Gut Dysbiosis and Arrhythmogenesis: The Potential Role of Microbial Alterations and Small Intestinal Bacterial Overgrowth in Cardiac Arrhythmias

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript is missing so many things from so many aspects try to correct and resubmit. Here is the report after focuses of this article, missing information are-

1. The title of the manuscript is good, but this manuscript mainly focuses on general dysbiosis. Try to change the title of this work like ''General Dysbiosis and Cardiac Electrophysiology''.  or any relevant title of this work and revise.
2. The introduction part it looks good, but it contains more general information regarding microbiota, but it's not directly link to the cardiac arrhythmias, try to focus on that and minimize it.
3. Avoid mechanistic repetitions like TMAO, SCFAs, LPS, Leaky gut and Inflammation.
4. This review majorly needs to focus on table separating includes more human studies observations, add interventional studies along with FMT major animal models and finally any mechanistic and latest in-vitro findings.
5. Coming to the clarity evidence strength- must include sample size along with diet, genetics and age group also.
6. Many sections of this study focus on individually try to link them accordingly.
7. Finally, within this manuscript the discussion should include major and vital mechanistic pathways and must avoid gaps in knowledge and this study will add future potential list of biomarkers and strategies of therapeutic targets.
8. Authors must concentrate on discussion and revise the conclusion. 

Comments on the Quality of English Language

Quality of English language in this review is good.

Author Response

Comment 1: The title of the manuscript is good, but this manuscript mainly focuses on general dysbiosis. Try to change the title of this work like ''General Dysbiosis and Cardiac Electrophysiology''.  or any relevant title of this work and revise.
Response 1: We sincerely thank the reviewer for this helpful comment. We have revised and refined the manuscript and believe that the current title is now appropriate and better reflects the scope of the work.

Comment 2: The introduction part it looks good, but it contains more general information regarding microbiota, but it's not directly link to the cardiac arrhythmias, try to focus on that and minimize it.
Response 2: We are grateful for this valuable suggestion. In response, we have reduced the general background information and revised the introduction.

Comment 3: Avoid mechanistic repetitions like TMAO, SCFAs, LPS, Leaky gut and Inflammation
Response 3: We thank the reviewer for this insightful comment. Redundant mechanistic descriptions have been minimized throughout the manuscript.

Comment 4: This review majorly needs to focus on table separating includes more human studies observations, add interventional studies along with FMT major animal models and finally any mechanistic and latest in-vitro findings.
Response 4: We appreciate this constructive suggestion. We have introduced a table summarizing the observational studies we were able to identify.

Comment 5: Coming to the clarity evidence strength- must include sample size along with diet, genetics and age group also.
Response 5: We thank the reviewer for this important comment. We have added a table including as many study details as were available in the original publications.

Comment 6: Many sections of this study focus on individually try to link them accordingly.
Response 6: We are grateful for this helpful comment. We have revised and reorganized the sections to improve coherence and better link related topics.

Comment 7: Finally, within this manuscript the discussion should include major and vital mechanistic pathways and must avoid gaps in knowledge and this study will add future potential list of biomarkers and strategies of therapeutic targets.
Response 7: We sincerely thank the reviewer for this valuable suggestion. The discussion has been expanded.

Comment 8: Authors must concentrate on discussion and revise the conclusion. 
Response 8: We appreciate this constructive comment. In response, we have revised the conclusions and further strengthened the discussion.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a well-researched and timely manuscript addressing the important relationship between gut microbiota and cardiac arrhythmias. The content is generally accurate and covers key mechanisms, including the roles of metabolites like TMAO and SCFAs, inflammatory pathways, and autonomic modulation. However, in its current form, the manuscript functions as a competent general summary of established concepts. To become a more impactful and novel contribution, it requires a sharper thematic focus, deeper mechanistic integration from a nutritional biochemistry perspective, and clearer positioning against existing reviews in this crowded field.

• The introduction is lengthy and could be streamlined. Consider moving some of the detailed microbiota composition and classification to later sections.
• The terms "dysbiosis," "SIBO," "LIBO," "SIFO," "IMO" are introduced but not consistently defined or differentiated in later sections. Consider a dedicated subsection or table early in the manuscript to clarify these terms.
• "Gut–heart axis" is mentioned but could be more explicitly defined and framed as a conceptual model guiding the review.
• Section 2 ("Potential Mechanisms") is dense and covers multiple overlapping concepts (e.g., inflammation, barrier dysfunction, metabolites). Consider subdividing into clearer themes like Gut Barrier Dysfunction and Translocation/ Microbial Metabolites (TMAO, SCFAs, LPS)/ Immune and Inflammatory Pathways /Autonomic and Neural Modulation
• Section 3 on arrhythmias is well-organized but could benefit from clearer transition paragraphs linking dysbiosis to specific arrhythmia types.
• Consider a summary table or figure early in the manuscript illustrating the key pathways linking dysbiosis to arrhythmias.
• The manuscript touches on drug–microbiota interactions but could expand on this given the relevance to arrhythmia management (e.g., amiodarone metabolism, warfarin, DOACs). Consider a dedicated subsection for a more detailed discussion.

o          Discuss microbiota-arrhythmia links across ages: infants (SIDS), adults (AF), elderly (aging microbiota, frailty + AF). Tie in developmental origins of health and disease (DOHaD) with early microbiota shaping later arrhythmia risk.

o          Critique current microbiota assessment tools (breath tests, metagenomics) in cardiology trials. Propose how machine learning could integrate microbiota data with ECG/imaging for arrhythmia prediction.

