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  1. Evaluation Summary:

    This manuscript is of interest to cardiovascular and developmental biologists as it describes Dnajb6 as a novel gene linked with Sick Sinus Syndrome. The claims are mostly supported by observations using zebrafish dnajb6b trap line and Dnajb6 heterozygous mouse models. However, the paper would be strengthened be clarification of some experimental aspects and a discussion of the potential connection of DNAJB6 to the Sick Sinus Syndrome in humans.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    The authors took advantage of an existing protein-trap resource in zebrafish to identify genes important for normal pacemaker function in adults. They generated a collection of lines with mutation in genes that expressed at reasonably high levels in the heart and assess their ECG. They identified 3 candidates with increased incidence of sinus arrest and focused on validation of dnajb6b. The dnjb6b mutant fish display other defects including enhanced response to atropine and carbacol and bradycardia. They show that dnajb6b is expressed in a subset of cells in the sinus node in zebrafish. In mouse sinus node, DNAJB6 expressing cells have low expression of TBX3 and its target HCN4. In addition, Dnajb6b+/- mice also display similar phenotypes. Analysis of pacemaker function in ex vivo mouse hearts by high-resolution fluorescent optical mapping of action potentials revealed that the number of leading pacemakers in Dnajb6b+/- hearts is decreased in the sinus node, with a concomitant increase in the auxiliary pacemakers. RNAseq analysis of the right atrial tissues detected expression changes in ion channels and genes involved in Ca2+ handling and Wnt signaling. Overall, the results support the conclusion that DNAJB6 is important for proper sinus node function, thus adding it to the short list of sick sinus syndrome genes. However, the manuscript has several weaknesses.

    Weakness
    The manuscript does not address the mechanism by which decreased DNAJB6B causes sick sinus syndrome. For example, it is unknown if DNAJB6B functions cell autonomously or non-cell autonomously in the sinus node. The RNAseq analysis identified changes in ion channels in the right atrial tissues of 1-year old mice, cellular electrophysiology of the sinus node cells was not assessed.

    The manuscript does not address why the zebrafish homozygous mutants are adult viable while the mouse homozygotes are embryonic lethal. The insertion of the GBT411 disrupt dnajb6b(L) but not dnajb6b(S), while the mouse mutation deletes the entire gene. Does this difference partially explain the difference?

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  3. Reviewer #2 (Public Review):

    In this manuscript, the authors expand upon previous work describing development of a protein trap library made with the gene-break transposon. This library was screened to identify lines displaying gene trap expression in the heart (zebrafish insertional cardiac mutant collection). A pilot screen of these lines using adult ECG phenotypes identifies dnajb6b as a new gene important for cardiac rhythm. Using the GBT/dnajb6b zebrafish line, Ding et al. find a proportion of aged homozygous mutant fish (1.5-2 years) present sinus arrest episodes and reduced heart rate. Treating GBT411/dnajb6b mutant adults with compounds revealed aberrant responses to autonomic stimuli, and sinus arrest episodes were induced following verapamil exposure, providing evidence that GBT411/dnajb6b as an arrhythmia mutant. This conclusion could be better supported by presenting specific ECG parameters to characterize the conduction defect more thoroughly. The authors then report that Dnajb6+/- adult mice recapitulate some of the phenotypes observed in zebrafish, including sinus arrest and AV blocks, as well as impaired (although different) responses to autonomic stimuli. The authors describe that these are features of sick sinus syndrome in the absence of cardiomyopathy phenotypes in either the zebrafish or mouse lines. However, overall cardiac morphology is not well described for either the GBT411/dnajb6b or Dnajb6+/- models.

    To further support a role for Dnajb6 in sinoatrial node dysfunction, the authors performed optical mapping of action potentials from isolated mouse atrial tissue. These data reveal that Dnajb6+/- cultures exhibit ectopic pacemakers outside of the sinoatrial node, including within the atrial wall and inter-atrial septum. These data also show prolongation of SAN recovery time at baseline and following autonomic stimulation, further suggesting SAN dysfunction. RNA-sequencing experiments of DNAjb6+/- adult right atrial tissue showed differentially expressed genes encoding Ca2+ handling related proteins, ion channels, and WNT pathway related proteins. As these genes are involved in the cardiac conduction system, the authors suggest these pathways as molecular mechanisms underlying SSS phenotypes in Dnajb6 models.

    Sick sinus syndrome is a relatively rare arrhythmia most commonly found in older populations. Therefore, it has been challenging to establish clinically relevant models and there is a limited understanding of mechanisms of SSS pathogenesis. One particular strength of this manuscript is the ECG phenotype-based forward screen of the gene-breaking transposon (GBT)-based gene trap library in aged animals. This pilot study provides proof-of-concept that this screening approach is well suited to identify regulators of cardiac function in adults and genes linked to adult diseases like SSS.

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  4. Reviewer #3 (Public Review):

    Sick Sinus Syndrome (SSS) is a heart rhythm disorder that often occurs in the older population. The underpinning mechanism of SSS is largely unknown. The Xu lab has previously reported the zebrafish insertion cardiac (ZIC) mutant collection that consists of 35 gene trap lines with cardiac expression. In this manuscript, the authors employed electrocardiography to screen the ZIC collection and found that loss of function of cyth3a, vapal and dnajb6b led to cardiac defects resembling SSS. They then performed more in-depth analyses to examine how dnajb6 loss would impact the heart in both zebrafish and mouse models. The authors made several interesting observations. The authors showed that cardiac cells with high HCN4 and Tbx3 expression have relatively low dnajb6 expression, suggesting differential transcriptional regulations of these genes in the heart. Electrophysiology and optical mapping analyses revealed potential defects in pacemaker activities in the absence of dnajb6 and bulk RNA-seq analysis detected altered expression of multiple ion channels and wnt signaling genes. While these data support a link between dnajb6 and SSS, the manuscript would be greatly strengthened with additional quantitative measurements and mechanistic studies.

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