Multi-Omics Profiling of Hypertrophic Cardiomyopathy Reveals Altered Mechanisms in Mitochondrial Dynamics and Excitation–Contraction Coupling
, Alexandre C. Pereira 3, Kallyandra Padilha 4, Matthew Lawton 1, Weiwei Lin 1, Raghuveera Goel 1, Ivan Luptak 5, Valentina Perissi 1, Christine E. Seidman 3,6,7, Jonathan Seidman 3, Michael T. Chin 8, Christopher Chen 2,9 and Andrew Emili 1,*,†
Round 1
Reviewer 1 Report
The paper describes the multi-omics analysis of hypertrophic cardiomyopathy.
The experimental methods are consisted of global proteomics with TMT, phosphoproteomics, and 13C-labeled tracing metabolomics. And so the molecular alteration analysis are no problems and very high quality method.
Samples are consisted of both cells and patient specimens.
The results whose pathways in mytochondrial and calcium altered are also very impressive.
I have no ideas for improvements in revision.
Author Response
Reviewer 1 Suggestions: The paper describes the multi-omics analysis of hypertrophic cardiomyopathy.
The experimental methods are consisted of global proteomics with TMT, phosphoproteomics, and 13C-labeled tracing metabolomics. And so the molecular alteration analysis are no problems and very high quality method.
Samples are consisted of both cells and patient specimens.
The results whose pathways in mitochondrial and calcium altered are also very impressive.
I have no ideas for improvements in revision.
Response: We thank the reviewer for their time, assistance, and thoughtful feedback in this process.
Reviewer 2 Report
The research article “Multi-omics profiling of hypertrophic cardiomyopathy reveals altered mechanisms in mitochondrial dynamics and excitation- contraction coupling by Moore et. al., provides a wide landscape of Hypertrophic cardiomyopathy.
Hypertrophic cardiomyopathy disease is associated with sudden cardiac death in young adults and is often seen as one of most the commonly inherited cardiomyopathies. Multi-omics studies not only provide detailed insights about the disease, but also help in the identifying the correlation between alterations observed and the clinical prognosis. Authors have investigated factors that may drive the pathogenesis by performing a quantitative multi-omics (proteomic, phosphoproteomic, metabolomic) analysis. The study profiles differential features linked to distinct molecular mechanisms regulating mitochondrial homeostasis as well as stage-specific metabolic and excitation-coupling maladaptation. This study broadens our understanding of early and stage specific changes associated with development of the disease.
The paper can be accepted in its current format. However, future studies validating these results would be interesting.
Author Response
Response: We thank the reviewer for their time, assistance, and thoughtful feedback in this process.
Reviewer 3 Report
Reviewer comments on IJMS manuscript "Multi-omics profiling of hypertrophic cardiomyopathy reveals altered mechanisms in mitochondrial dynamics and excitation-contraction coupling” by Jarrod Moore et al.
In their manuscript "Multi-omics profiling of hypertrophic cardiomyopathy….” the authors describe an experimental comparison of an early-stage hiPSC-based HCM model (MYH7R402Q+/-) with late-stage samples from HCM patient myectomies on a) proteomic, b) phosphor-proteomic and c) metabolomic levels. As corroborating evidence, Ca2+ imaging data from the early stage model is presented and discussed.
Technical quality. The manuscript is overall of excellent technical quality and detail. Text and figures are very clear and well-structured, the data and the authors’ arguments laid out clearly, and the discussion detailed and to-the-point. Finding are discussed against a rich body of literature. I would like to specifically commend the authors for this!
Similarly, the quality and statistical evaluation of the presented experiments fully corresponds to the state-of-the-art and is done on a very high level, which is reflected in number of significant proteins and phosphorylation sites available for discussion.
Relevance. High quality studies of major cardiac diseases such as HCM on the molecular level are still surprisingly scarce. Consequently, the data presented here provide a rich and comprehensive resource, and making the data available for the scientific community will provide a valuable resource for future experimentation.
From a results point of view, it is clear that the early-stage HCM fails to fully predict or even recapitulate processes in the late-stage myectomy samples, even to the point where regulatory mechanisms such as PLN-mediated SERCA2a-regulation appear to be reversed. While this may be seem as a depreciation of the use of hiPSC-based disease models, it is een more important to make this knowledge available to the scientific public. In this regard, I would encourage the authors to state this point even more clearly in the manuscript.