• The conclusion could be strengthened by summarizing key mechanistic insights and highlighting gaps in current knowledge (e.g., lack of human intervention trials, need for standardized microbiota assessment).
• Consider adding a paragraph on therapeutic implications and future research directions (e.g., personalized microbiota modulation, biomarker development).
• Avoid repetition (e.g., multiple statements on inflammation and barrier dysfunction).
• Ensure consistent use of italicization for bacterial enera (e.g., Faecalibacterium, Ruminococcus).
• Ensure consistent use of abbreviations (e.g., TMAO, SCFAs, LPS) and define upon first use.

In-Depth Comments

• The manuscript would benefit from a more defined central thesis. Currently, it surveys the broad "gut-heart axis." To stand out, it should explicitly position Small Intestinal Bacterial Overgrowth (SIBO) as a primary clinical archetype of dysbiosis. This condition offers a distinct pathophysiological model characterized by proximal gut inflammation, competitive nutrient consumption, and direct metabolite release into the portal system, differentiating it from colonic dysbiosis. Structuring the narrative around SIBO's unique implications for arrhythmogenesis would provide a cohesive and novel framework.
• The connection between malabsorption and arrhythmia needs elaboration. Please detail how deficiencies in magnesium, potassium, and calcium—common in SIBO—directly impair specific cardiac ion channels. For instance, hypomagnesemia can reduce the outward potassium current through the hERG channel, delaying repolarization and prolonging the QT interval, thereby elevating the risk for torsades de pointes. Citing clinical studies where electrolyte repletion reduces arrhythmia burden would solidify this argument.
• The discussion on fiber should move from general benefits to quantitative and mechanistic specificity. Please cite human intervention studies that link specific daily fiber intakes (e.g., >35g) to measurable increases in cardioprotective SCFAs like butyrate and propionate in systemic circulation. Furthermore, distinguish between fiber types: beta-glucan may primarily elevate propionate, while resistant starch is a potent driver of butyrate production by specific taxa like Roseburia.
• When discussing TMAO, explicitly list its major dietary precursors: egg yolks, red meat, liver, and certain saltwater fish. Crucially, address the clinical paradox: choline is an essential nutrient for liver and brain function, yet its microbial metabolism can be detrimental. The manuscript should discuss how individual genetic variations in enzymes like FMO3 influence TMAO production, hinting at the need for personalized dietary guidance regarding choline-rich foods.
• Expand on how omega-3 fatty acids exert prebiotic-like effects. They are incorporated into bacterial membranes, reducing the endotoxicity of lipopolysaccharides (LPS). They also promote the growth of Lactobacillus and Bifidobacterium, which can suppress pro-inflammatory proteobacteria. This microbial shift contributes to the well-documented reduction in systemic levels of IL-6 and TNF-α, thereby potentially mitigating the inflammatory substrate for atrial fibrillation.
• Introduce a holistic dietary pattern analysis, using the Mediterranean diet as a prime example. Describe how its synergistic components—high in polyphenols, fiber, and unsaturated fats—collectively enhance microbial diversity and the abundance of SCFA-producing species like Faecalibacterium prausnitzii. Reference longitudinal cohort data (e.g., from the PREDIMED study) that links adherence to this pattern with a lower incidence of atrial fibrillation, connecting diet structure to clinical outcome.
• Elaborate on the consequences of SIBO-induced vitamin B12 deficiency. Bacterial consumption of B12 in the small intestine impairs the function of methionine synthase. This leads to the accumulation of homocysteine, a molecule that promotes endothelial dysfunction, activates cardiac fibroblasts, and induces oxidative stress. This pathway provides a direct mechanistic link between a microbial-driven deficiency and the structural remodeling of the atria.
• Incorporate recent findings on dietary sodium's extra-renal effects. High salt intake has been shown to reduce levels of beneficial Lactobacillus species in the gut. This loss alleviates a natural suppression on the differentiation of T-helper 17 (TH17) cells, leading to increased IL-17 production, systemic inflammation, and hypertension—a major driver of atrial fibrillation.
• Address the emerging controversy surrounding artificial sweeteners. Studies indicate that compounds like sucralose and saccharin can induce glucose intolerance by altering gut microbiota composition, favoring pro-inflammatory Enterobacteriaceae. This microbial shift and the resultant metabolic dysfunction could indirectly influence cardiac autonomic tone and arrhythmia risk, an area warranting further investigation.
• Introduce the concept that the bioactivity of many dietary polyphenols (e.g., in berries, tea, nuts) is dependent on microbial metabolism. Gut bacteria transform parent compounds into absorbable metabolites like urolithins (from ellagitannins) with enhanced anti-inflammatory and antioxidant properties. This process directly links dietary intake to the production of systemically active compounds that can improve endothelial function and myocardial resilience.
• Present a balanced view of very-low-carbohydrate diets. While they may reduce beneficial Bifidobacterium due to fiber restriction, they also dramatically decrease the abundance of LPS-producing bacterial families. The net effect on systemic inflammation and arrhythmia risk is therefore complex and context-dependent, potentially offering benefit in specific scenarios (e.g., severe metabolic syndrome) but carrying unknown long-term risks for gut and cardiovascular health.
• Cite preclinical evidence that common dietary emulsifiers like polysorbate-80 and carboxymethylcellulose can degrade the protective intestinal mucus layer, enhance bacterial adhesion to the epithelium, and induce low-grade inflammation and metabolic dysregulation in animal models. This provides a plausible mechanistic link between the consumption of ultra-processed foods and a pro-arrhythmic systemic state.
• Differentiate between classes of prebiotics. For example, inulin and fructooligosaccharides (FOS) are highly selective for stimulating Bifidobacterium, while resistant starch (RS) is a preferred substrate for butyrate-producing genera like Eubacterium rectale. Since different SCFAs have distinct cardiac effects, this specificity is crucial for designing targeted nutritional interventions.
• Elevate the discussion by referencing specific probiotic strains with documented cardiovascular effects in human trials. Examples include Lactobacillus reuteri NCIMB 30242 for cholesterol management and Lactobacillus rhamnosus GG for enhancing gut barrier integrity. This moves the narrative from general concepts to clinically substantiated, strain-specific applications.
