Review Reports - Epigenetic Regulation and Molecular Mechanisms in Cardiovascular Diseases: A Review of Recent Advances and Therapeutic Implications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the review titled “Epigenetic Regulation and Molecular Mechanisms in Cardiovascular Diseases: A Review of Recent Advances and Therapeutic Implications,” Młynarska et al. explain how different types of epigenetic mechanisms have been explored as potential treatments for cardiovascular diseases. The importance of non-genetic factors in cardiovascular pathophysiology is well described and i I especially found Section 4, titled “Molecular Mechanisms Linking Epigenetics and CVD,” to be very really clear and useful.

The authors did a good job explaining the complexity of epigenetic landscapes and describing the different types of epigenetic modifications.

-monor comments:

The authors should add, in the introduction, a small section describing the different pathophysiological mechanisms that lead to CVD. This would clarify the relevance of the various epigenetic targets—such as inflammation and fibrosis—which are listed by the authors as targets for potential epigenetic therapies.

In Section 5.1, “Epigenetic Drug Candidates,” the authors should expand the discussion related to HDAC and BET inhibition. Recent promising studies in TAC mice and HFpEF mouse models clearly demonstrate the potential therapeutic effects of JQ1 and other BRD inhibitors, and including these findings would strengthen this section.

Author Response

We sincerely thank the Reviewer for the positive and encouraging comments.

1. The authors should add, in the introduction, a small section describing the different pathophysiological mechanisms that lead to CVD. This would clarify the relevance of the various epigenetic targets—such as inflammation and fibrosis—which are listed by the authors as targets for potential epigenetic therapies.

We thank the Reviewer for this valuable suggestion. In response, we have added a concise section to the Introduction outlining the major pathophysiological mechanisms underlying cardiovascular diseases, including endothelial dysfunction, chronic inflammation, oxidative stress, vascular remodeling, and myocardial fibrosis. This addition provides the necessary biological context and clarifies the relevance of epigenetic targets, such as inflammatory and fibrotic pathways, discussed later in the manuscript as potential therapeutic intervention points.

2. In Section 5.1, “Epigenetic Drug Candidates,” the authors should expand the discussion related to HDAC and BET inhibition. Recent promising studies in TAC mice and HFpEF mouse models clearly demonstrate the potential therapeutic effects of JQ1 and other BRD inhibitors, and including these findings would strengthen this section.

We thank the Reviewer for this insightful and constructive suggestion. Section 6.1 - “Epigenetic Drug Candidates” (paragraphs 8 and 9) has been expanded to incorporate recent preclinical evidence on BET inhibition in both TAC and HFpEF mouse models. Specifically, we now discuss mechanistic and functional studies on JQ1 and the selective BET inhibitor apabetalone, highlighting their cardioprotective effects in pressure overload-induced remodeling as well as HFpEF-associated inflammation and diastolic dysfunction.

