manuscript side-by-side with the male data on the same scale to allow comparison of effect sizes on similar assays. Minor comments:
    5. Please change the title: "inflammatory cardiomyopathy" is a poorly defined term and would suggest myocarditis or inflammatory cell infiltrates, which are not shown in the manuscript. In addition, the only discussion of inflammation is through the innate immunity pathway in the RNA-seq data, with no real further follow-up.
    6. Line 39, Abstract: "ANT" needs to be in brackets/parenthesis
    7. Figure 1M: It would be good to see a higher magnification image showing fibrosis in the trichrome stain.
    8. Line 180, "gender" should more properly read "sex".
    9. At line 321, the authors state that there are no changes in mitochondrial elongation, however, Figure 3D seems to suggest that mitochondrial area is decreased in MKO cells. Is this an error or are the authors suggesting that the data in 3D is not significant? How was elongation measured?
    10. At line 335, the authors state that MTFP1 KO mitochondria were not protected from fragmentation, this is supported by the data in Figures 3G-H. However, to my eye, it appears that the mitochondria from the KO cells were far more fragmented in response to hydrogen peroxide. Is this data not significant?

    Significance

    This paper is novel in that it constitutes the first description of a mouse cardiac knockout of MTFP1, a poorly studied protein previously thought to be involved in mitochondrial fission. Previously MTFP1 has been described in knockdown cells (Aung et al. J Cell Mol Med. 2017 Dec; 21(12)) and the current paper builds upon this research. The current paper demonstrates that MTDP1 is important for cardiac function, but intriguingly, does not share the prior in vitro phenotypes related to mitochondrial fission, suggesting that it may have some other physiological function. Most of the methods shown are standard, though there are some quite novel machine learning-based analyses of imaging data. The paper is quite thorough and of relevance to a wide range of investigators interested in cardiac mitochondrial function, mitochondrial kinetics (fusion/fission), and cell death mechanisms more broadly. Our field of expertise is in cardiac mitochondrial function. The ML computational tools are very interesting, but these are not our expertise.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript entitled "Mitochondrial fission process 1 (MTFP1) controls bioenergetic efficiency and prevents inflammatory cardiomyopathy and heart failure in mice" by Donnarumma and collaborators investigate the role of MTFP1 in the heart in vivo. Mice with a cardiac-specific deletion of Mtfp1 were generated and fully characterized. Structure-function analyses were performed prior to and at the onset of the cardiomyopathy and show that homozygous Mtfp1 ko mice develop DCM progressing to heart failure and death in middle age, associated with increased fibrosis. RNAseq data revealed a severe impairment of metabolic processes including reduced oxidative phosphorylation, TCA cycle and mitochondrial gene expression. Mitochondrial respiration was significantly reduced in mitochondria isolated from Mtfp1 ko mice, while global mitochondrial proteins and activity of the Krebs cycle remained normal. Further assessment of a variety of processes revealed an increase in proton leak through ANT as the major contributor to the mitochondrial defects and cardiac dysfunction in Mtfp1 ko mice. Cardiomyocytes isolated from Mtfp1 ko mice were also more sensitive to stress-induced apoptosis and to mPTP opening. The major conclusion of the study is that contrary to previous reports documenting a role of MTFP1 in mitochondrial fission, MTFP1 does not regulate mitochondria morphology but rather is essential for cardiac energy balance. This is substantiated by mass spectrometry experiments which identify mitochondrial proteins of the complex I/IV and proteins regulating mPTP as MTFP1 partners.

    Overall, this is an elegant study with an impressive amount of work performed in isolated mitochondria and in vivo before and at the onset of DCM. Results are important because they challenge previous findings that established a role of MTFP1 in mitochondrial fission and therefore reveal another function of MTFP1. To rigorously establish how MTFP1 regulates cardiac bioenergetics, additional experiments are needed and are listed below. In particular, the use of wildtype mice as control is concerning because some transgenic lines of Myh6-Cre+ develop DCM. Also, experiments addressing MTFP1 as an essential fission protein should be performed in adult ventricular myocytes isolated from Mtfp1 ko mice to show consistency with experiments performed in MEFs.

    Major comments:

    One major concern is with the control mice, which appear to be wildtype (Myh6-Cre+/+ Mtfp1 LoxP/LoxP). The proper control group should be Myh6-Cretg/+. This is important because some models of Myh6-Cre+ mice develop DCM including mitochondrial dysfunction (Buerger et al., J Card Failure 2006; Hall et al., Am J Physiol Heart Circ Physiol 2011). At a minimum, the most critical assays evaluating mitochondrial function should be performed using Myh6-Cre+ as control to verify that they do not develop pathological cardiac remodeling.

    The observation that Mtfp1ko mice show a complete loss of the protein by Western blot analysis is intriguing because it suggests that Mtfp1 is only expressed in ventricular myocytes and not in the other cells populating the heart. Can you please comment on this?

    Was Seahorse analysis from ventricular myocytes isolated from Mtfp1ko performed in parallel with the analysis in MEF and U2OS cells? This should be done to establish the cell specific defects observed in cardiac mitochondria lacking Mtfp1.

