Review Reports - The Landscape of SERCA2 in Cardiovascular Diseases: Expression Regulation, Therapeutic Applications, and Emerging Roles

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review article "The landscape of SERCA2 in cardiovascular diseases: Expression regulation, therapeutic applications and emerging roles", is a very comprehensive review of the role of SERCA2 in cardiovascular disease.

With a few minor revisions, I feel that this will be a review that is will be an excellent review that is very useful to the field.

Minor points

Line 16 clinical treatments are focused on the alleviation of treatments.

Line 25 (and several other places in the review), the authors conflate gene expression and function. Some modifications effect the level of expression whereas other modification impact the function and turnover the protein

Throughout, there are many redudancies that if reduced would make the review more readable.

In the introduction: The review of cardiovascular risk is good but should be more focused how altered calcium handling impacts cardiovascular risk and less on general cardiovascular risk.

line 78 there is an extra space

Summary section: There is a formatting error (line spacing).

lines 258-281 Sumoization is not a word. I think they mean sumoylation. Also SUMO does not need to be capitalized in SUMOylation.

line 268. should read "these modifications are disease-stage specific.

There are similar redundancies, grammatical, and spelling errors throughout, but aside from a careful revision, I feel that no major changes are required.

Author Response

Q1: Line 16 clinical treatments are focused on the alleviation of treatments.

A1: We sincerely appreciate your valuable comments! We fully agree with your comments. We have revised the content in Line 16, and the original text has been modified to read as follows: Clinical treatments are focused on the alleviation of treatments.

Q2: Line 25 (and several other places in the review), the authors conflate gene expression and function. Some modifications effect the level of expression whereas other modification impact the function and turnover the protein

A2: We sincerely appreciate your valuable comments! We fully agree with your comments. To clarify the distinct impacts of different modifications on gene expression and function, we have revised the content in Line 25, with the original text amended to: This review systematically explores the regulatory mechanisms of SERCA2 expression and its functional regulation—including transcriptional regulation, post-translational modifications, and protein-protein interactions—and further investigates its pathological roles in cardiovascular diseases as well as its potential as a therapeutic target.

Q3: Throughout, there are many redudancies that if reduced would make the review more readable.

A3: We sincerely appreciate your valuable comments! We have already deleted the duplicated content.

Q4: In the introduction: The review of cardiovascular risk is good but should be more focused how altered calcium handling impacts cardiovascular risk and less on general cardiovascular risk.

A4: We sincerely appreciate your valuable comments! In lines 114-128, we described the coupling relationship between hydrogen ion flux and calcium ion flux in the function of SERCA2: In particular, in the heart, SERCA2a takes up Ca2+ and returns it to the sarcoplasmic reticulum after each contrac-tile maneuver completed by cardiomyocytes [26]. This process involves the realization of a typical “E1-E2 conforma-tional cycle”: the E1 structure of SERCA2a has a high affinity for Ca2+ and its binding site (Asp351/Asp703) is ex-posed to the sarcoplasmic reticulum. However, the E2 structure has a lower affinity for Ca2+ and is located in the lumen of the sarcoplasmic reticulum. After cardiomyocyte diastole, Ca2+ is released from troponin into the cyto-plasm and binds to E1 (the binding site is located at an aspartic acid residue), followed by phosphorylation of E1 with the involvement of ATP (Ca2+-E1-P), which results in a conformational change from E1 to E2 (Ca2+- E2-P) [27], and the E2 structure becomes an ADP-insensitive intermediate state [28]. This process allows the binding site for Ca²⁺ to be localized in the sarcoplasmic reticulum. Due to the low affinity of E2 for Ca2+, Ca2+ is released into the sarcoplasmic reticulum upon dissociation from E2, accompanied by the conversion of E2 to E1 and on to the next cycle [29]. The proper functioning of its function is essential for the systolic and diastolic cycles of the heart.

Q5: line 78 there is an extra space

A5: We sincerely appreciate your valuable comments! We fully agree with your comments. We have removed the extra spaces.

Q6: Summary section: There is a formatting error (line spacing).

A6: We sincerely appreciate your valuable comments! We fully agree with your comments. We have adjusted the line spacing of the summary section to 1.5 lines.

Q7: lines 258-281 Sumoization is not a word. I think they mean sumoylation. Also SUMO does not need to be capitalized in SUMOylation.

A7: We sincerely appreciate your valuable comments! We fully agree with your comments. We have corrected the term Sumoization to Sumoylation throughout the manuscript, and we have revised all uppercase "SUMO" in SUMOylation to lowercase.

Q8: line 268. should read "these modifications are disease-stage specific.

A8: We sincerely appreciate your valuable comments! We fully agree with your comments. To highlight the importance of these modifications in different disease stages, we have revised the content in Line 268 to read as follows: These findings not only elucidate the critical and disease-stage-specific role of sumoylation modification in maintaining SERCA2a function, but also provide potential therapeutic targets for the treatment of cardiovascular diseases.

Q9: There are similar redundancies, grammatical, and spelling errors throughout, but aside from a careful revision, I feel that no major changes are required.

