Review Reports - Mitochondrial Targeting by Elamipretide Improves Myocardial Bioenergetics Without Translating into Functional Benefits in HFpEF

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Elamipretide is a mitochondria-targeted agent. Heart failure with preserved ejection fraction (HFpEF) is frequently accompanied by mitochondrial dysfunction and disturbances in cardiolipin metabolism. Based on this premise, the authors explored the therapeutic potential of Elamipretide for HFpEF, which constitutes a meaningful research. Despite yielding negative results, this study still provides valuable guidance for subsequent investigations in this field.

Comments:

As noted by the authors in the Limitations section, this study was primarily based on a single animal model, and the conclusions drawn may therefore lack generalizability. It is recommended that the Discussion section be supplemented with referencesof clinical studies reporting on mitochondrial function and cardiolipin metabolism in HFpEF, particularly reports derived from experiments using clinical specimens. By integrating these literature reports, the authors should analyze the potential underlying mechanisms that explain why Elamipretide can ameliorate mitochondrial function，but did not confer corresponding improvements in cardiac function.
Elamipretide was originally developed for mitochondrial disorders such as Barth syndrome. Please mention, in the Introduction section, whether similar reports of adverse reaction exacerbation or suboptimal therapeutic efficacy have been documented in its preclinical or clinical investigations for other diseases; a comprehensive discussion and analysis of this issue should also be included in the Discussion section.
The experiment did not include a positive control drug group, which resulted in the lack of a reliable assessment of the model's validity. Please provide statements and discussions on this issue.

Author Response

We thank the reviewers for their careful evaluation of our manuscript and for the constructive and insightful comments. To the best of our knowledge, we have addressed all points raised below and revised the manuscript accordingly, which we believe has improved its clarity and quality.

Reviewer 1

Comments and Suggestions for Authors

Comments:

As noted by the authors in the Limitations section, this study was primarily based on a single animal model, and the conclusions drawn may therefore lack generalizability. It is recommended that the Discussion section be supplemented with references of clinical studies reporting on mitochondrial function and cardiolipin metabolism in HFpEF, particularly reports derived from experiments using clinical specimens. By integrating these literature reports, the authors should analyze the potential underlying mechanisms that explain why Elamipretide can ameliorate mitochondrial function，but did not confer corresponding improvements in cardiac function.

We thank the reviewer for this important question.

Only limited direct evidence based on human myocardial samples is currently available regarding mitochondrial function and cardiolipin (CL) metabolism in HFpEF. The few studies addressing cardiolipin metabolism in human samples primarily focus on HFrEF, as comparable data from HFpEF myocardial tissue are currently lacking. In these studies, cardiolipin levels, particularly CL 72:8, were found to be significantly reduced in myocardial tissue from patients with HFrEF (Sparagna et al., 2007; Chatfield et al., 2019; Smeir et al., 2021) and were associated with impaired mitochondrial function (Chatfield et al., 2019). In response to Ela treatment, mitochondrial oxygen flux and complex activity were significantly improved, likely through improved coupling of the mitochondrial supercomplex. Notably, Ela improved respiration in intact mitochondria isolated from failing human hearts, suggesting that its effect may be restricted to relatively preserved mitochondria and limited by the severity of mitochondrial injury (Chatfield et al., 2019). Neither cardiolipin composition nor cardiolipin quantity were altered by Ela treatment, which is consistent with our results. However, exposure to Ela was limited to a few hours, which does not allow conclusions regarding chronic treatment effects.

An additional text has now been added to the discussion section (line 281-291 and 303-318).

Elamipretide was originally developed for mitochondrial disorders such as Barth syndrome. Please mention, in the Introduction section, whether similar reports of adverse reaction exacerbation or suboptimal therapeutic efficacy have been documented in its preclinical or clinical investigations for other diseases; a comprehensive discussion and analysis of this issue should also be included in the Discussion section.