• Explain the synergistic logic behind synbiotic formulations: the prebiotic component acts as a targeted fuel source to enhance the survival, colonization, and metabolic output of the co-administered probiotic. Reference clinical trials where synbiotic combinations have demonstrated superior efficacy in reducing inflammatory biomarkers like hs-CRP compared to probiotics or prebiotics alone in patients with cardiometabolic risk factors.
• Discuss how intermittent fasting regimens influence the gut ecosystem, notably by increasing the abundance of Akkermansia muciniphila, a bacterium associated with improved gut barrier function and metabolic health. Link these dietary patterns to improvements in parasympathetic tone (evidenced by increased heart rate variability) and reduced oxidative stress, which may collectively lower susceptibility to autonomically triggered arrhythmias.
• Elaborate on the non-skeletal role of Vitamin D. Activation of the Vitamin D Receptor (VDR) in intestinal epithelial cells upregulates the expression of tight junction proteins (claudin, occludin). Therefore, Vitamin D deficiency—common in malabsorptive states—compromises intestinal barrier integrity, facilitating the translocation of pro-inflammatory molecules like LPS and creating a vicious cycle relevant to arrhythmogenesis.
• Describe the enterosalivary circuit for nitric oxide (NO) generation. Dietary nitrates from leafy greens and beets are absorbed, concentrated in saliva, and reduced to nitrite by oral bacteria. Upon swallowing, nitrite is converted to NO in the acidic stomach and systemically. This microbiota-dependent pathway boosts bioactive NO, improving endothelial function, vasodilation, and potentially stabilizing the myocardial electrical substrate, particularly under ischemic conditions.
• Introduce the concept of enterotypes—stable community structures like Bacteroides-dominant or Prevotella-dominant profiles. Suggest that an individual's enterotype may predict their cardiometabolic response to dietary interventions. For instance, a Prevotella-dominant individual might derive greater arrhythmia risk reduction from a high-fiber intervention than a Bacteroides-dominant individual, pointing toward future personalized nutrition strategies.
• Deepen the analysis of ultra-processed foods beyond low fiber content. Their lack of fermentable substrate may shift microbial metabolism toward the breakdown of endogenous proteins, increasing the production of deleterious metabolites like p-cresol sulfate and indoxyl sulfate. These uremic toxins are independently associated with endothelial dysfunction, oxidative stress, and fibrosis, all key players in arrhythmogenesis.
• Discuss how dysbiosis and intestinal inflammation can specifically impair the absorption of copper and zinc. Copper deficiency can lead to dilated cardiomyopathy and electrical abnormalities, while zinc deficiency impairs antioxidant defense (as a cofactor for superoxide dismutase). Furthermore, excessive zinc supplementation can induce copper deficiency, highlighting the importance of balance and the role of gut health in mineral homeostasis for cardiac function.
• Address the complexity of caffeine. It is metabolized by gut bacterial enzymes, and its byproducts may influence host physiology. Furthermore, as an adenosine receptor antagonist, caffeine's long-term impact on atrial tissue is nuanced; while adenosine has anti-fibrotic properties, acute caffeine intake is not consistently linked to increased AF risk in epidemiological studies, suggesting a U-shaped relationship that may be influenced by individual microbial metabolism.
• Explain that AGEs, formed during high-temperature cooking, can alter gut microbiota composition and increase intestinal permeability. Upon absorption, AGEs bind to their receptor (RAGE) on vascular and cardiac cells, triggering oxidative stress and pro-inflammatory signaling. This pathway is particularly pertinent in the context of diabetes, linking diet, microbiota, and the enhanced arrhythmia risk seen in diabetic cardiomyopathy.
• Highlight that the cardiovascular benefits of soy may depend on the host's gut microbial capacity to convert daidzein into equol, a more potent antioxidant. Only a subset of the population harbors equol-producing bacteria. This phenotype could confer significant protection against oxidative atrial damage, suggesting that dietary recommendations for soy may need to be personalized based on microbial metabolism.
• Discuss how erratic eating patterns can desynchronize the circadian rhythms of both the host and the gut microbiota. This dyssynchrony is associated with dysbiosis, impaired glucose metabolism, and altered autonomic nervous system activity, potentially increasing nocturnal sympathetic tone and the risk of arrhythmias. Regular meal timing may serve as a simple intervention to stabilize these rhythms.
• In the context of SIBO, explain that certain overgrown bacteria can produce histamine, while intestinal inflammation can impair the activity of the diamine oxidase (DAO) enzyme needed for its breakdown. The resultant systemic histamine excess can cause symptoms like tachycardia, palpitations, and coronary vasospasm, which can mimic or exacerbate underlying cardiac arrhythmias, presenting a direct clinical link.
• While acknowledging the efficacy of the low FODMAP diet for SIBO symptom relief, provide a critical perspective on its long-term use. The restrictive phase drastically reduces fermentable substrates, potentially diminishing SCFA production and microbial diversity. The manuscript should emphasize the importance of the supervised reintroduction phase to personalize the diet and minimize potential long-term cardiovascular risks associated with a permanently low-fiber, low-prebiotic intake.
• Expand on how dysbiosis disrupts one-carbon metabolism involving folate, choline, and betaine. This disruption leads to hyperhomocysteinemia and alters the availability of S-adenosylmethionine (SAM), the universal methyl donor for DNA and histone methylation. This can epigenetically modify the expression of genes involved in cardiac fibrosis, hypertrophy, and ion channel function, creating a direct molecular link between microbial metabolism and gene regulation in the heart.
• Note that certain gut bacteria can convert dietary linoleic acid into various isomers of conjugated linoleic acid (CLA). Specific CLA isomers (e.g., trans-10, cis-12) have demonstrated anti-inflammatory and anti-fibrotic properties in animal models of cardiovascular disease. This represents a clear example of a beneficial, microbiota-dependent metabolite derived from dietary fat.
• Clarify the complex fate of dietary tryptophan. Only a minor fraction is used for serotonin synthesis. The majority is metabolized by gut bacteria via competing pathways: the kynurenine pathway (often pro-inflammatory) and the indole pathway (producing ligands for the aryl hydrocarbon receptor, which regulates immune and barrier function). The balance of these microbial metabolites significantly influences systemic immune tone and inflammation, thereby affecting the cardiac substrate for arrhythmias.