Reviewer 2 Report

Comments and Suggestions for Authors

Lack of systematicity (Major methodological limitation)
The article completely lacks a "Materials and Methods" section or a description of the literature search strategy. It is necessary to indicate the databases (PubMed, Scopus, Web of Science) searched, the keywords used (MeSH terms), and the inclusion/exclusion criteria (e.g., "only articles from the last 5-10 years"). If this is missing, it cannot be ruled out that the authors selected only articles that support their thesis, omitting contradictory data.
The authors discuss direct epigenetic drugs (DNMT and HDAC inhibitors), which are still far from clinical use in cardiology due to toxicity. However, they completely overlook the fact that standard drugs (statins, metformin, SGLT2 inhibitors - dapagliflozin/empagliflozin) have proven epigenetic effects. For example, SGLT2 inhibitors affect sirtuin-1 (SIRT1) and AMPK-dependent pathways by altering histone acetylation. This is important for clinicians, as it is the most clinically applicable aspect of epigenetics right now.
Section 3.1 (lines 275–278) only mentions m6A methylation. In 2024–2025, the focus shifted to other RNA modifications: m5C (5-methylcytosine), m1A, ac4C (cytidine acetylation), and tRNA modifications. Their role in myocardial hypertrophy and regeneration is currently being actively studied. This information should be added.
The authors mention scRNA-seq (line 339), but barely touch on scATAC-seq (single-cell chromatin accessibility). What's missing: Studies demonstrating cell-specific enhancers in the endothelium of different vascular beds (aorta vs. coronary arteries), which explains why atherosclerosis affects specific sites. This is important for understanding the site-specificity of diseases.
The topic of clonal hematopoiesis (CHIP) is briefly mentioned in the introduction (lines 75–76). The role of specific driver mutations (DNMT3A, TET2, ASXL1) in activating the NLRP3 inflammasome in macrophages is not addressed. This direct link between somatic mutations and plaque inflammation requires a separate subsection. The authors describe HDAC inhibitors (lines 673–688) as promising cardioprotectors, based primarily on mouse models. In oncology, the use of pan-HDAC inhibitors (vorinostat, romidepsin) is associated with serious cardiotoxicity (QT prolongation, ventricular arrhythmias, thrombocytopenia). The applicability of these drugs to cardiology, where patients are treated lifelong (rather than in courses, as in chemotherapy), is highly questionable. The authors mention "toxicity" only in general terms, without focusing on the specific cardiac risks identified in oncotrials.
Mechanism of action of RNA drugs (section 5.2). Controversial point: Discussion of Inclisiran (lines 747–765) as an "epigenetic" therapy. Inclisiran is an siRNA that induces degradation of PCSK9 mRNA in the cytoplasm (a process called RNA interference). Strictly speaking, this is post-transcriptional gene silencing, not classical epigenetics (chromatin modification or DNA methylation in the nucleus), although it is included in the RNA-based therapeutics section. It is important to clearly distinguish between gene silencing and epigenetic modulation.
Metabolic-Epigenetic Crosstalk. The relationship is described superficially (section 4.1 and the mention of butyrate in 469). A description of how Krebs cycle intermediates (alpha-ketoglutarate, succinate, fumarate) act as cofactors or inhibitors for TET demethylases and JmjC demethylases of histones is missing. This is a fundamental mechanism linking mitochondrial dysfunction in heart failure with epigenetic gene silencing.
It is recommended to add a "Search Strategy / Methodology" section: Clearly describe how articles were selected to eliminate bias. Expand the Discussion section: Discuss the delivery challenge. RNA drugs (like inclisiran) work well in the liver (GalNAc conjugates), but delivery to the heart (cardiomyocytes) remains an unsolved problem (LNPs accumulate in the liver). This is the main limitation of cardiac gene therapy. Critically evaluate the safety of HDACi (risk of arrhythmias). Include the "Indirect Epigenetic Modulators" table: Add data on how metformin and SGLT2i affect the epigenome. This will increase the article's citation rate among clinicians. Expand the CHIP section: Describe the mechanism: TET2 loss-of-function → promoter hypermethylation → increased IL-1beta/IL-6 expression → atherosclerosis. Visualization: Figure 1 (mentioned in the text but not shown in detail) should show not just "arrows," but the integration of metabolic signals and epigenetic enzymes (e.g., Acetyl-CoA as a donor for HATs). Summary: The article provides a good overview of the topic, but for expert-level use, it lacks biochemical detail, a critical analysis of clinical trial failures, and coverage of the "indirect" effects of currently used drugs.

Author Response

Lack of systematicity (Major methodological limitation)
1. The article completely lacks a "Materials and Methods" section or a description of the literature search strategy. It is necessary to indicate the databases (PubMed, Scopus, Web of Science) searched, the keywords used (MeSH terms), and the inclusion/exclusion criteria (e.g., "only articles from the last 5-10 years"). If this is missing, it cannot be ruled out that the authors selected only articles that support their thesis, omitting contradictory data.

We thank the Reviewer for this comment. A Materials and Methods section has now been added, specifying the databases searched (PubMed, Scopus, Web of Science), keywords, time frame, and inclusion/exclusion criteria. We clarify that this is a narrative review, and that relevant evidence, including consistent and conflicting findings where available, was considered.

