PLoS ONE
Public Library of Science
Aging-associated sinus arrest and sick sinus syndrome in adult zebrafish
Volume: 15, Issue: 5
DOI 10.1371/journal.pone.0232457
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### Notes

Abstract

Because of its powerful genetics, the adult zebrafish has been increasingly used for studying cardiovascular diseases. Considering its heart rate of ~100 beats per minute at ambient temperature, which is very close to human, we assessed the use of this vertebrate animal for modeling heart rhythm disorders such as sinus arrest (SA) and sick sinus syndrome (SSS). We firstly optimized a protocol to measure electrocardiogram in adult zebrafish. We determined the location of the probes, implemented an open-chest microsurgery procedure, measured the effects of temperature, and determined appropriate anesthesia dose and time. We then proposed an PP interval of more than 1.5 seconds as an arbitrary criterion to define an SA episode in an adult fish at ambient temperature, based on comparison between the current definition of an SA episode in humans and our studies of candidate SA episodes in aged wild-type fish and Tg(SCN5A-D1275N) fish (a fish model for inherited SSS). With this criterion, a subpopulation of about 5% wild-type fish can be considered to have SA episodes, and this percentage significantly increases to about 25% in 3-year-old fish. In response to atropine, this subpopulation has both common SSS phenotypic traits that are shared with the Tg(SCN5A-D1275N) model, such as bradycardia; and unique SSS phenotypic traits, such as increased QRS/P ratio and chronotropic incompetence. In summary, this study defined baseline SA and SSS in adult zebrafish and underscored use of the zebrafish as an alternative model to study aging-associated SSS.

Yan, Li, Bu, Jiao, Zhang, Le, Cao, Li, Ding, Xu, and Barbuti: Aging-associated sinus arrest and sick sinus syndrome in adult zebrafish

## 1. Introduction

A mammalian heart strictly controls the rhythm of the beat to meet the demand of the body for circulation. Heart rhythm is primarily initiated by the automatic beating of pacemaker cells located in the sinoatrial node (SAN), a specialized area in the upper right chamber of a mammalian heart. The initial innate electrical potential transmits from the SAN to the atrioventricular node (AVN) and finally passes to the His-Purkinje system. This well-controlled rhythmic contraction is modulated both positively by sympathetic nerves and negatively by parasympathetic nerves[1]. Sick sinus syndrome (SSS) is one type of heart rhythm disorder that affects the sinoatrial node; the disorder is also called sinus node dysfunction (SND) [2]. The causes of SSS can be divided into both intrinsic and extrinsic factors that disrupt the SAN function. Intrinsic causes include age-related degenerative fibrosis, ion channel dysfunction, and remodeling of the SAN. Extrinsic factors include the use of certain pharmacologic agents, metabolic disturbances, and autonomic dysfunctions that exacerbate SSS. A critical index for diagnosing SSS is sinus arrest (SA), which can be determined with surface electrocardiography (ECG). In humans, a PP or RR interval more than 1.6 seconds is considered a candidate episode for SA, more than 2.0 seconds is widely accepted as a definitive SA episode [3], and more than 3.0 seconds is considered a criterion for implanting a pacemaker [4]. Other phenotypic traits for SSS include sinus bradycardia, sinoatrial exit block, tachycardia-bradycardia syndrome, decreased P amplitude, increased QRS/P ratio, dizziness, and syncope [510]. SSS is the major reason for installation of a pacemaker and the common cause of sudden cardiac death [11]. Aging is a well-known risk factor for SSS, and the prevalence of SSS in elderly people can reach 1 in 600 [2, 12], likely due to age-related degenerative fibrosis of the sinus node [2, 13]. Although genetic contributions to SSS have been recognized, only a few genetic factors have been identified. Sodium voltage-gated channel alpha subunit 5 (SCN5A) and hyperpolarization-activated cyclic nucleotide-gated channels 4 (HCN4 ) are two well-established disease causative genes for SSS [14, 15].