 

Author Response

Comment 1: The introduction is lengthy and could be streamlined. Consider moving some of the detailed microbiota composition and classification to later sections
Response 1: We are deeply grateful for your valuable suggestion. Following your recommendation, we have shortened the introduction.

Comment 2: The terms "dysbiosis," "SIBO," "LIBO," "SIFO," "IMO" are introduced but not consistently defined or differentiated in later sections. Consider a dedicated subsection or table early in the manuscript to clarify these terms.
Response 2: We decided not to elaborate on the other types of dysbiosis (SIFO, LIBO, IMO), as this is not the primary topic of our paper. Instead, we have included the definitions of these conditions in a figure as a brief explanation of the terms mentioned in the text.

Comment 3: "Gut–heart axis" is mentioned but could be more explicitly defined and framed as a conceptual model guiding the review.
Response 3: We thank the reviewer for this valuable comment. This has now been incorporated into the manuscript.

Comment 4: Section 2 ("Potential Mechanisms") is dense and covers multiple overlapping concepts (e.g., inflammation, barrier dysfunction, metabolites). Consider subdividing into clearer themes like Gut Barrier Dysfunction and Translocation/ Microbial Metabolites (TMAO, SCFAs, LPS)/ Immune and Inflammatory Pathways /Autonomic and Neural Modulation
Response 4: In response, Section 2 was comprehensively revised and reorganized to better delineate the key mechanisms linking gut dysbiosis with cardiac electrophysiology. An introductory paragraph was added to explicitly outline the main mechanistic domains discussed in this section. The content was subsequently restructured into distinct thematic subsections addressing gut barrier dysfunction and microbial translocation, microbiota-derived metabolites, immune and inflammatory pathways, and autonomic and neuronal modulation. In addition to structural reorganization, several mechanistic aspects were expanded and clarified.

A final integrative subsection was added to place these mechanisms in the broader context of heart failure and the bidirectional gut–heart axis. No original concepts were removed; rather, the revisions aimed to improve conceptual flow, reduce overlap between mechanisms, and strengthen the mechanistic links relevant to cardiac electrophysiology.

Comment 5: Section 3 on arrhythmias is well-organized but could benefit from clearer transition paragraphs linking dysbiosis to specific arrhythmia types.
Response 5: We thank the reviewer for this comment. We have made revisions to improve the clarity of the text.

Comment 6: Consider a summary table or figure early in the manuscript illustrating the key pathways linking dysbiosis to arrhythmias.
Response 6: We thank the reviewer for the suggestion. We have added additional text in these sections to improve clarity and flow; the figure summarizing key pathways was already included earlier in the manuscript.

Comment 7: The manuscript touches on drug–microbiota interactions but could expand on this given the relevance to arrhythmia management (e.g., amiodarone metabolism, warfarin, DOACs). Consider a dedicated subsection for a more detailed discussion.
Response 7: Thank you very much for your insightful comment. We truly appreciate your suggestion regarding the discussion of drug–microbiota interactions in the context of arrhythmia treatment. After careful consideration, we have decided to remove the specific mention of medications from the manuscript, as this topic is highly complex and extensive, warranting a more focused treatment in a separate, dedicated study.

Comment 8: Discuss microbiota-arrhythmia links across ages: infants (SIDS), adults (AF), elderly (aging microbiota, frailty + AF). Tie in developmental origins of health and disease (DOHaD) with early microbiota shaping later arrhythmia risk.
Response 8: Thank you for your insightful comment. Aspects related to older adults and adults have already been mentioned, and we have now supplemented the discussion by including considerations for younger populations to provide a more comprehensive overview.

Comment 9: Critique current microbiota assessment tools (breath tests, metagenomics) in cardiology trials. Propose how machine learning could integrate microbiota data with ECG/imaging for arrhythmia prediction.
Response 9: Thank you. We added a brief note on current microbiota assessment tools (breath tests, metagenomics) and suggested that machine learning could combine microbiota data with ECG/imaging to help predict arrhythmias.

Comment 10: The conclusion could be strengthened by summarizing key mechanistic insights and highlighting gaps in current knowledge (e.g., lack of human intervention trials, need for standardized microbiota assessment).
Response 10: Thank you. We strengthened the conclusion by summarizing key mechanisms, noting knowledge gaps, and adding a short paragraph on therapeutic implications and future research directions.

Comment 11: Consider adding a paragraph on therapeutic implications and future research directions (e.g., personalized microbiota modulation, biomarker development).
Response 11: Thank you. We strengthened the conclusion by summarizing key mechanisms, noting knowledge gaps, and adding a short paragraph on therapeutic implications and future research directions.

Comment 12: Avoid repetition (e.g., multiple statements on inflammation and barrier dysfunction).
Response 12: Thank you for this comment. 