A9: We sincerely appreciate your valuable comments! We have corrected the similar redundancies, grammatical, and spelling errors in the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Wu and co-authors submitted an updated literature review manuscript on SERCA2's expression regulation, role in major cardiovascular diseases, and current therapeutic options. Some specific highlights of the manuscript include a timeline of key findings surrounding the role of SERCA2, sequential discussion of SERCA2 expression/function (transcriptional regulation, PTMs, protein-protein interactions), SERCA2's role in atherosclerosis, and direct & indirect therapeutic strategies involving small molecular activators and gene therapy. With examples indicated below, my only recommendation for improvement is to perform another thorough editing of the manuscript for typos.

Line 74, "SERCA2 oxidative..."; likely meant "oxidation"

Line 358, "XBP1s) pathway..."

Line 403, "...and Phosphorylation of SERCA2..."

Line 434, "...in vivo. ang II..."

Line 457, "...aorta. proportion..."

Line 463, "...LDL-/-..."; This mouse is actually an LDLR knockout and should be indicated as "LDLR-/-".

Line 495, "...NADP. sorbitol..."

Line 504, "...ROS.ROS..."

Lines 524-525, "...insulin resistance endoplasmic reticulum..."; was there supposed to be a comma and/or an "and" in there somewhere?

Line 583, "microRNAs(miRNAs, miR); likely need to capitalize as "MicroRNAs" while allowing a single space from the parentheses.

Line 587, "miR-25..."; again, likely need to capitalize as "MiR-25".

Author Response

Q1: Wu and co-authors submitted an updated literature review manuscript on SERCA2's expression regulation, role in major cardiovascular diseases, and current therapeutic options. Some specific highlights of the manuscript include a timeline of key findings surrounding the role of SERCA2, sequential discussion of SERCA2 expression/function (transcriptional regulation, PTMs, protein-protein interactions), SERCA2's role in atherosclerosis, and direct & indirect therapeutic strategies involving small molecular activators and gene therapy. With examples indicated below, my only recommendation for improvement is to perform another thorough editing of the manuscript for typos.

Line 74, "SERCA2 oxidative..."; likely meant "oxidation"

Line 358, "XBP1s) pathway..."

Line 403, "...and Phosphorylation of SERCA2..."

Line 434, "...in vivo. ang II..."

Line 457, "...aorta. proportion..."

Line 463, "...LDL-/-..."; This mouse is actually an LDLR knockout and should be indicated as "LDLR-/-".

Line 495, "...NACl..."

Line 504, "...ROS.ROS..."

Lines 524-525, "...insulin resistance endoplasmic reticulum..."; was there supposed to be a comma and/or an "and" in there somewhere?

Line 583, "microRNAs(miRNAs, miR); likely need to capitalize as "MicroRNAs" while allowing a single space from the parentheses.

Line 587, "miR-25..."; again, likely need to capitalize as "MiR-25".

A1: We sincerely appreciate your valuable comments! We fully agree with your comments. We have corrected all the aforementioned errors. In this revised version, all the modifications have been highlighted in red to facilitate your quick and efficient review. We would like to express our sincere gratitude to you again for your generous guidance.

Reviewer 3 Report

Comments and Suggestions for Authors

The review article by Wu et al. aims to summarize the alterations observed in SERCA2 expression and activity in cardiovascular diseases (CVD) along with the regulatory mechanisms in these processes. Also, the manuscript provides an overview of therapeutic strategies targeting SERCA2. Although the manuscript intends to cover various aspects of SERCA2 research, the enthusiasm is lowered due to the lack of adequate information in certain areas and use of inaccurate terminology in various parts of the text. Below are the major concerns.

1) Several studies have shown that a major mechanism of HNO (nitroxyl)-induced SERCA2a activation involves the modification of phospholamban (PLN) cysteine residues by HNO and the consequent relief of PLN inhibition on SERCA2a. In fact, many papers point to the lack of HNO-induced effects on SERCA2a in the absence of PLN. These studies should be included also. The authors may consider the relevant references for in-depth information [e.g. Antioxid Redox Signal. 2013, 19(11):1185-97; J Gen Physiol. 2019, 151(6):758-770; Biochemistry 2008, 47(50):13150-2].

2) HNO donors should be mentioned under the section titled "Advances in treatment strategies".

3) Table 1: Regarding the studies on the mechanism of SERCA2 in CVD, findings related to SERCA2a/PLN complex should be considered, especially in the case of heart failure (HF).

4) Abstract: "This review systematically examines the regulatory mechanisms governing SERCA2 expression—such as transcriptional control, post-translational modifications, and protein-protein interactions...". Post-translational modifications and protein-protein interactions are mainly related to SERCA2 activity and does not necessarily mean they impact expression levels. Similarly, section 3 is titled as "The regulatory mechanisms of SERCA2 expression", however it contains a subsection "3.3. Mechanisms of post-translational modification regulation at the protein level". The title of section 3 and the abstract should be edited to represent the content accurately.

5) Lines 249-251: Please clarify "nitrosative modifications promote S-glutathionylation." The paper cited includes HNO (nitroxyl)-induced modification on SERCA2a. HNO does not result in a nitrosative modification on cysteines. (Actually, the cited paper points to direct modification of SERCA2a cysteine by HNO.)

