Cardiolipin deficiency disrupts electron transport chain to drive steatohepatitis

Reviewer #1 (Public review):

Summary:

The manuscript by Brothwell and colleagues describes a central role for hepatic cardiolipin deficiency in MASH. The authors identify cardiolipin as a mediator of two long-standing problems in the field: how dysregulated lipid metabolism relates to altered mitochondrial metabolism during MASLD, and what the innate changes are in the steatotic liver that cause the increased respiration. The authors identified reduced liver cardiolipin in humans with MASH and in a variety of mouse models with MASH. When they knocked out hepatic cardiolipin synthesis, mice developed steatosis and inflammation. These mice also recapitulated the elevated hepatic oxidative metabolism and oxidative stress found in obese humans with MASLD. Some of the in vivo functional data related to glucose homeostasis and substrate metabolism could be stronger, and interpretation of the in vitro flux data needs some clarification, but in both cases, the data are not essential to the main conclusions of the manuscript. Overall, the study offers compelling evidence that cardiolipin is reduced in MASLD and that impaired cardiolipin synthesis is sufficient to recapitulate many features of MASLD.

Strengths:

The main strengths of the study are:

(1) The identification of reduced cardiolipin levels in the liver of humans with MASLD and in a variety of mouse models of MASLD.

(2) The finding that loss of cardiolipin synthesis recapitulates steatosis and inflammation in MASH.

(3) The finding that loss of cardiolipin increases mitochondrial respiration, ROS production, and fat oxidation (in a separate hepatocyte cell line), again recapitulates several previous studies in obese humans with MASLD.

(4) Evidence, though less definitive, that cardiolipin deficiency promotes electron leak by disrupting respiratory supercomplexes and preventing CoQ reduction.

Weaknesses:

(1) Figure 3A-D tries to make the point that liver CLS KO causes defects in substrate handling in vivo, based on glucose and pyruvate tolerance tests. The KO mice have a blunted response to a glucose tolerance test, but the pyruvate tolerance test showed very little (almost no) effect on glucose levels in either WT or LKO mice. The small blunting of the response in the LKO is impossible to interpret (if it's real), since the ability to clear glucose is also increased, and no tracers were used. It might be useful to monitor pyruvate and lactate levels during the experiment. However, this reviewer doesn't think the data is essential to prove the authors' main points.

(2) After presenting convincing evidence that respiration is elevated in isolated mitochondria from CLS KO liver, the authors follow up the findings by investigating whether 13C-palmitate and 13C-glucose oxidation are altered by CLS knockdown in murine Hepa1-6 cells (Figure 4). A few comments are worth mentioning about Figure 4:

a. It is not clear why the authors chose to use a hepatoma cell line rather than primary hepatocytes from LKO mice. The latter would be more convincing, since there could be important differences in metabolism between hepatoma cells and hepatocytes (e.g., preference for fatty acids vs glucose). Nevertheless, I think the approach is sufficient to test the general effect of loss of CLS on substrate metabolism.

b. The authors use the M+2 enrichments of TCA cycle intermediates to infer rates of oxidation of [U-13C]palmitate or [U-13C]glucose. It is important to note that this kind of data reports fractional carbon sources (i.e., substrate preference) rather than rates of oxidation. For example, data from the 13C-palmitate experiment indicates that the CLS KD cells increase the fractional contribution from 13C palmitate (compared to glucose, for example) to the TCA cycle, but the actual rate of palmitate oxidation is not implicit in the data. However, it is reasonable to suggest that, in combination with the increased rates of O2 consumption observed in isolated mitochondria, this data supports increased fat oxidation.

c. I have some concern that the [U-13C]glucose experiment is more complicated to interpret than the description implies. I'm not sure what happens in this cell line, but in the liver, most labeling from pyruvate (i.e., originating from glucose in this case) enters the TCA cycle via pyruvate carboxylase, with smaller amounts entering via PDH (depending on the nutritional state). Since one could expect pyruvate carboxylase to contribute M+3 labeled TCA cycle intermediates initially, and M+2 on the first turn of the cycle, it's hard to conclude what the data indicates about glucose oxidation. The authors could generalize the conclusion by framing the TCA cycle enrichment data as the contribution of glucose carbons and noting in Figure 4A that pyruvate carbons can enter the TCA cycle via PDH or pyruvate carboxylase, without attempting to assign their relative contributions. There are better ways to do it, but it's a small nuance here since the authors aren't making a critical point about the pathways.

Reviewer #2 (Public review):

In this study, the authors show that alterations in the lipid composition of the inner mitochondrial membrane, particularly changes in cardiolipin (CL) content, lead to defects in electron transport, supercomplex formation, and oxidative stress. Using liver-specific CLS knockout mice, which are characterized by dysfunctional capacity for cardiolipin synthesis, the authors highlight an underappreciated role for CL in MASH pathology. Overall, this is an interesting study highlighting the importance of functional/physiological electron transport (and in this context, electron leakage) in MASH pathophysiology. Despite that, this manuscript has several weaknesses that require attention.

(1) For all LKO studies, it is stated that the decrease in hepatic CL is causal for the observed phenotype. However, it is evident that many other lipids are impacted by CLS KO, including a marked increase in hepatic PG. In this respect, the authors show no evidence that the observed metabolic phenotype is indeed due to the reduction in CL and not to other accompanying changes.

(2) In the results, the authors highlight that 'MASLD has been shown to alter the total cellular lipidome in liver.' Given that this study focused on CL, it would be useful to include specific studies that pointed to changes in hepatic CL content in MASLD/MASH/fibrosis.