The zebrafish (Danio rerio ) is emerging as a powerful vertebrate model for studying cardiovascular diseases, largely because of its amenability to large scale genetic studies, enabling rapid discovery of new genetic factors [16]. The electric wiring system in a zebrafish heart has remarkable similarities to that in a mammalian heart. Its sinoatrial node is a ring-like structure between the sinus venosus and the atrium [17, 18], and the node seems to use pacemaking mechanisms similar to those in mammals [19]. The heart is innervated and controlled by both sympathetic and parasympathetic nerve fibers [18]. Single-channel surface ECG technology has shown that the zebrafish has heart rate (HR) and ECG patterns similar to those in humans [20]. Electrophysiologic indices, including shape and duration of action potential, resemble those in humans more than those in the mouse [16, 2123]. A spectrum of rhythmic diseases has been successfully modeled in zebrafish, including long and short QT-interval syndrome and atrial fibrillation [24]. The SCN5A gene encodes the α subunit of the cardiac sodium (Na+) channel, NaV1.5. A missense mutation, D1275N, in the SCN5A gene is associated with cardiac conduction disease, sinus node dysfunction, and arrhythmia [14]. Transgenic overexpression of SCN5A-D1275N in zebrafish led to bradycardia, conduction-system abnormalities and premature death, which could be considered a zebrafish model for inherited SSS [25].

To further establish the zebrafish as a model for heart rhythm disorders, we describe the optimization of an ECG protocol using a commercial available ECG system for adult fish system. A series of experiments led us to propose a PP interval of more than 1.5 seconds as a criterion for an SA episode in adult zebrafish. With this criterion, we found that a small percentage of wild-type (WT) fish have SA episodes even when they are young, and the percentage increases significantly during the aging process. This subpopulation of fish manifests SSS phenotypic traits, such as bradycardia, increased QRS/P ratio, aberrant response to atropine, some of which are distinct from the Tg(SCN5A-D1275N) model. In summary, our study established zebrafish as an alternative vertebrate model for studying aging-associated SA and SSS.

## 2. Materials and methods

### 2.1 Zebrafish husbandry

Adult WT zebrafish from three different strains (WIK, NIHGR, TU) were used for the study. All zebrafish were maintained in a controlled environment at a temperature of 28°C with a light-dark cycle of about 14 hours-10 hours. All animal study procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and the Mayo Clinic Institutional Animal Care and Use Committee (approved protocol number A17610).

### 2.2 Anesthesia

The anesthetic agent used in this study was tricaine, which is the only anesthetic approved for certain aquatic species by the US Food and Drug Administration and the most widely used sedative and anesthetic for zebrafish. The solution of pH 7.0-adjusted tricaine (MS-222, Sigma) at the typical anesthesia concentrations (200 mg/L, 0.02%) for zebrafish was dissolved in E3 medium (containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) and was used to immobilize the fish.

### 2.3 Microsurgery to open the pericardium sac

Microsurgery has been reported to boost and to reduce background noise of the ECG signal [26, 27]. The operations were conducted typically 1 week before ECG. The zebrafish were anesthetized with 0.02% tricaine for 3 minutes before subjected to microsurgery on a dented sponge. With use of a dissecting microscope, tweezers were used to puncture the skin on the surface of the heart and to make a small incision through the skin. The silvery epithelial layer of the hypodermis was torn gently with the tip of the tweezers to expose the heart visually. After the surgery was completed, usually within 1–2 minutes, fish were returned to tank water for recovery. After 1 week, the surface skin wound was healed, but the pericardial sac was not; as a result, the cardiac electrical current could flow through with minimal attenuation.

### 2.4 Adult zebrafish ECG recording

Unless specified, ECG recording was performed at ambient temperature which was maintained at 24°C. Before subjected to ECG recording, tanks holding adult fish were acclimated for about an hour after transferred from the circulating fish facility to the laboratory bench. When a different temperature other than ambient temperature is needed, a temperature-controlled chamber set-up was used. The temperature-controlled chamber was made by covering the ECG recording system with a foam box. The ECG machine was held on top of a heating plate controlled by a heating machine. Temperature within the chamber could range from 22°C to 32°C, as measured by a thermometer inside the chamber. ECG signals were then obtained with the ECG recording and analysis System, according to instructions (ZS-200, iWorx Systems, Inc). ECG signals were amplified and filtered at 0.5 Hz high pass and 200 Hz low pass. After being anesthetized with 0.02% tricaine for 6 minutes, the fish were transferred onto a dented sponge to stably hold them upside down. A few drops of E3 medium were added to the fish to maintain moisture on the fish surface. ECG signals were recorded for 2 minutes before the fish were transferred to E3 water for recovery.