6) Section 6. Summary (lines 644-647): “Exploring the interactions between SERCA2 and other calcium regulator proteins (e.g., PLB, RyR, etc.): Examine the interactions between SERCA2 and other calcium regulatory proteins, such as PLB and ryanodine receptors (RyR), to achieve a more comprehensive understanding of cardiac calcium signaling. This holistic perspective will enhance our knowledge of calcium homeostasis and its impact on cardiac function.” There is a significant amount of literature available on SERCA2 and PLN (and other calcium regulatory proteins). Although further research is definitely needed, the authors should briefly summarize the existing research in the relevant sections.

7) Lines 292-293: Please clarify the statement. What do the authors mean by "...resulted in a significant decrease in phosphorylation and oligomerization of the SERCA2a Thr484 site, ...".

8) Lines 286-293: The authors mention SERCA2a oligomerization, however it wasn't included in "Summary of SERCA2" section, where SERCA2 structure and function is discussed.

9) Lines 390-392: Please edit the statement to avoid confusion: “The specific mechanism by which SERCA2a ubiquitination enhances the function of SERCA2a has not yet been fully elucidated, and it is possible that SERCA2a function may be enhanced by blocking other post-translational modifications, such as acetylation or increasing ATPase activity.”

10) Please check the terminology throughout the text and figures/tables [e.g. PLN "oligomerization" instead of "aggregation". PLN is known to be in equilibrium between its monomeric and oligomeric forms.] Also, in several instances, the authors refer to “C674 inactivation”. "Irreversible oxidation" or "oxidation" is not synonymous with "inactivation".

11) Lines 466-467: Please clarify or elaborate the following: “… SERCA2 dysfunction interfered with intracellular calcium disorders, ...".

12) In several instances, the specific isoform of SERCA2 was not mentioned. Please mention it if the information is available.

Minor:

1) “PLN” and “PLB” alternate throughout manuscript. Although both abbreviations can be used for phospholamban, it'll be easier for the reader if the abbreviation is consistent throughout the text.

2) Lines 131-133: Please include the relevant references.

3) Lines 497-498: Please include a reference for the following sentence: “On the other hand, elevated NADH levels also increase superoxide levels.”

Comments on the Quality of English Language

Please edit the manuscript text and figures to avoid repetitions and/or typos (e.g. lines 416-419).

Author Response

A1: We sincerely appreciate your valuable comments! We are extremely grateful for your contribution to the professional content of our manuscript. We have added the relevant content in lines 255-262 and cited the aforementioned literature provided by you: Meanwhile, Sivakumaran et al. [59] reported that HNO can also augment SERCA2a activity and cardiomyocyte function by facilitating redox-dependent oligomerization of PLN. In cardiomyocytes, HNO maintains PLN in an oligomeric conformation through disulfide bond-mediated mechanisms; this process decreases the level of free PLN monomers with inhibitory activity, thereby relieving the inhibitory effect of PLN on the calcium pump [60]. Furthermore, utilizing ¹⁵N-edited nuclear magnetic resonance (NMR) spectroscopy, Keceli et al. [61] identified that HNO exerts its cardioprotective effect by targeting cysteine 41 (Cys41) and cysteine 46 (Cys46) residues of PLN to improve cardiac function. Collectively, these redox-sensitive modifications underscore the sophisticated and intricate regulatory mechanisms governing SERCA2a activity.

2) HNO donors should be mentioned under the section titled "Advances in treatment strategies".

A2: We sincerely appreciate your valuable comments! We have supplemented 250 to 255 related contents: Notably, Lokuta et al. [58] found approximately 2-fold elevated nitrotyrosine levels in cardiac tissues of dilated cardiomyopathy (DCM) patients compared to controls and confirmed SERCA2a as the major nitration target protein by mass spectrometry, which may be related to long-term exposure to peroxynitrite. In addition, Lancel et al. [19] demonstrated that nitroxyl (HNO) activates SERCA in cardiomyocytes via glutathiolation of cysteine 674, which elevates the calcium transport efficiency of the enzyme and ultimately ameliorates the contractile and relaxant functions of cardiomyocytes.

3) Table 1: Regarding the studies on the mechanism of SERCA2 in CVD, findings related to SERCA2a/PLN complex should be considered, especially in the case of heart failure (HF).

A3: We sincerely appreciate your valuable comments! We fully agree with your comments. We have made corrections to the content of Table 1. We have changed the article for the year 2009 to: The role of SERCA2a/PLN complex, Ca(2+) homeostasis, and anti-apoptotic proteins in determining cell fate.

A4: We sincerely appreciate your valuable comments! We have revised the abstract sentence from "This review systematically examines the regulatory mechanisms governing SERCA2 expression" to "This review systematically explores the regulatory mechanisms of SERCA2 expression and its functional regulation".Title of Chapter 3 has been revised to "The Regulatory Mechanisms of SERCA2 Expression and Its Functional Regulation".

A5: We sincerely appreciate your valuable comments! The original text does not mean "nitrogenation modification promotes the ionization of S-glutamate". We have removed the incorrect statement and corrected the following text as follows: In addition, Lancel and colleagues [18] found that nitroxyl (HNO) can activate SERCA in cardiomyocytes through glutathiolation of cysteine 674, thereby enhancing its calcium transport efficiency and ultimately improving the contractile and relaxant functions of cardiomyocytes.