The authors discuss direct epigenetic drugs (DNMT and HDAC inhibitors), which are still far from clinical use in cardiology due to toxicity. However, they completely overlook the fact that standard drugs (statins, metformin, SGLT2 inhibitors - dapagliflozin/empagliflozin) have proven epigenetic effects. For example, SGLT2 inhibitors affect sirtuin-1 (SIRT1) and AMPK-dependent pathways by altering histone acetylation. This is important for clinicians, as it is the most clinically applicable aspect of epigenetics right now.

We thank the Reviewer for this important and clinically relevant comment. Section 6.1 has been expanded to acknowledge that, in addition to direct epigenetic inhibitors, several widely used cardiovascular drugs exert indirect but functionally relevant epigenetic effects. We now discuss the epigenetic actions of SGLT2 inhibitors, statins, and metformin, focusing on modulation of SIRT1- and AMPK-dependent pathways as clinically relevant epigenetic mechanisms associated with routinely used cardiovascular therapies. These additions are presented in the final paragraph of Section 6.1.

3. Section 3.1 (lines 275–278) only mentions m6A methylation. In 2024–2025, the focus shifted to other RNA modifications: m5C (5-methylcytosine), m1A, ac4C (cytidine acetylation), and tRNA modifications. Their role in myocardial hypertrophy and regeneration is currently being actively studied. This information should be added.

Thank you for this valuable suggestion. We have updated Section 3.1 with information on other RNA modifications.

The authors mention scRNA-seq (line 339), but barely touch on scATAC-seq (single-cell chromatin accessibility). What's missing: Studies demonstrating cell-specific enhancers in the endothelium of different vascular beds (aorta vs. coronary arteries), which explains why atherosclerosis affects specific sites. This is important for understanding the site-specificity of diseases.

We appreciate this constructive comment. We have revised the manuscript to incorporate scATAC-seq data.

The topic of clonal hematopoiesis (CHIP) is briefly mentioned in the introduction (lines 75–76). The role of specific driver mutations (DNMT3A, TET2, ASXL1) in activating the NLRP3 inflammasome in macrophages is not addressed. This direct link between somatic mutations and plaque inflammation requires a separate subsection.

We thank the Reviewer for this valuable comment. To adress this point, we have added subsection 5.2.7. describing the mechanistic link between CHIP-associated mutations (DNMT3A, TET2, ASXL1) and cardiovascular inflammation. The revised text explains how TET2 loss-of-function impairs DNA demethylation, leading to inflammatory gene dysregulation in macrophages, activation of the NLRP3 inflammasome, increased IL-1β secretion, and accelerated atherosclerosis, supported by experimental and pharmacological evidence. This addition establishes a direct connection between somatic hematopoietic mutations and plaque inflammation.

The authors describe HDAC inhibitors (lines 673–688) as promising cardioprotectors, based primarily on mouse models. In oncology, the use of pan-HDAC inhibitors (vorinostat, romidepsin) is associated with serious cardiotoxicity (QT prolongation, ventricular arrhythmias, thrombocytopenia). The applicability of these drugs to cardiology, where patients are treated lifelong (rather than in courses, as in chemotherapy), is highly questionable. The authors mention "toxicity" only in general terms, without focusing on the specific cardiac risks identified in oncotrials.

We thank the Reviewer for this valuable comment. To address this point, we have revised the manuscript to more clearly delineate the current stage of evidence regarding HDAC inhibition in cardiovascular disease (Section 6.1, 6 paragraph). The revised text now explicitly states that the reported cardioprotective effects of HDAC inhibitors are based predominantly on preclinical studies using cultured cardiomyocytes and animal models. In addition, we have expanded the discussion to acknowledge clinical safety considerations reported for HDAC inhibitors in oncological use, including electrocardiographic abnormalities (such as QT interval prolongation), reports of ventricular arrhythmias in some settings, and class-associated hematological adverse events, which may limit their translational applicability to cardiovascular indications, particularly in the context of long-term therapy. These clarifications were introduced to provide a more balanced and cautious interpretation of the available evidence.