Comment 13: Ensure consistent use of italicization for bacterial enera (e.g., Faecalibacterium, Ruminococcus).
Response 13: We thank the reviewer for this helpful comment. We have ensured consistent italics for all bacterial genus names.

Comment 14: Ensure consistent use of abbreviations (e.g., TMAO, SCFAs, LPS) and define upon first use.
Response 14: We appreciate this suggestion. Abbreviations have been standardized and defined at first mention throughout the manuscript.

In-Depth Comments

Comment 15: The manuscript would benefit from a more defined central thesis. Currently, it surveys the broad "gut-heart axis." To stand out, it should explicitly position Small Intestinal Bacterial Overgrowth (SIBO) as a primary clinical archetype of dysbiosis. This condition offers a distinct pathophysiological model characterized by proximal gut inflammation, competitive nutrient consumption, and direct metabolite release into the portal system, differentiating it from colonic dysbiosis. Structuring the narrative around SIBO's unique implications for arrhythmogenesis would provide a cohesive and novel framework.
Response 15: We thank the reviewer for this valuable suggestion. We have revised the manuscript accordingly to incorporate this focus.

Comment 16: The connection between malabsorption and arrhythmia needs elaboration. Please detail how deficiencies in magnesium, potassium, and calcium—common in SIBO—directly impair specific cardiac ion channels. For instance, hypomagnesemia can reduce the outward potassium current through the hERG channel, delaying repolarization and prolonging the QT interval, thereby elevating the risk for torsades de pointes. Citing clinical studies where electrolyte repletion reduces arrhythmia burden would solidify this argument.
Response 16: We thank the Reviewer for the careful assessment of our manuscript and the valuable comments provided. We have addressed the remarks by expanding the section on the link between malabsorption and arrhythmogenesis, including mechanisms by which deficiencies of magnesium, potassium, and calcium impair cardiac ion channels. In accordance with the Reviewer’s suggestion, we have incorporated clinical data demonstrating that electrolyte supplementation, particularly magnesium, reduces arrhythmia burden.

Comment 17: The discussion on fiber should move from general benefits to quantitative and mechanistic specificity. Please cite human intervention studies that link specific daily fiber intakes (e.g., >35g) to measurable increases in cardioprotective SCFAs like butyrate and propionate in systemic circulation. Furthermore, distinguish between fiber types: beta-glucan may primarily elevate propionate, while resistant starch is a potent driver of butyrate production by specific taxa like Roseburia.
Response 17: Thank you for this valuable comment. We have revised the manuscript accordingly and added a more quantitative and mechanistic discussion, including human intervention studies linking higher daily fiber intake (>35 g/day) with increased circulating SCFAs, as well as a clearer distinction between fiber types (e.g. beta-glucan vs. resistant starch).

Comment 18: When discussing TMAO, explicitly list its major dietary precursors: egg yolks, red meat, liver, and certain saltwater fish. Crucially, address the clinical paradox: choline is an essential nutrient for liver and brain function, yet its microbial metabolism can be detrimental. The manuscript should discuss how individual genetic variations in enzymes like FMO3 influence TMAO production, hinting at the need for personalized dietary guidance regarding choline-rich foods.
Response 18: Thank you for your comment. We have made revisions to include the dietary sources of TMAO as well as the genetic factors influencing the production of this metabolite.

Comment 19: Expand on how omega-3 fatty acids exert prebiotic-like effects. They are incorporated into bacterial membranes, reducing the endotoxicity of lipopolysaccharides (LPS). They also promote the growth of Lactobacillus and Bifidobacterium, which can suppress pro-inflammatory proteobacteria. This microbial shift contributes to the well-documented reduction in systemic levels of IL-6 and TNF-α, thereby potentially mitigating the inflammatory substrate for atrial fibrillation.
Response 19: We have incorporated the suggested changes regarding omega-3 fatty acids and their beneficial effects on gut microbiota and inflammation.

Comment 20: Introduce a holistic dietary pattern analysis, using the Mediterranean diet as a prime example. Describe how its synergistic components—high in polyphenols, fiber, and unsaturated fats—collectively enhance microbial diversity and the abundance of SCFA-producing species like Faecalibacterium prausnitzii. Reference longitudinal cohort data (e.g., from the PREDIMED study) that links adherence to this pattern with a lower incidence of atrial fibrillation, connecting diet structure to clinical outcome.
Response 20: Thank you for this suggestion. We have expanded the discussion to include dietary pattern analysis, using the Mediterranean diet as an example, and described how its synergistic components are associated with increased gut microbiota diversity and SCFA-producing taxa.

Comment 21: Elaborate on the consequences of SIBO-induced vitamin B12 deficiency. Bacterial consumption of B12 in the small intestine impairs the function of methionine synthase. This leads to the accumulation of homocysteine, a molecule that promotes endothelial dysfunction, activates cardiac fibroblasts, and induces oxidative stress. This pathway provides a direct mechanistic link between a microbial-driven deficiency and the structural remodeling of the atria.
Response 21: Thank you for this comment. We have expanded the manuscript to more clearly address the consequences of SIBO-related vitamin B12 deficiency

Comment 22: Incorporate recent findings on dietary sodium's extra-renal effects. High salt intake has been shown to reduce levels of beneficial Lactobacillus species in the gut. This loss alleviates a natural suppression on the differentiation of T-helper 17 (TH17) cells, leading to increased IL-17 production, systemic inflammation, and hypertension—a major driver of atrial fibrillation.
Response 22: Thank you for this comment. We have incorporated recent evidence on the extra-renal effects of dietary sodium.