A6: We sincerely appreciate your valuable comments! We fully agree with your comments. We have added an overview of the existing literature on SERCA2 and PLN-related studies in lines 253-262: In addition, Lancel et al. [19] demonstrated that nitroxyl (HNO) activates SERCA in cardiomyocytes via glutathiolation of cysteine 674, which elevates the calcium transport efficiency of the enzyme and ultimately ameliorates the contractile and relaxant functions of cardiomyocytes. Meanwhile, Sivakumaran et al. [59] reported that HNO can also augment SERCA2a activity and cardiomyocyte function by facilitating redox-dependent oligomerization of PLN. In cardiomyocytes, HNO maintains PLN in an oligomeric conformation through disulfide bond-mediated mechanisms; this process decreases the level of free PLN monomers with inhibitory activity, thereby relieving the inhibitory effect of PLN on the calcium pump [60]. Furthermore, utilizing ¹⁵N-edited nuclear magnetic resonance (NMR) spectroscopy, Keceli et al. [61] identified that HNO exerts its cardioprotective effect by targeting cysteine 41 (Cys41) and cysteine 46 (Cys46) residues of PLN to improve cardiac function. Collectively, these redox-sensitive modifications underscore the sophisticated and intricate regulatory mechanisms governing SERCA2a activity.

7) Lines 292-293: Please clarify the statement. What do the authors mean by "...resulted in a significant decrease in phosphorylation and oligomerization of the SERCA2a Thr484 site, ...".

A7: We sincerely appreciate your valuable comments! We have corrected the incorrect statement in the original text to: Quan and colleagues [72] further demonstrated that the second kinase domain of striated muscle preferentially expressed protein kinase (SPEG) phosphorylates SERCA2a at Thr484, thereby promoting SERCA2a oligomerization and enhancing SR/ER Ca²⁺ reuptake in both cultured cells and primary neonatal murine cardiomyocytes.

8) Lines 286-293: The authors mention SERCA2a oligomerization, however it wasn't included in "Summary of SERCA2" section, where SERCA2 structure and function is discussed.

A8: We sincerely appreciate your valuable comments! We have provided supplementary information on the oligomerization of SERCA2a in lines 135-137: Notably, Blackwell et al. [31] further demonstrated that SERCA2a can form inherent homodimers in living cells, and crucially, the formation of such homodimers is not regulated by the conformational states of SERCA2a, PLN binding, or PLN phosphorylation.

A9: We sincerely appreciate your valuable comments! We fully agree with your comments. We have removed the confusing content. We would like to express our sincere gratitude to you again for your generous guidance.

A10: We sincerely appreciate your valuable comments! We appreciate the reviewer’s insightful comment on the term “inactivation.” We acknowledge that “inactivation” can be ambiguous in different research contexts. In this study, the term “inactivation of SERCA2a” specifically refers to the reversible loss of its calcium-transporting activity caused by altered interaction with phospholamban, rather than the irreversible inactivation (i.e., complete destruction of the protein structure or function) commonly used in pathogen-related studies. To avoid confusion, we have revised the relevant descriptions in the manuscript by adding the modifier “irreversible oxidative inactivation” before “inactivation” (see Line 355-361). We hope this clarification addresses the reviewer’s concern.

11) Lines 466-467: Please clarify or elaborate the following: “… SERCA2 dysfunction interfered with intracellular calcium disorders, ...".

A11: We sincerely appreciate your valuable comments! We provided detailed supplementary explanations for the relevant content in lines 482 to 492: Tong et al. [111] generated a SERCA2 C674S knock-in (SKI) mouse model, in which the cysteine at position 674 of SERCA2 was substituted with serine (S674) to mimic the irreversible oxidative activity loss of C674 under pathological conditions. Their investigations demonstrated that SERCA2 dysfunction results in intracellular Ca²⁺ accumulation and activates Ca²⁺-dependent signaling pathways, such as the calcineurin-mediated nuclear factor of activated T-cells (NFAT) and nuclear factor κB (NFκB) pathways [89], while also downregulating peroxisome proliferator-activated receptor γ (PPARγ) [153] and inducing endoplasmic reticulum (ER) stress [112]. Mechanistically, SERCA2 dysfunction accelerates aortic aneurysm and atherosclerosis by triggering oxidative stress in arterial smooth muscle cells (ASMCs), and concurrently facilitates ASMC phenotypic transformation via the activation of ERK1/2 and angiotensin II/AT1R signaling [111]. Notably, inhibition of oxidative stress in ASMCs alleviates angiotensin II-induced aortic aneurysm and atherosclerosis caused by SERCA2 dysfunction, thereby providing novel insights and potential therapeutic targets for the management of atherosclerosis.

12) In several instances, the specific isoform of SERCA2 was not mentioned. Please mention it if the information is available.

A12: We sincerely appreciate your valuable comments! Since some of the original studies cited in our manuscript (e.g., references [88, 89, 96]) did not specify the exact SERCA2 isoform involved in their research, we have retained the term 'SERCA2' to maintain consistency with the original literature. It is worth noting that SERCA2a is the main isoform expressed in arterial smooth muscle cells (ASMCs) and is widely considered the key functional isoform regulating vascular Ca²⁺ homeostasis and remodeling in studies on aortic aneurysm and atherosclerosis.

Minor:

A1: We sincerely appreciate your valuable comments! We fully agree with your comments. We have standardized the abbreviation of "phospholamban" in the manuscript to PLN.We would like to express our sincere gratitude to you again for your generous guidance.