Mechanism of action of RNA drugs (section 5.2). Controversial point: Discussion of Inclisiran (lines 747–765) as an "epigenetic" therapy. Inclisiran is an siRNA that induces degradation of PCSK9 mRNA in the cytoplasm (a process called RNA interference). Strictly speaking, this is post-transcriptional gene silencing, not classical epigenetics (chromatin modification or DNA methylation in the nucleus), although it is included in the RNA-based therapeutics section. It is important to clearly distinguish between gene silencing and epigenetic modulation.

We thank the Reviewer for this important conceptual clarification. We agree that inclisiran acts through post-transcriptional gene silencing mediated by RNA interference rather than classical epigenetic mechanisms involving chromatin modification or DNA methylation. To address this point, we have added a clarifying sentence in Section 6.2 explicitly distinguishing RNA interference-based therapies from epigenetic regulation at the chromatin level.

Metabolic-Epigenetic Crosstalk. The relationship is described superficially (section 4.1 and the mention of butyrate in 469). A description of how Krebs cycle intermediates (alpha-ketoglutarate, succinate, fumarate) act as cofactors or inhibitors for TET demethylases and JmjC demethylases of histones is missing. This is a fundamental mechanism linking mitochondrial dysfunction in heart failure with epigenetic gene silencing.

We thank the Reviewer for this constructive comment. Requested informations has been added to the article to section 4.1.

It is recommended to add a "Search Strategy / Methodology" section: Clearly describe how articles were selected to eliminate bias.

Expand the Discussion section: Discuss the delivery challenge. RNA drugs (like inclisiran) work well in the liver (GalNAc conjugates), but delivery to the heart (cardiomyocytes) remains an unsolved problem (LNPs accumulate in the liver). This is the main limitation of cardiac gene therapy. Critically evaluate the safety of HDACi (risk of arrhythmias). Include the "Indirect Epigenetic Modulators" table: Add data on how metformin and SGLT2i affect the epigenome. This will increase the article's citation rate among clinicians.

We thank the Reviewer for this constructive comment. The translational limitations of RNA-based therapies have been further discussed in Section 6.2 in last paragraph, where we now explicitly address challenges related to tissue-specific delivery, highlighting that current siRNA platforms (including GalNAc conjugates and lipid-based carriers) achieve efficient hepatic targeting, whereas delivery to cardiomyocytes remains an unresolved limitation for cardiac gene therapy. The safety of HDAC inhibitors has been critically re-evaluated in Section 6.1, with an expanded discussion of cardiotoxicity reported in oncology, including electrocardiographic abnormalities and arrhythmic risk, which may constrain long-term cardiovascular applications. Finally, in response to the Reviewer’s suggestion, we have added a dedicated summary table of indirect epigenetic modulators (Table 1), detailing how widely used cardiometabolic drugs, including metformin and SGLT2 inhibitors, influence epigenetic regulation through metabolic and signaling pathways. Together, these additions strengthen the translational perspective of the manuscript and improve its clinical relevance.

Expand the CHIP section: Describe the mechanism: TET2 loss-of-function → promoter hypermethylation → increased IL-1beta/IL-6 expression → atherosclerosis. Visualization: Figure 1 (mentioned in the text but not shown in detail) should show not just "arrows," but the integration of metabolic signals and epigenetic enzymes (e.g., Acetyl-CoA as a donor for HATs).

We thank the Reviewer for this valuable comment. To address this point, we have expanded the CHIP section by adding a new subsection describing the mechanistic link between clonal hematopoiesis- associated mutations and cardiovascular inflammation. Specifically, we explain how loss-of-function mutations in TET2 impair DNA demethylation, leading to promoter hypermethylation of inflammation-related genes, increased activation of the NLRP3 inflammasome, enhanced secretion of pro-inflammatory cytokines such as IL-1β (and IL-6), and accelerated atherosclerosis.

In addition, Figure 1 has been revised to better illustrate these mechanisms. The updated figure now depicts the integration of metabolic signals with epigenetic regulation, including the role of metabolites such as acetyl-CoA as donors for histone acetyltransferases, and their impact on inflammatory gene expression, rather than showing only simplified directional arrows.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors did a great job and responded well and to the point to most of my key comments.