Novelty. The manuscript is of sufficient novelty. While the methodologies employed here are not novel in themselves, the use of novel algorithms to link proteome to metabolome such as MOMENTA has to my best knowledge not yet been successfully applied to cardiac research.
I am therefore happy to recommend the manuscript for publication with only a few minor requests for change or comment:
11) The atrial, ventricular and septal proteomes are distrinctly different (Doll et al., 2017). hiPSCs can today be differentiated into e.g. atrial or ventricular cardiomoytes (Cyganek et al., 2018). Can the authors comment on what is known about the employed differentiation procedure in this regard? Are the differentiated myocytes sufficiently similar to septal tissue?
22) Figure 2, proteomics and phosphoproteomic data: was normality of the data assessed either by test or at least visually, to justify the use of t-testing?
33) Figure 2 panel B, leftside plots: the sides of the volcano plots are ‘upregulated in HCM’ or ‘Downregulated in HCM’. Since these data are from the stem cell model, this should rather be labeled ‘up in MYH7R402Q+/-‘ or ‘down in MYH7R402Q+/-‘
44) Figure 2 and Results section 2.2 p.6: the authors discuss a number of proteins (and later in the manuscript, phosphorylation sites’, however the discussed proteins are not labeled in Fig 2 and therefore difficult to assess for the reader. This should be changed.
55) Figure 33 – is there a reason to not display an overview of the GSEA analysis from the myectomy samples, in parallel to panel A) for the hiPSC samples? This would be of interest to have in the main figure here, and should be added
66) Table 1 – the MYH7 mutations observed in the patient samples are not the same as the single mutation used for the hiPSC model. How much is known about the different impact of these different mutations? This should be shortly discussed and justified. I am aware that it is not always possible to achieve genetic consistency in these setups, however the authors should comment on potential differences.
77) Results section 2.7 p.12 – the authors discuss PLN phosphorylation on S17 and T17 as more or less the same thing, however the modifications actual play different roles in SERCA2a regulation – there is quite some bod of work on this from the past 5 years. The authors shold differentiate here, especially in light of the observed difference in Ca2+ homeostasis phosphoproteome changes between the early and the late model.
88) Data availability, p.22: the PRIDE accession numbers should by now be available, they are usually given out a few days after submission, with the data staying under password protection until the manuscript is published. The authors should supply the accessions as a condition for having the manuscript accepted.
Once these minor corrections are addressed, the manuscript should be in good shape for publication in IJMS!
Author Response
We thank Reviewer 3 for carefully reviewing our manuscript, and for the detailed analysis of each section. We hope these changes are satisfactory.
11) Our protocol is a based on Lian et al., which generated primarily ventricular cardiomyocytes, with a smaller subpopulation of atrial cells. We predict a similar composition[1].
Given this fact, we would argue that these cells are closely related to the septal ventricular cardiomyocytes harvested from the patients.
22) The normality of the data was assessed by intensity expression boxplots and density vs intensity plots for the proteome and phosphoproteome of all samples. These intensity plots were visualized before and after normalization. All plots are attached as a supplementary figure (Supplementary Figure 1).
33) This is now updated.
44) We thank the reviewer for this point. Many of these points are labeled in Figure 3A (the smaller heat maps), though we added labels from this section to Figure 2.
55) This is a very fair point. We felt that symmetry of the figure was disrupted by another large GSEA plot. We added a supplementary figure of this plot (Supplementary Figure 2).
66) Yes, we studied an array of MYH7 mutations. We added a section to the discussion (line 504-506) addressing this limitation.
77) We made a clearer distinction between the two events in the discussion (line 605) by focusing on the differences in upstream kinases.
88) The pride accession number was assigned and added to manuscript with the proper login information.
References:
- Lian, X.; Hsiao, C.; Wilson, G.; Zhu, K.; Hazeltine, L.B.; Azarin, S.M.; Raval, K.K.; Zhang, J.; Kamp, T.J.; Palecek, S.P. Cozzarelli Prize Winner: Robust Cardiomyocyte Differentiation from Human Pluripotent Stem Cells via Temporal Modulation of Canonical Wnt Signaling. Proceedings of the National Academy of Sciences 2012, 109, E1848–E1857, doi:10.1073/pnas.1200250109.