2) Lines 131-133: Please include the relevant references.

A2: We sincerely appreciate your valuable comments! We have supplemented the references related to the content. We would like to express our sincere gratitude to you again for your generous guidance.

3) Lines 497-498: Please include a reference for the following sentence: “On the other hand, elevated NADH levels also increase superoxide levels.”

A3: We sincerely appreciate your valuable comments! We fully agree with your comments. We have supplemented the references related to the content. We would like to express our sincere gratitude to you again for your generous guidance.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript provides a solid framework; however, it requires updates to ensure recency, improvements in clarity, and expansions in certain areas. Below are specific comments:

1. The introduction cites 2021 WHO data (17.9 million CVD deaths) and projects 23 million by 2030, but as of late 2025, more recent global estimates are available (e.g., WHO 2024 updates show ~18.6 million deaths in 2023). The authors are suggested to revise these statistics and economic figures (e.g., $1.2 trillion expenditure) to reflect current data for accuracy.

2. The manuscript has several awkward phrasings and minor grammatical issues (e.g., "With rapid socioeconomic development and lifestyle changes, the global burden of cardiovascular diseases (CVDs) is increasing" could be more concise; inconsistencies in tense like "has been recognized" vs. "plays a pivotal role"). Proofread thoroughly or use professional editing to improve readability and flow.

3. The therapeutic section discusses AAV1/SERCA2a gene therapy (e.g., CUPID trials) and small molecules like CDN1163. Still, it omits 2024-2025 developments, such as the role of NEXN in promoting SERCA2a to combat vascular calcification and alarin-mediated regulation of RyR2/SERCA2 in heart failure models. Expanding this section with 5-10 recent references to strengthen the "Emerging Roles" aspect is highly recommended.

4. While SERCA2a, 2b, and 2c are described, the text could better differentiate their roles in vascular tissues. Add a table comparing isoform expression patterns, affinities, and CVD implications to improve visualization

5. The heart failure section focuses on reduced SERCA2a expression (30-40%), but recent 2025 reviews highlight conflicting data in HFpEF (heart failure with preserved ejection fraction), where SERCA2a levels may not always decrease. Discuss this variability and potential mechanisms to provide a balanced view.

6. Figure 1 (overview of advances) is a helpful timeline, but ensure all milestones (e.g., 2023-2025 entries like SERCA2a activation in obesity-related dysfunction) are updated. Also, cross-reference figures more explicitly in the text, and verify that all citations are complete and accessible.

7. The style of references should be carefully revised to meet the guidelines of Biomolecules.

Author Response

The manuscript provides a solid framework; however, it requires updates to ensure recency, improvements in clarity, and expansions in certain areas. Below are specific comments:

The introduction cites 2021 WHO data (17.9 million CVD deaths) and projects 23 million by 2030, but as of late 2025, more recent global estimates are available (e.g., WHO 2024 updates show ~18.6 million deaths in 2023). The authors are suggested to revise these statistics and economic figures (e.g., $1.2 trillion expenditure) to reflect current data for accuracy.

A1: We sincerely appreciate your valuable comments! We fully agree with your comments. To reflect the accuracy of the current data, we have updated and corrected the data in the article as follows: According to the World Health Organization (WHO), approximately 18.6 million people died from CVD in 2023, accounting for 31% of all global deaths (WHO, 2024). Of these, ischemic heart disease (9.7 million cases) and stroke (6.1 million cases) are the leading causes of death. We would like to express our sincere gratitude to you again for your generous guidance.

The manuscript has several awkward phrasings and minor grammatical issues (e.g., "With rapid socioeconomic development and lifestyle changes, the global burden of cardiovascular diseases (CVDs) is increasing" could be more concise; inconsistencies in tense like "has been recognized" vs. "plays a pivotal role"). Proofread thoroughly or use professional editing to improve readability and flow.

A2: We sincerely appreciate your valuable comments! We fully agree with your comments. We have already corrected the sentences with grammatical errors: Driven by rapid socioeconomic progress and changing lifestyles, the global burden of cardiovascular diseases (CVDs) continues to escalate, with surging morbidity and mortality rates imposing a severe threat to public health. We would like to express our sincere gratitude to you again for your generous guidance.

The therapeutic section discusses AAV1/SERCA2a gene therapy (e.g., CUPID trials) and small molecules like CDN1163. Still, it omits 2024-2025 developments, such as the role of NEXN in promoting SERCA2a to combat vascular calcification and alarin-mediated regulation of RyR2/SERCA2 in heart failure models. Expanding this section with 5-10 recent references to strengthen the "Emerging Roles" aspect is highly recommended.