Comment 23: Address the emerging controversy surrounding artificial sweeteners. Studies indicate that compounds like sucralose and saccharin can induce glucose intolerance by altering gut microbiota composition, favoring pro-inflammatory Enterobacteriaceae. This microbial shift and the resultant metabolic dysfunction could indirectly influence cardiac autonomic tone and arrhythmia risk, an area warranting further investigation.
Response 23: Thank you for this comment. We have revised the manuscript accordingly and implemented the suggested changes.

Comment 24: Introduce the concept that the bioactivity of many dietary polyphenols (e.g., in berries, tea, nuts) is dependent on microbial metabolism. Gut bacteria transform parent compounds into absorbable metabolites like urolithins (from ellagitannins) with enhanced anti-inflammatory and antioxidant properties. This process directly links dietary intake to the production of systemically active compounds that can improve endothelial function and myocardial resilience.
Response 24: Thank you for the comment. We have added revisions discussing polyphenol bioactivity in the context of microbial metabolism.

Comment 25: Present a balanced view of very-low-carbohydrate diets. While they may reduce beneficial Bifidobacterium due to fiber restriction, they also dramatically decrease the abundance of LPS-producing bacterial families. The net effect on systemic inflammation and arrhythmia risk is therefore complex and context-dependent, potentially offering benefit in specific scenarios (e.g., severe metabolic syndrome) but carrying unknown long-term risks for gut and cardiovascular health.
Response 25: Thank you for this comment. We have added a balanced perspective on very low-carbohydrate diets, acknowledging both the potential reduction in beneficial fiber-dependent taxa and the possible decrease in LPS-producing bacteria.

Comment 26: Cite preclinical evidence that common dietary emulsifiers like polysorbate-80 and carboxymethylcellulose can degrade the protective intestinal mucus layer, enhance bacterial adhesion to the epithelium, and induce low-grade inflammation and metabolic dysregulation in animal models. This provides a plausible mechanistic link between the consumption of ultra-processed foods and a pro-arrhythmic systemic state.
Response 26: Thank you. We have added evidence that emulsifiers like polysorbate-80 and carboxymethylcellulose can disrupt the gut mucus, promote inflammation, and contribute to pro-arrhythmic effects.

Comment 27: Differentiate between classes of prebiotics. For example, inulin and fructooligosaccharides (FOS) are highly selective for stimulating Bifidobacterium, while resistant starch (RS) is a preferred substrate for butyrate-producing genera like Eubacterium rectale. Since different SCFAs have distinct cardiac effects, this specificity is crucial for designing targeted nutritional interventions.
Response 27: We appreciate the suggestion. We have distinguished prebiotic types, noting that inulin and FOS preferentially stimulate Bifidobacterium, while resistant starch supports butyrate producers such as Eubacterium rectale, which is relevant for designing targeted interventions.

Comment 28: Elevate the discussion by referencing specific probiotic strains with documented cardiovascular effects in human trials. Examples include Lactobacillus reuteri NCIMB 30242 for cholesterol management and Lactobacillus rhamnosus GG for enhancing gut barrier integrity. This moves the narrative from general concepts to clinically substantiated, strain-specific applications.
Response 28: We have incorporated your suggestion by specifically citing probiotic strains with documented cardiovascular effects, such as Lactobacillus reuteri NCIMB 30242 for cholesterol regulation and Lactobacillus rhamnosus GG for intestinal barrier enhancement.

Comment 29: Explain the synergistic logic behind synbiotic formulations: the prebiotic component acts as a targeted fuel source to enhance the survival, colonization, and metabolic output of the co-administered probiotic. Reference clinical trials where synbiotic combinations have demonstrated superior efficacy in reducing inflammatory biomarkers like hs-CRP compared to probiotics or prebiotics alone in patients with cardiometabolic risk factors.
Response 29: We thank the reviewer for this comment. We have clarified this aspect in the manuscript by incorporating the suggested changes.

Comment 30: Discuss how intermittent fasting regimens influence the gut ecosystem, notably by increasing the abundance of Akkermansia muciniphila, a bacterium associated with improved gut barrier function and metabolic health. Link these dietary patterns to improvements in parasympathetic tone (evidenced by increased heart rate variability) and reduced oxidative stress, which may collectively lower susceptibility to autonomically triggered arrhythmias.
Response 30: Thank you for this comment. We have incorporated a general discussion on intermittent fasting, including its effects on gut microbiota composition (notably Akkermansia), gut barrier function and the potential implications for arrhythmia susceptibility.

Comment 31: Elaborate on the non-skeletal role of Vitamin D. Activation of the Vitamin D Receptor (VDR) in intestinal epithelial cells upregulates the expression of tight junction proteins (claudin, occludin). Therefore, Vitamin D deficiency—common in malabsorptive states—compromises intestinal barrier integrity, facilitating the translocation of pro-inflammatory molecules like LPS and creating a vicious cycle relevant to arrhythmogenesis.
Response 31: Thank you for this comment. We have added a brief discussion on the mechanism of vitamin D, highlighting its effects on intestinal barrier integrity via VDR activation, and the potential consequences of deficiency for LPS translocation and arrhythmia risk.

Comment 32: Describe the enterosalivary circuit for nitric oxide (NO) generation. Dietary nitrates from leafy greens and beets are absorbed, concentrated in saliva, and reduced to nitrite by oral bacteria. Upon swallowing, nitrite is converted to NO in the acidic stomach and systemically. This microbiota-dependent pathway boosts bioactive NO, improving endothelial function, vasodilation, and potentially stabilizing the myocardial electrical substrate, particularly under ischemic conditions.
Response 32: Thank you. We added a note on enterosalivary NO, emphasizing its microbiota-dependent role in endothelial function, vasodilation, and cardiac diseases.