We appreciate the reviewer’s insightful comment. We have now added a corresponding passage on the original application of Ela in Barth syndrome to the Introduction section (lines 54–56). We also extended the Discussion section, by addressing adverse events in clinical Ela studies of HFrEF and Barth syndrome under Section 3.5 “Clinical Implications” (line 353-362)

The experiment did not include a positive control drug group, which resulted in the lack of a reliable assessment of the model's validity. Please provide statements and discussions on this issue.

We acknowledge that no pharmacological positive control was included in the study. However, no established positive control treatment is currently available for HFpEF. Animals therefore received either Ela or vehicle (NaCl), and model validity was assessed based on established HFpEF characteristics of the ZSF1 model (baseline data and previous studies by our and other groups).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates the effects of the mitochondria-targeted peptide elamipretide (Ela) in a well-established preclinical HFpEF model (female obese ZSF1 rats). The authors provide a comprehensive and methodologically rigorous evaluation of cardiac mitochondrial function, myocardial structure, hemodynamics, and vascular properties. The study is carefully executed, the dataset is extensive, and the negative/neutral findings are reported transparently.

Importantly, the work addresses a clinically relevant and timely question, namely whether improving mitochondrial bioenergetics can translate into functional benefit in established HFpEF. The conclusion that enhanced mitochondrial respiration does not improve diastolic function or reverse maladaptive remodeling is scientifically valuable and contributes meaningfully to the field.

Overall, the manuscript is of high technical quality, but several conceptual, interpretative, and presentation issues should be addressed before publication.

1. Please expand the discussion on: Whether the observed respiratory improvement reflects increased oxygen consumption without improved ATP coupling (i.e., efficiency). The possible role of increased oxidative stress, especially given the upregulation of NOX2 and macrophage infiltration. The absence of data on ATP production, P/O ratio, or ROS emission, which would be critical to interpret the functional relevance of the respiratory findings.

2. Please consider: Explicitly discussing whether Ela might exacerbate pressure load, inflammation, or maladaptive remodeling in late-stage HFpEF. Clarifying whether these effects could be secondary to increased myocardial energy demand or altered mitochondrial–ROS signaling.

3. The manuscript does not sufficiently address why Ela failed to affect cardiolipin remodeling, despite its known cardiolipin-binding properties. Please clarify whether Ela is expected to: Stabilize existing cardiolipin only, or Influence cardiolipin synthesis/remodeling pathways (e.g., tafazzin activity).

4. Only female ZSF1 rats were studied. Given the strong sex-specific phenotype of HFpEF. Please expand the discussion on how these findings might be sex-dependent. Clarify whether mitochondrial dysfunction, cardiolipin depletion, or Ela responsiveness differ between sexes based on available literature. This is particularly relevant for clinical translation.

Author Response

Reviewer 2

Comments and Suggestions for Authors

Overall, the manuscript is of high technical quality, but several conceptual, interpretative, and presentation issues should be addressed before publication.

Please expand the discussion on: Whether the observed respiratory improvement reflects increased oxygen consumption without improved ATP coupling (i.e., efficiency). The possible role of increased oxidative stress, especially given the upregulation of NOX2 and macrophage infiltration. The absence of data on ATP production, P/O ratio, or ROS emission, which would be critical to interpret the functional relevance of the respiratory findings.

We thank the reviewer for this important comment. Measurements of mitochondrial ATP production, coupling efficiency (P/O ratio), and ROS emission were not within the methodological and organizational scope of the present study and were therefore not assessed. Future studies incorporating direct bioenergetic and redox measurements will be important to further refine the interpretation of mitochondrial functional changes, particularly in the context of inflammatory and oxidative signaling. We have now explicitly acknowledged this limitation in the revised manuscript (line 378-382)