A3: We sincerely appreciate your valuable comments! We fully agree with your comments. We have supplemented the development information that was omitted in the report for the years 2024-2025: Jiang et al. [133] established a homozygous NEXN-knockout cardiomyocyte model by combining CRISPR/Cas9 gene-editing technology with the directed differentiation of human induced pluripotent stem cells (hiPSCs). Their findings demonstrated that NEXN⁻/⁻ hiPSC-CMs exhibited a hypertrophic phenotype, accompanied by a marked reduction in SERCA2a expression and impaired Ca²⁺ handling in NEXN⁻/⁻ hiPSCs. Notably, treatment with the metabolism-enhancing agent levocarnitine and SERCA2a Activator 1 was shown to effectively ameliorate the energy metabolism of NEXN⁻/⁻ hiPSCs. In a separate study, Zhang et al. [134] reported that overexpression of the long non-coding RNA (lncRNA) ZFAS1 in otherwise healthy mice induced cardiac dysfunction analogous to that observed in a murine model of myocardial infarction. At the molecular level, they revealed that ZFAS1 directly binds to the SERCA2a protein, thereby suppressing both its activity and expression. Critically, knockdown of ZFAS1 reversed the inhibitory effect of this lncRNA on SERCA2a function. Unlike lncRNAs, Pan et al. [135] performed transcriptome analysis following circRYR2 knockdown and found that this intervention led to decreased levels of SERCA2 and calsequestrin (CSQ), as well as altered phosphorylation of PLN. However, overexpression of circRYR2 in hiPSC-CMs did not affect the total protein abundance of SERCA2, indicating that circRYR2 modulates SERCA2 activity through mechanisms independent of altering its synthesis or degradation rates. Collectively, these observations highlight the need for further investigations into the specific molecular mechanisms underlying the interactions between circRYR2, SERCA2, and its regulatory factors (PLN and NCX1) under both physiological and pathological conditions. We would like to express our sincere gratitude to you again for your generous guidance.

While SERCA2a, 2b, and 2c are described, the text could better differentiate their roles in vascular tissues. Add a table comparing isoform expression patterns, affinities, and CVD implications to improve visualization

A4: We sincerely appreciate your valuable comments! We fully agree with your comments. We added a table at line 169 to compare the expression patterns, affinities and CVD effects of the isomers. We would like to express our sincere gratitude to you again for your generous guidance.

The heart failure section focuses on reduced SERCA2a expression (30-40%), but recent 2025 reviews highlight conflicting data in HFpEF (heart failure with preserved ejection fraction), where SERCA2a levels may not always decrease. Discuss this variability and potential mechanisms to provide a balanced view.

A5: We sincerely appreciate your valuable comments! We fully agree with your comments. We have made supplementary explanations regarding the relevant content in lines 394 to 400: However, emerging evidence [93], highlights significant variability in SERCA2a expression in heart failure with preserved ejection fraction (HFpEF), which accounts for 50% or more of all HF cases. Unlike the consistent downregulation in HFrEF, SERCA2a protein and mRNA levels in HFpEF patients are not uniformly decreased—some studies report no significant change compared to healthy controls, while a subset show mild upregulation or focal reduction in specific myocardial regions [94]. This variability challenges the traditional view that SERCA2a downregulation is a universal hallmark of HF and reflects the heterogeneous pathophysiology of HFpEF.

We would like to express our sincere gratitude to you again for your generous guidance.

Figure 1 (overview of advances) is a helpful timeline, but ensure all milestones (e.g., 2023-2025 entries like SERCA2a activation in obesity-related dysfunction) are updated. Also, cross-reference figures more explicitly in the text, and verify that all citations are complete and accessible.

A6: We sincerely appreciate your valuable comments! We fully agree with your comments. We have updated the table in Figure 1. We would like to express our sincere gratitude to you again for your generous guidance.

The style of references should be carefully revised to meet the guidelines of Biomolecules.

A7: We sincerely appreciate your valuable comments! We fully agree with your comments. We have uniformly revised the format of the references. We would like to express our sincere gratitude to you again for your generous guidance.

Reviewer 5 Report

Comments and Suggestions for Authors

I have enjoyed reading and evaluating this timely and quite comprehensive review manuscript concerning the functional roles of SERCA2, a calcium pump, in selected cardiovascular diseases. As presented it is informative but incomplete and therefore in need of supplementation, clarification and removal of redundant sections as described below. My suggestions include the following:

Although the text is extensive and quite comprehensive, key primary References have been omitted. Three examples are:

In the section concerning small peptide modulators of SERCA2, the original work of the Olson group needs to be cited and described much more clearly. Two papers that need to be described are:

Newly Discovered Micropeptide Regulators of SERCA Form Oligomers but Bind to the Pump as Monomers.Singh DR, Dalton MP, Cho EE, Pribadi MP, Zak TJ, Šeflová J, Makarewich CA, Olson EN, Robia SL. J Mol Biol. 2019 Nov 8;431(22):4429-4443. doi: 10.1016/j.jmb.2019.07.037. Epub 2019 Aug 23. PMID: 31449798

Widespread control of calcium signaling by a family of SERCA-inhibiting micropeptides.Anderson DM, Makarewich CA, Anderson KM, Shelton JM, Bezprozvannaya S, Bassel-Duby R, Olson EN. Sci Signal. 2016 Dec 6;9(457):ra119. doi: 10.1126/scisignal.aaj1460. PMID: 27923914

Similarly, and equally importantly, the text concerning the roles of SERCA2 in diabetic cardiomyopathy need to be more clearly and convincingly reported by specific citations to literature from the Dillmann group and the Howarth group. Key papers that I have in mind are listed below.

Diabetic Cardiomyopathy. Dillmann WH. Circ Res. 2019 Apr 12;124(8):1160-1162. doi: 10.1161/CIRCRESAHA.118.314665. PMID: 30973809.