Comment 33: Introduce the concept of enterotypes—stable community structures like Bacteroides-dominant or Prevotella-dominant profiles. Suggest that an individual's enterotype may predict their cardiometabolic response to dietary interventions. For instance, a Prevotella-dominant individual might derive greater arrhythmia risk reduction from a high-fiber intervention than a Bacteroides-dominant individual, pointing toward future personalized nutrition strategies.
Response 33: Thank you. We have added a note on enterotypes as stable gut microbiota patterns that may influence individual responses to diet.

Comment 34: Deepen the analysis of ultra-processed foods beyond low fiber content. Their lack of fermentable substrate may shift microbial metabolism toward the breakdown of endogenous proteins, increasing the production of deleterious metabolites like p-cresol sulfate and indoxyl sulfate. These uremic toxins are independently associated with endothelial dysfunction, oxidative stress, and fibrosis, all key players in arrhythmogenesis.
Response 34: Thank you. We have added that low fiber may shift gut microbial metabolism toward protein degradation, increasing uremic toxins (p-cresol, indoxyl sulfate).

Comment 35: Discuss how dysbiosis and intestinal inflammation can specifically impair the absorption of copper and zinc. Copper deficiency can lead to dilated cardiomyopathy and electrical abnormalities, while zinc deficiency impairs antioxidant defense (as a cofactor for superoxide dismutase). Furthermore, excessive zinc supplementation can induce copper deficiency, highlighting the importance of balance and the role of gut health in mineral homeostasis for cardiac function.
Response 35: Thank you for this comment. We have revised the manuscript to clarify how dysbiosis and intestinal inflammation can impair the absorption of copper and zinc, and highlighted the cardiovascular implications of their deficiencies, as well as the potential interaction between excessive zinc supplementation and copper status.

Comment 36: Address the complexity of caffeine. It is metabolized by gut bacterial enzymes, and its byproducts may influence host physiology. Furthermore, as an adenosine receptor antagonist, caffeine's long-term impact on atrial tissue is nuanced; while adenosine has anti-fibrotic properties, acute caffeine intake is not consistently linked to increased AF risk in epidemiological studies, suggesting a U-shaped relationship that may be influenced by individual microbial metabolism.
Response 36: We thank the reviewer for this comment and have added a discussion of the complex effects of coffee and caffeine in the revised manuscript.

Comment 37: Explain that AGEs, formed during high-temperature cooking, can alter gut microbiota composition and increase intestinal permeability. Upon absorption, AGEs bind to their receptor (RAGE) on vascular and cardiac cells, triggering oxidative stress and pro-inflammatory signaling. This pathway is particularly pertinent in the context of diabetes, linking diet, microbiota, and the enhanced arrhythmia risk seen in diabetic cardiomyopathy.
Response 37: Thank you for the comment. We have added a section explaining that advanced glycation end-products (AGEs) can alter the gut microbiota, increase intestinal permeability, and, upon absorption, bind to RAGE receptors, triggering oxidative stress and inflammation.

Comment 38: Highlight that the cardiovascular benefits of soy may depend on the host's gut microbial capacity to convert daidzein into equol, a more potent antioxidant. Only a subset of the population harbors equol-producing bacteria. This phenotype could confer significant protection against oxidative atrial damage, suggesting that dietary recommendations for soy may need to be personalized based on microbial metabolism.
Response 38: Thank you very much for this valuable comment. We have now incorporated this aspect into the manuscript.

Comment 39: Discuss how erratic eating patterns can desynchronize the circadian rhythms of both the host and the gut microbiota. This dyssynchrony is associated with dysbiosis, impaired glucose metabolism, and altered autonomic nervous system activity, potentially increasing nocturnal sympathetic tone and the risk of arrhythmias. Regular meal timing may serve as a simple intervention to stabilize these rhythms.
Response 39: We greatly appreciate this insightful comment and have now integrated this point into the manuscript.

Comment 40: In the context of SIBO, explain that certain overgrown bacteria can produce histamine, while intestinal inflammation can impair the activity of the diamine oxidase (DAO) enzyme needed for its breakdown. The resultant systemic histamine excess can cause symptoms like tachycardia, palpitations, and coronary vasospasm, which can mimic or exacerbate underlying cardiac arrhythmias, presenting a direct clinical link.
Response 40: Thank you for your comment. We have now included in the discussion the role of histamine in SIBO and its potential effects on heart rhythm.

Comment 41: While acknowledging the efficacy of the low FODMAP diet for SIBO symptom relief, provide a critical perspective on its long-term use. The restrictive phase drastically reduces fermentable substrates, potentially diminishing SCFA production and microbial diversity. The manuscript should emphasize the importance of the supervised reintroduction phase to personalize the diet and minimize potential long-term cardiovascular risks associated with a permanently low-fiber, low-prebiotic intake.
Response 41: We thank the reviewer for this suggestion. We have identified various studies on the effects of the low FODMAP diet and have included a summary in the revised manuscript.

Comment 42: Expand on how dysbiosis disrupts one-carbon metabolism involving folate, choline, and betaine. This disruption leads to hyperhomocysteinemia and alters the availability of S-adenosylmethionine (SAM), the universal methyl donor for DNA and histone methylation. This can epigenetically modify the expression of genes involved in cardiac fibrosis, hypertrophy, and ion channel function, creating a direct molecular link between microbial metabolism and gene regulation in the heart.
Response 42: We have included a brief mention of the impact of gut dysbiosis on one-carbon metabolism and epigenetic regulation in the revised manuscript.

Comment 43: Note that certain gut bacteria can convert dietary linoleic acid into various isomers of conjugated linoleic acid (CLA). Specific CLA isomers (e.g., trans-10, cis-12) have demonstrated anti-inflammatory and anti-fibrotic properties in animal models of cardiovascular disease. This represents a clear example of a beneficial, microbiota-dependent metabolite derived from dietary fat.
Response 43: We thank the reviewer for this comment. We have incorporated the aspect of linoleic acid in the discussion of animal models.

