Type-1 Ryanodine Receptor Plays an Important Role in Cardiac Hypertrophy and Heart Failure by Increasing Type-2 Ryanodine Receptor-Mediated Calcium Release

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Overview

This manuscript investigates the potential role of the Type-1 ryanodine receptor (RyR1) in the heart by measuring cardiac hypertrophy, cardiac function, and mechanistic changes in cardiac-specific RyR1 overexpression mice. It provides a comprehensive examination of RyR1's functional dynamics, with particular emphasis on its expression in cardiac myocytes from both animal models and human patients with various forms of cardiac hypertrophy and heart failure. The topic is compelling, and the methodologies employed are effective. I recommend this manuscript for publication, with a few suggestions for the authors:

1. As described in the manuscript, increased intracellular Ca2+ signaling is a major contributor to the development of cardiac hypertrophy. Additionally, RyR1 mRNA expression is significantly elevated in cardiac myocytes from both animal models and human patients with various forms of cardiac hypertrophy and heart failure. The author attempts to rescue the embryonic lethality of RyR2 knockout (KO) mice; however, overexpression of RyR1 (RyR1 OE) does not rescue this lethality. I am curious whether the author has examined the endogenous expression of RyR1 in cardiac-specific RyR1 overexpression mice. I suggest that the author include a discussion of the structural function and regulatory differences between endogenous RyR1 and RyR2 in the introduction and discussion sections to better highlight their distinct roles.
2. RyR2 channels are oxidized, nitrosylated, and hyperphosphorylated by PKA, resulting in “leaky” channels in failing hearts. These leaky RyR2 channels contribute to the depletion of Ca2+ from the sarcoplasmic reticulum (SR), and the leaking Ca2+ depolarizes cardiomyocytes, triggering fatal arrhythmias. Although the current investigation of the heart in RyR1-overexpressing mice is clear, I recommend also performing rhythm analysis on cardiac-specific RyR1-overexpressing mice.
3. I suggest that the author include hematoxylin-eosin (HE) staining and immunohistochemical analysis of RyR1 and RyR2 in RyR1 overexpression (OE) mice in Figure 1.
4. There may be a typographical error in line 115 and 308; please check.

Author Response

Dear Reviewer 1, We extremely appreciate your kindest evaluations of our manuscript. The comments and suggestions are all very helpful; as such, we have carefully addressed each of them, as shown below. The major changes in the text are written in blue.

Comments 1: I suggest that the author include a discussion of the structural function and regulatory differences between endogenous RyR1 and RyR2 in the introduction and discussion sections.

Responses 1: We fully agree with this excellent suggestion; thus, we have made appropriate changes to better highlight their distinct roles in the Introduction and Discussion sections.

Comments 2: Although the current investigation of the heart in RyR1-overexpressing mice is clear, I recommend also performing rhythm analysis on cardiac-specific RyR1-overexpressing mice.

Responses 2:  In response to this great recommendation, we performed the suggested analysis. The results were included in Table 2, showing the increased heart rate, QRS duration, and QT interval.

Comments 3: I suggest that the author include hematoxylin-eosin (H&E) staining and immunohistochemical analysis of RyR1 and RyR2 in RyR1 overexpression (OE) mice in Figure 1.

Responses 3:  As suggested, we did additional H&E staining. The new results were included in Figure 1, further indicating the increased cardiomyocyte cross-sectional area in RyR1 OE mice.

Comments 4: There may be a typographical error in line 115 and 308; please check.

Responses 4:  We made suitable changes, Thanks.

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, authors tried to explore a role of RYR1 and RYR2 in Cardiac pathology.

They should that RYR1 cardiac overexpressed mice develop HCM and heart failure, which is mediated by increased RYR2 calcium leak mediated by Mitochondrial ROS production. Authors have also showed that TAC mice also had RYR1 overexpression, and increased expression of HCM markers genes, similar to what observed in RYR1 overexpressed mice.

RYR1 overexpression did not rescue the RYR2 knockout lethal effect.

Comments:

I wish to see below two experiments, which may explain a bit more about this pathophysiology.

1)  You generated RYR1 overexpressed mice with RYR2 complete knock-out. But it did not rescue the RYR2 knock out lethality.

Recently, we have seen that heterozygous loss of function RYR2 leads to CRDS (Catecholamine Release Deficiency Syndrome) opposite to CPVT (due to gain of function mutation in RYR2). Have you checked the phenotypes in mice with overexpressed RYR1 and heterozygous loss of one RYR2 allele ?

2) You have mentioned that increased Calcium leak in cytoplasm happens due to RYR2 leak, inhibiting the RYR2 by Riluzole.  What happens when a cell line with RYR2 gain of function mutation is treated with Riluzole ? Does it also inhibit the RYR2 channel in such mutants ?

Author Response

Dear Reviewer 2, We extremely appreciate your kindest evaluations of our manuscript. The comments and suggestions are all very helpful; as such, we have carefully addressed each of them, as shown below. The major changes in the text are written in blue.

Comments 1: Recently, we have seen that heterozygous loss of function RyR2 leads to CRDS. Have you checked the phenotypes in mice with overexpressed RyR1 and heterozygous loss of one RyR2 allele?

Responses 1: We have just started this interesting series of experiments. Results may take several months to emerge. Nevertheless, the new findings from these experiments will be included in our next manuscript on the functional importance of RyR2 in RyR1 OE mice. In the meantime, we have added further deliberations for clarity in the Discussion section.

Comments 2: What happens when a cell line with RyR2 gain of function mutation is treated with Riluzole?

Responses 2: We believe that treatment with riluzole will produce similar inhibitory effects in a cell line with RyR2 gain of function mutation, as illustrated in Figure 8. These studies are currently underway, with results expected in a few months. Thank you very much again!

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I have no further comments. 

Author Response

Dear Academic Editor,

I am really delighted to receive your further excellent clear and specific comments about our re-revised manuscript. Herein, I have meticulously addressed each of your comments, as described below. The major changes are written in blue in the text for your convenience. Thank you very much again!

Comments 1:  The Methods should be written in a clear, objective, and past-tense format.

Responses 1:  I fully agree with this excellent comment; thus, we have made all appropriate changes. For instance, we have revised the sentence from “We also performed…” to a more standard form such as “Histological staining of mouse heart tissue was performed…”).

In addition, we have avoided interpretive or narrative phrases; for instance the phrase “thereby inspiring us to perform…” has been removed.

Comments 2: Please improve clarity and precision in the description of experimental procedures.

Responses 2: In response to this fantastic comment, we have made all necessary changes to improve clarity and precision in the description of experimental procedures. For example, we have clarified the histological analysis and specified how cardiomyocyte size was quantified (e.g., cross-sectional area).

Comments 3: The description of electrocardiographic analysis is currently too general.

Responses 3: Following this outstanding comment, we have specified which ECG parameters were measured and analyzed (e.g., heart rate, PR interval, QRS duration, QT/QTc interval, arrhythmia incidence, etc.).