Excess protein O-GlcNAcylation and the progression of diabetic cardiomyopathy. Fricovsky ES, Suarez J, Ihm SH, Scott BT, Suarez-Ramirez JA, Banerjee I, Torres-Gonzalez M, Wang H, Ellrott I, Maya-Ramos L, Villarreal F, Dillmann WH. Am J Physiol Regul Integr Comp Physiol. 2012 Oct 1;303(7):R689-99. doi: 10.1152/ajpregu.00548.2011. Epub 2012 Aug 8. PMID: 22874425

Alterations in Energy Metabolism, Mitochondrial Function and Redox Homeostasis in GK Diabetic Rat Tissues Treated with Aspirin. John A, Amiri L, Shafarin J, Tariq S, Adeghate E, Howarth FC, Raza H. Life (Basel). 2022 Jan 12;12(1):104. doi: 10.3390/life12010104. PMID: 35054496

Effects of obesity and diabesity on heart rhythm in the Zucker rat.Sultan A, Jacobson M, Adeghate E, Oulhaj A, Shafiullah M, Qureshi A, Howarth FC. Clin Exp Pharmacol Physiol. 2021 May;48(5):735-747. doi: 10.1111/1440-1681.13473. Epub 2021 Feb 20. PMID: 33609055

Calcium Signaling in the Ventricular Myocardium of the Goto-Kakizaki Type 2 Diabetic Rat.Al Kury L, Smail M, Qureshi MA, Sydorenko V, Shmygol A, Oz M, Singh J, Howarth FC. J Diabetes Res. 2018 Apr 10;2018:2974304. doi: 10.1155/2018/2974304. eCollection 2018. PMID: 29850600

In addition, when revising your manuscript it is important to bring out two functional principles that the present text fails to develop. The first is whether or not hydrogen ion fluxes are coupled to pump-mediated calcium fluxes in the context of SERCA2 function. Second, you make little or no mention of the ways in which intracellular calcium homeostasis and SERCA2 function are relevant to atrial and/or ventricular rhythm disturbances in the settings of any of the cardiovascular diseases that you have chosen to describe.

Your manuscript is quite clear and is logically presented. However, it is lengthy and part of the reason for that is redundancy. Please review the sections between lines 97 and 101, as well as lines 331 to 337 to remove redundancy. In doing so please choose an alternate term for your expression 'myocardial contraction/diastole'. This is an unconventional term and its meaning is ambiguous.

Finally, most of your review is quite clear but a lengthy and key section is not. Attached find this section with my handwritten suggestions for additions, clarifications and overall improvements.

Any papers recommended in the report are for reference only. They are not mandatory. You may cite and reference other papers related to this topic.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Adequate but could/must be improved as described above and outlined in the attachment.

Author Response

Q1: Although the text is extensive and quite comprehensive, key primary References have been omitted. Three examples are:

In the section concerning small peptide modulators of SERCA2, the original work of the Olson group needs to be cited and described much more clearly. Two papers that need to be described are:

A1: We sincerely appreciate your valuable comments! Thank you very much for the literature you provided, which has supplemented our manuscript. We have added the corresponding content in lines 308-319 and cited the literature you provided：Additionally, the Olson group [80] performed a bioinformatics screen of the mouse genome to identify open reading frames (ORFs) potentially containing SERCA-binding motifs homologous to those of myoregulin (MLN),P LN, and SLN. This screen led to the discovery of two genes encoding uncharacterized transmembrane micropeptides, both harboring SERCA-binding motifs, which were ultimately named endoregulin (ELN) and another regulator (ALN), respectively. Subsequently, the Olson group [81] further evaluated the regulatory effects of multiple micropeptides—including PLB, SLN, ELN, dwarf open reading frame (DWORF), MLN, and ALN—on SERCA. These analyses employed co-immunoprecipitation and fluorescence resonance energy transfer (FRET) to quantify micropeptide oligomerization and SERCA binding. Results showed that all micropeptides, except DWORF, reduced the apparent affinity of SERCA for Ca²⁺. Interestingly, DWORF exhibits unique oligomerization/SERCA-binding properties: unlike other SERCA-interacting micropeptides that exert inhibitory effects, DWORF does not suppress SERCA activity upon binding [82]; instead, it promotes Ca²⁺ uptake by competitively displacing PLB [83], which stands in stark contrast to the inhibitory functions of other SERCA-binding micropeptides.

Q2: Similarly, and equally importantly, the text concerning the roles of SERCA2 in diabetic cardiomyopathy need to be more clearly and convincingly reported by specific citations to literature from the Dillmann group and the Howarth group. Key papers that I have in mind are listed below.

Diabetic Cardiomyopathy. Dillmann WH. Circ Res. 2019 Apr 12;124(8):1160-1162. doi: 10.1161/CIRCRESAHA.118.314665. PMID: 30973809.

A2: We sincerely appreciate your valuable comments! Thank you very much for the literature you provided, which has supplemented our manuscript. We have added the corresponding content in lines 515-521 and cited the literature you provided：The Dillmann group [117] systematically investigated the temporal changes in the expression levels of SERCA2a and PLN in a mouse model of type 2 diabetes mellitus (DM2). At 4 months after DM2 induction, SERCA2a protein levels were significantly decreased, and this reduction was associated with a lowered ratio of phosphorylated PLN (p-PLN) to total PLN (p-PLN/PLN). Additionally, PLN is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and the level of PLN O-GlcNAcylation was markedly elevated in DM2 mice. The study further revealed an inverse correlation between p-PLN levels and O-GlcNAcylated PLN levels in DM2 mice. O-GlcNAcylation was shown to interfere with the phosphorylation of PLN, leading to the accumulation of non-phosphorylated PLN, which in turn exacerbates systolic dysfunction [118].