Comment 44: Clarify the complex fate of dietary tryptophan. Only a minor fraction is used for serotonin synthesis. The majority is metabolized by gut bacteria via competing pathways: the kynurenine pathway (often pro-inflammatory) and the indole pathway (producing ligands for the aryl hydrocarbon receptor, which regulates immune and barrier function). The balance of these microbial metabolites significantly influences systemic immune tone and inflammation, thereby affecting the cardiac substrate for arrhythmias.
Response 44: We thank the reviewer for this comment. We have incorporated the aspect of dietary tryptophan metabolism into the manuscript.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

After thoroughly checking the revised version of manuscript is good now.

Author Response

We would like to sincerely thank you for your time and effort in thoroughly reviewing the revised version of our manuscript. We greatly appreciate your positive assessment and are pleased to hear that you find the manuscript to be satisfactory in its current form.

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to commend the authors for their thorough and diligent work. The revised manuscript shows clear improvement over the previous version. However, my review has identified several areas where the authors’ stated responses do not fully align with the changes reflected in the text. To ensure the manuscript is complete and fully responsive to peer review, I recommend the following final revisions prior to acceptance:

Comment 3: Although the response indicates this was added, the manuscript still lacks an explicit, early definition of the "gut-heart axis" as a guiding conceptual framework. Please include a concise definition in the introduction to anchor the narrative.

Comment 7: Omitting this topic weakens the manuscript. Given its direct relevance to arrhythmia management (e.g., metabolism of amiodarone, warfarin, DOACs), I strongly recommend reinstating and briefly expanding this discussion in a dedicated subsection.

Comment 9: The response mentions a “brief note,” but this is not visible in the text. Please add the promised content, a critical overview of current microbiota assessment tools (breath tests, metagenomics) in cardiology, along with a forward-looking statement on the potential of machine learning to integrate microbiota data with ECG/imaging for predictive purposes.

Comment 17: To fully address this point, please incorporate quantitative data from human intervention studies (e.g., daily fiber intake >35g associated with increased circulating SCFAs) and clarify mechanistic distinctions between fiber types (e.g., beta-glucan vs. resistant starch) and their effects on specific bacterial taxa (e.g., Roseburia, Eubacterium rectale).

Minor Enhancements for Precision: Please expand the new text for Comments 19 (Omega-3), 25 (Low-Carb Diets), 29 (Synbiotic Logic), and 33 (Enterotypes) to match the level of mechanistic detail and clinical context provided elsewhere. For example:

• Comment 19: Explicitly note how omega-3 incorporation reduces LPS endotoxicity.

• Comment 29: Explain the synergistic “fuel source” rationale of synbiotics and cite relevant clinical trial outcomes.

• Comment 33: Briefly describe how enterotypes may predict differential responses to dietary interventions.

 

 

Author Response

Comment 3: Although the response indicates this was added, the manuscript still lacks an explicit, early definition of the "gut-heart axis" as a guiding conceptual framework. Please include a concise definition in the introduction to anchor the narrative.
Response 3: Following your suggestions, we have expanded the section in the Introduction describing the gut–heart axis to clearly emphasize the significance of the disturbances occurring in the course of SIBO and their impact on the cardiovascular system.

Comment 7: Omitting this topic weakens the manuscript. Given its direct relevance to arrhythmia management (e.g., metabolism of amiodarone, warfarin, DOACs), I strongly recommend reinstating and briefly expanding this discussion in a dedicated subsection.
Response 7: We thank the Reviewer for this comment. In accordance with the suggestion, we have reinstated this topic and added a dedicated subsection briefly discussing the relevance of these drugs to arrhythmia management, including their metabolism. 

Comment 9: The response mentions a “brief note,” but this is not visible in the text. Please add the promised content, a critical overview of current microbiota assessment tools (breath tests, metagenomics) in cardiology, along with a forward-looking statement on the potential of machine learning to integrate microbiota data with ECG/imaging for predictive purposes.
Response 9: We apologize for the oversight. This content was already included in the previous version; however, we did not clearly indicate its location. For clarity, we have now highlighted the relevant fragment in yellow in the Discussion section. 

Comment 17: To fully address this point, please incorporate quantitative data from human intervention studies (e.g., daily fiber intake >35g associated with increased circulating SCFAs) and clarify mechanistic distinctions between fiber types (e.g., beta-glucan vs. resistant starch) and their effects on specific bacterial taxa (e.g., Roseburia, Eubacterium rectale).
Response 17: Section 5.1 has been revised to add previously missing quantitative data from human intervention studies on dietary fiber intake and SCFA levels. In addition, we expanded the discussion of fiber type–specific effects by including β-glucan and inulin, while the effects of resistant starch were already described in the original manuscript.

Comment 19: Explicitly note how omega-3 incorporation reduces LPS endotoxicity.
Response 19: In accordance with the reviewer’s suggestions, we have expanded the manuscript to include a discussion of omega-3 fatty acid supplementation in the context of LPS-induced toxicity. 

Comment 29: Explain the synergistic “fuel source” rationale of synbiotics and cite relevant clinical trial outcomes.
Response 29: We have further highlighted the role of prebiotics as a metabolic energy source for colonizing microbial strains, supported by specific clinical evidence demonstrating the beneficial effects of their administration.

Comment 33: Briefly describe how enterotypes may predict differential responses to dietary interventions.
Response 33: Thank you for this valuable comment. We agree that enterotypes may play an important role in predicting differential responses to various dietary interventions. In the revised manuscript, we have clarified this aspect and emphasized that our discussion focuses specifically on dietary fiber as a key intervention.