    Mitochondrial morphology under normal or stress condition was assessed in MEF, which have very distinct characteristics than primary cardiac cells. The experiment using oligomycin, rotenone and CCCP should be performed in ventricular myocytes isolated from Mtfp1 ko mice, to rigorously reach the conclusion that MTFP1 is not essential for mitochondrial fission.

    Related to that, is-it possible that while total levels of mitochondrial fission and fusion proteins are similar in Mtfp1 ko and wt mice, their phosphorylated forms may be different?

    Figure 4: Cell death in Mtfp1 ko and control cardiomyocytes is measured using supervised ML-assisted high throughput live-cell imaging (Cretin et al., 2021). This result should be substantiated by additional apoptosis assays.

    Cell death assay are performed by treating cardiomyocytes isolated from Mtfp1 ko and wt mice with the cardiotoxic anthracycline doxorubicin (DOX). The dose DOX of 60 microM is extremely high. Can cell death be observed at lower concentrations of DOX?

    Minor comments:

    Line 349: there is a typo. Please replace "we sought investigate whether MTFP1 loss specifically..." with "we sought to investigate whether MTFP1 loss specifically..."

    Line 417: What the authors mean is that "the modest level of over-expression did not negatively impact cardiac function in vivo (Figure S5B-C)".

    Line 490-500: this is a very long sentence. Please break it down into 2 sentences to ease the reading.

    Significance

    The role of MTFP1 has been investigated in isolated cells where conflicting results were reported in the literature. The in vivo role of MTFP1 in the heart is currently unknown. RNAseq and a panoply of approaches assessing mitochondrial structure and function, before symptomatic DCM occurs, provide important insights on early events causing the cardiomyopathy. This study is potentially conceptually innovative and could reveal a new role of MTFP1 in maintaining energy metabolism in the heart as well as in other organs.

    Referee Cross-commenting

    I have read the comments of the other 3 reviewers in details. Like reviewer 3 and 4, I believe that the study is very well performed and provides new knowledge on the role of MTFP1 on cardiac energetics, assuming that the control mice do not develop DCM. I agree with the issues they identified. Regarding the issues raised by reviewer 1, especially concerning the lack of mechanistic insights, I actually thought that the full characterization of the Mtfp1 cko mouse model before and at the onset of the cardiomyopathy showing a strong cardiac phenotype, the RNAseq data showing alteration of metabolic genes and the detailed experiments performed in isolated mitochondria and isolated cells including rescue experiments, provide strong evidence that Mtfp1 regulates energy metabolism. That being said, I agree that direct causality could be better demonstrated by adding siRNA experiments to knockdown Mtfp1 and see if it can recapitulate the adverse effects seen in Mtfp1 ko mice. This was attempted in MEFs and U2OS cells, which did not show the expected results. I would perform this experiment in cardiac cells, which is the relevant cell type to investigate underlying mechanisms. Adding causality experiments would strengthen the study even more.

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    Referee #1

    Evidence, reproducibility and clarity

    The study investigated the role of mitochondrial fission process 1 (MTFP1) on cardiac structure and function. MTFP1 deletion in the heart resulted in adult-onset dilated cardiomyopathy (DCM), reduced membrane potential, and increased non-phosphorylation-dependent respiration. MTFP1 deletion also increased the sensitivity to programmed cell death, which was accompanied by an opening of the mitochondrial permeability transition pore (mPTP) in vitro. Thus, the authors conclude that MTFP1 influences mitochondrial coupling and cell death sensitivity.

    I have the following concerns regarding the study and its main conclusions:

    Major concerns:

    1- While the study challenges previous reports regarding the role of MTFP1 in mitochondrial fission, the study is descriptive and does not provide any mechanistic insights delineating the impact of MTFP1 on cardiac energy metabolism and cell death.

    2- The significance of the RNA sequencing data is not clear, and the authors need to put these changes in context and explain how these changes may fit in the study context. It is also not clear why the authors decided to only comment on the changes in Nppa and Nppb levels?

    3- It is not clear how MTFP1 influences bioenergetic efficiency, and the authors do not prove any evidence to suggest that this might be the case.

    4- In Figure 2F, there is a decrease in the expression of ATP5A complex in the cMKO mitochondria, which could explain the changes in state 4 respiration and membrane potential. The authors need to delineate how MTFP1 could influence the activity of the ATP5 complex.

    5- In Figure S4, the author should report the baseline measurements of LV function and structure pre-doxorubicin treatment to ensure no significant difference in these parameters occurred prior to the treatment protocol.

    6- How does MTFP1 modify PTP activity? More work is needed to characterize this effect.

    7- Co-immunoprecipitation data in figure S5 are confusing and have no clear significance. Therefore, the authors need to discuss the significance of these changes and how they might be relevant in the study context.

    Minor:

    • Line 222, "wholesale" > whole cell

    Significance

    Significance: This study challenges existing dogma, although the data is not convincing enough to make this challenge convincing.

    Referee Cross-commenting

    I have read the comments of the other 3 reviewers, and I agree with their comments. This is an interesting study, that if adequately revised would make an important contribution to the literature.

  6. Version published to 10.1101/2021.10.21.465262v1 on bioRxiv