Q3: In addition, when revising your manuscript it is important to bring out two functional principles that the present text fails to develop. The first is whether or not hydrogen ion fluxes are coupled to pump-mediated calcium fluxes in the context of SERCA2 function. Second, you make little or no mention of the ways in which intracellular calcium homeostasis and SERCA2 function are relevant to atrial and/or ventricular rhythm disturbances in the settings of any of the cardiovascular diseases that you have chosen to describe.

A3: We sincerely appreciate your valuable comments!These suggestions are of great significance in guiding the improvement of the scientific and comprehensive nature of this review. Regarding the two core scientific issues that you pointed out, namely the coupling relationship between hydrogen ion flux and calcium ion flux in the function of SERCA2, and the association between the function of SERCA2, intracellular calcium homeostasis and cardiac rhythm disorders. In lines 114-128, we described the coupling relationship between hydrogen ion flux and calcium ion flux in the function of SERCA2: In particular, in the heart, SERCA2a takes up Ca2+ and returns it to the sarcoplasmic reticulum after each contrac-tile maneuver completed by cardiomyocytes [26]. This process involves the realization of a typical “E1-E2 conforma-tional cycle”: the E1 structure of SERCA2a has a high affinity for Ca2+ and its binding site (Asp351/Asp703) is ex-posed to the sarcoplasmic reticulum. However, the E2 structure has a lower affinity for Ca2+ and is located in the lumen of the sarcoplasmic reticulum. After cardiomyocyte diastole, Ca2+ is released from troponin into the cyto-plasm and binds to E1 (the binding site is located at an aspartic acid residue), followed by phosphorylation of E1 with the involvement of ATP (Ca2+-E1-P), which results in a conformational change from E1 to E2 (Ca2+- E2-P) [27], and the E2 structure becomes an ADP-insensitive intermediate state [28]. This process allows the binding site for Ca²⁺ to be localized in the sarcoplasmic reticulum. Due to the low affinity of E2 for Ca2+, Ca2+ is released into the sarcoplasmic reticulum upon dissociation from E2, accompanied by the conversion of E2 to E1 and on to the next cycle [29]. The proper functioning of its function is essential for the systolic and diastolic cycles of the heart.

Q4: Your manuscript is quite clear and is logically presented. However, it is lengthy and part of the reason for that is redundancy. Please review the sections between lines 97 and 101, as well as lines 331 to 337 to remove redundancy. In doing so please choose an alternate term for your expression 'myocardial contraction/diastole'. This is an unconventional term and its meaning is ambiguous.

A4: We sincerely appreciate your valuable comments! We have removed the repetitive statements and corrected the incorrect expressions.

Q5: Finally, most of your review is quite clear but a lengthy and key section is not. Attached find this section with my handwritten suggestions for additions, clarifications and overall improvements.

Any papers recommended in the report are for reference only. They are not mandatory. You may cite and reference other papers related to this topic.

A5: We would like to express our sincere gratitude for your invaluable comments and suggestions. A special thank you goes to you for providing the handwritten revision notes for our manuscript.In this revised version, all the modifications have been highlighted in red to facilitate your quick and efficient review. Your professional guidance has significantly enhanced the scientific rigor and accuracy of our manuscript. We would like to extend our deepest appreciation to you once again!

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have sufficiently addressed the majority of my comments.

Minor:

Please replace the term "irreversible oxidative inactivation" used throughout the text with the appropriate chemical/biochemical terminology (i.e., "irreversible oxidation"). (Activation/inactivation can be used for an enzyme, but not for an amino acid or amino acid residue of an enzyme.). Also, the wording in Fig. 3 ("SERCA2 Cys674 inactivated" etc.) should be edited.

Author Response

Q1: Please replace the term "irreversible oxidative inactivation" used throughout the text with the appropriate chemical/biochemical terminology (i.e., "irreversible oxidation"). (Activation/inactivation can be used for an enzyme, but not for an amino acid or amino acid residue of an enzyme.). Also, the wording in Fig. 3 ("SERCA2 Cys674 inactivated" etc.) should be edited. A1: We sincerely appreciate your valuable comments! We are extremely grateful for your contribution to the professional content of our manuscript. We have replaced the phrase "irreversible oxidative inactivation" with "irreversible oxidation" wherever it appears in the manuscript, and we have also made the corresponding revision to the relevant description in Figure 3.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have addressed all my comments. It is now ready for publication in Biomolecules.

Author Response

We sincerely appreciate your thorough review and constructive feedback on our manuscript. It is with great pleasure that we note your confirmation that all your comments have been properly addressed. We are truly grateful for your professional guidance throughout the revision process, which has significantly enhanced the quality and rigor of our work. Thank you again for your time and efforts dedicated to evaluating our manuscript.

Reviewer 5 Report

Comments and Suggestions for Authors

Thank you for considering my comments and adding appropriate responses to the text of your R1 manuscript.

Author Response