(3) The initial human mitochondrial lipidomics studies show a reduction in mitochondrial CL and PG content. What was the content/expression of CL synthase and PGP synthase in these samples? If this cannot be assessed, is there any association of CLS or PGPS expression and MASLD/fibrosis (etc) in publicly available databases (e.g, GEP liver) that may explain the reduction in mitochondrial PG and CL content?

(4) The validation of MASH in patients (Figure 1B) is not convincing (ie., no quantification/scoring provided). NAS /fibrosis scoring (according to Kleiner) would help to define if all patients have indeed MASH, and what subset has fibrosis. Could the reduction in CL/PG content be (also) associated with fibrosis? In addition, Masson's Trichrome should be added to Figure 1B.

(5) In human lipidomics, the authors suggest that reductions are observed in tetralinoleoyl CL (Figure 1C). However, Figure 1C only shows the combined FA acyl chain length + unsaturation, therefore not allowing for FA-specific ID (unless such data are available from the LC/MS analysis).

(6) Figures 1 J/K/I. It is obvious that the background in all murine immunoblotting analysis has been altered. The authors should provide unaltered images for these immunoblots.

(7) For Figure 1, it is unclear what is meant by 'we performed all mitochondrial lipidomic analyses by quantifying lipids per mg of mitochondrial proteins'. Was the murine lipidomics carried out on fractionated mitochondria or whole liver? If whole liver, then how were the data corrected, particularly given that PG is not a mitochondria-specific lipid?

(8) While total CL content seems indeed decreased across the different mouse models, this is mostly due to 1-2 CL species showing a pronounced reduction, with the remainder being unaltered. This should at least be acknowledged in the results. This is similarly the case in the LKO livers.

(9) Figure 2. A secondary biochemical analysis of changes in lipid content should be provided, e.g., total triglyceride content, particularly given that the histology analysis does not show any major changes in hepatic lipid droplets/steatosis. In addition, the Masson's Trichrome staining shows almost no collagen deposition.

(10) Figure 3. 'CLS deletion modestly reduced glucose handling' should be reworded. The LKO mice show improved glucose tolerance (despite the MASH phenotype), which is not evident from the above wording.

(11) Looking at the mechanism behind the increase in hepatic steatosis, the authors state that lipid accumulation can occur due to increased lipogenesis, or dysfunctional VLDL secretion or beta oxidation, and subsequently assessed the relevant proteins/pathways. What about fatty acid uptake, which is also one of the four major pathways impacted in MASLD? This should be included in this assessment in Figure 3.

(12) For Figure 5A, it is simply stated 'CLS deletion promotes liver fibrosis in standard chow-fed condition', and it is unclear what is highlighted within the selected EM images and what the arrows refer to. The authors should clarify this within the text.

Reviewer #3 (Public review):

Summary:

Mitochondrial oxphos causes lipid accumulation, leading to MASH, although the mechanism has been poorly understood. In this study, Funai and colleagues identify that reductions in cardiolipin in the mitochondria cause disruptions in the electron transport chain. Knockout of cardiolipin synthase was sufficient to drive MASH phenotypes, increase respiratory capacity, and cause electron leak at complexes II and III. It is well established that loss of cardiolipin increases ROS. Studies to date have been performed on whole tissue lysates, but to rule out which changes in mitochondrial lipids are driven by changes in mitochondrial number versus lipid synthesis/turnover, the authors uniquely purified mitochondria from human and mouse livers in MASH and NASH models for this study. This study provides critical information to the field that will inevitably help us better understand the mechanisms underlying MASH and NASH onset. The evidence provided is both convincing and compelling. With further suggested revision experiments, this study has the potential to change our understanding of MASH and NASH pathogenesis.

Strengths:

The authors use a unique approach of lipidomics on purified mitochondria. They also analyze many distinct MASH models and provide a unique resource for the field of comprehensive lipidomics analysis of the different ways in which MASH can be induced. The use of human tissue elevates the impact/significance of the findings.

Weaknesses:

The data on the super complexes was the least compelling, and frankly, I do not think the authors needed those data to make a compelling argument! The authors should shift their focus more to the compelling electron leak data they have collected. If possible, it would also strengthen the work to include cardiolipin rescues on more of the experiments. Finally, expanding their explanations of the model systems would be very helpful for the readership.

Reviewer #4 (Public review):

Summary:

Here, the authors wish to shed light on factors that contribute to the development of liver disease in what used to be called 'the metabolic syndrome'. This is a human-health problem of considerable significance, and the insights they provide, namely the implication of a defect in mitochondrial cardiolipin (CL) content to the progression from metabolic dysfunction-associated steatotic liver disease to steatohepatitis, are plausible.

Strengths:

The experimental evidence proffered is derived from the observation of lower levels of (CL) in mitochondria from the liver of patients undergoing liver transplant or resection due to end-stage steatohepatitis compared with mitochondria derived from livers of patients with other conditions. This correlation is buttressed by observations made in mice with liver-selective compromise in CL synthesis and which suggest a pathological environment associated with mitochondrial dysfunction and enhanced oxidative stress, features deemed to play a role in the progression from steatotic liver disease to steatohepatitis.

The paper is well written, and the findings are well explained and superficially convincing.

Weaknesses:

It is unclear how much can be learned from compromising a key enzyme that produces a key mitochondrial lipid in a busy metabolic organ like the liver - isn't the discovery of a mitochondrial defect in such a context rather trivial? And how reliably can these findings be related to the human observations? Most importantly, the chain of causality implied by the title is unproven: the key question of whether or not (somehow) preventing the drop in cardiolipin content affects the course of steatohepatitis remains unanswered.