In our study, NOX2 expression, CD68 expression, and macrophage infiltration were comparable between the two obese groups and, with respect to CD68 expression, were increased compared with untreated lean animals, suggesting a similar level of oxidative and inflammatory stress in both obese groups. Thus, although Ela did not improve these parameters, it also did not further exacerbate them. We now clarified this aspect under 2.4. (line 191-195). According to this, we already stated the following in 3.3.: “Persistent structural and inflammatory remodeling likely limits the translation of mitochondrial improvement into functional benefit. Fibrosis and titin hypophosphorylation are established contributors to diastolic stiffness in HFpEF [17,25,26]. Similarly, macrophage infiltration and ROS overproduction exacerbate mitochondrial dysfunction and ECM remodeling, forming a reinforcing pathogenic loop [11,27,28].” (line 329-333)

Please consider: Explicitly discussing whether Ela might exacerbate pressure load, inflammation, or maladaptive remodeling in late-stage HFpEF. Clarifying whether these effects could be secondary to increased myocardial energy demand or altered mitochondrial–ROS signaling.

We appreciate this comment from the reviewer. In our study, apart from LVEDP, which was increased under treatment, filling pressure, inflammation, and maladaptive remodeling were comparable between untreated and Ela-treated obese rats. These findings suggest that Ela does not improve these parameters, but also does not exacerbate them. As discussed in the manuscript, the lack of beneficial effects may be related to the already advanced stage of HFpEF in our model and/or the treatment duration.

The manuscript does not sufficiently address why Ela failed to affect cardiolipin remodeling, despite its known cardiolipin-binding properties. Please clarify whether Ela is expected to: Stabilize existing cardiolipin only, or Influence cardiolipin synthesis/remodeling pathways (e.g., tafazzin activity).

We thank the reviewer for this important comment. Ela is generally considered to primarily stabilize existing cardiolipin, but studies investigating its effects on cardiolipin remodeling have reported variable outcomes depending on disease model and treatment duration. Allen et al. demonstrated improved mitochondrial respiration and mitigated cristae fragmentation by Ela in ventricular fibers of rats following ischemia–reperfusion, even though cardiolipin content remained reduced in the treatment group (Allen 2020). In contrast, three months of Ela treatment reversed abnormalities in mitochondrial dynamics in left ventricular tissue of dogs and humans with HFrEF, including restoration of cardiolipin content (Sabbah 2018). Little is known about cardiolipin remodeling under Ela in the setting of HFpEF. In Barth syndrome, which is associated with an HFpEF-like cardiomyopathy (reduced left ventricular volumes, preserved ejection fraction, and impaired active and passive relaxation and filling), long-term treatment with Ela resulted in functional improvements, correlated with an increased ratio of mature to immature cardiolipin species (Thompson 2024). By contrast, in shorter treatment durations (12 weeks), this ratio remained at baseline levels and failed to meet primary and secondary endpoints, including myocardial functional parameters (Thompson 2021). In our chronic HFpEF model, cardiolipin content and composition remained unchanged, suggesting that under conditions of established mitochondrial and structural remodeling, Ela’s stabilizing effects may be insufficient to induce measurable remodeling. We have now extended the Discussion section to explicitly address these points, including the differential effects of elamipretide on cardiolipin remodeling across models, treatment durations, and disease settings (Section 3.2, lines 303–318).

Only female ZSF1 rats were studied. Given the strong sex-specific phenotype of HFpEF. Please expand the discussion on how these findings might be sex-dependent. Clarify whether mitochondrial dysfunction, cardiolipin depletion, or Ela responsiveness differ between sexes based on available literature. This is particularly relevant for clinical translation.

As already noted in the Limitations section, only female ZSF1 rats were included in this study. To the best of our knowledge, there is currently no literature directly comparing mitochondrial dysfunction, cardiolipin depletion, or responsiveness to Ela between male and female HFpEF models. Any statement on potential sex-dependent effects would therefore be speculative. Based on this, we have now expanded the Limitations section to explicitly acknowledge that our findings may not be directly extrapolated to male animals, and that future studies including both sexes are warranted to assess potential sex-dependent effects and enhance translational relevance (Section 3.6, lines 372–375).

Author Response File: Author Response.pdf