### 2.5 Analysis of zebrafish ECG recording

A series of ECG variables, including HR, P-wave amplitude, R-wave amplitude, and PP and RR intervals were calculated with an in-house Matlab code [28]. The root mean square of the successive differences (RMSSD) [29] was used to evaluate the short-period (60 seconds) beat-to-beat variance of HR according to the following formula:

$\text{RMSSD (milliseconds)}=\sqrt{\frac{{\sum }_{i-1}^{n-1}\left(PPi+1-PPi\right)^2}{n-1}}$

Unlike the conventional route of administration in mammals, atropine was administered by gill titration because of the robust blood circulation in the zebrafish gill. The fish were anesthetized and held upside down on a dented sponge while ECG was performed. After a reliable ECG signal was obtained, 4 μg/g atropine (A0132, Sigma) was added to the gill region of the fish. The dose of the atropine was calculated as the weight of drug that is normalized by the body weight of the fish, and the duration of the atropine administration was determined experimentally.

### 2.7 Cardiac function assay

Cardiac function was quantified with a Vevo 3100 high-frequency imaging system (FUJIFILM VisualSonics, Inc) equipped with a 50-MHz linear array transducer (MX700). Zebrafish were anesthetized with 0.02% tricaine for 6 minutes and held upside down in a dented sponge. The MX700 transducer was positioned above the zebrafish to align sagittally to the heart. Images were acquired and processed with the Vevo LAB workstation. Data were acquired and processed as previously described [27]. End-diastolic volume (EDV) and end-systolic volume (ESV) were calculated using the biplane area-length formula: V = 2/3AAL*LAL, in which AAL is the ventricle area in the transverse plane (short axis) and LAL is the ventricle length in the longitudinal plane (long axis), and ejection fraction (EF) = (EDV −ESV)/EDV. For each index, measurements were obtained during 3 to 5 independent cardiac cycles per fish to determine average values. With B-mode imaging, a Doppler gate (window) was positioned downstream from the atrioventricular valve in the ventricular inflow region to interrogate inflow velocities. Pulsed-wave Doppler signals were also recorded for 3 to 5 independent cardiac cycles per fish. Signals of passive E wave (peak velocity of early ventricular filling) and active A wave (peak velocity of late ventricular filling), isovolumic contraction time (IVCT), ejection time (ET), and isovolumic relaxation time (IVRT) were measured to determine the myocardial performance index (MPI), as follows: MPI = (IVCT + IVRT) / ET.

### 2.8 Statistics

Either an unpaired 2-tailed Student t test or a nonparametric test was used to compare 2 groups, depending on whether parametric assumptions of normality and equality of variance were met. A one-way analysis of variance (or Kruskal-Wallis) test followed by a post hoc Tukey test was used to compare 3 or more groups. The χ2 test was used for rate comparison. P values less than 0.05 were considered statistically significant. Results are presented as mean±SD. All statistical analyses were conducted with GraphPad Prism 6 software (GraphPad Software).

## 3. Results

### 3.1 Reliable ECG signals can be obtained after optimizing probe location, conducting microsurgery, and minimizing effects of temperature and anesthesia

While acquiring ECG signal in adult zebrafish, we noted that different ECG patterns can be obtained when the electrodes are placed at different locations. If the 2 electrodes spanned the heart, an inverted QRS complex that is opposite to the P wave was obtained (Fig 1A, i). When the positive probe was aligned to the heart, typical P, QRS, and T waves were obtained (Fig 1A, ii). If the electrodes were moved further caudally, amplitudes of P, QRS, and T waves were attenuated (Fig 1A, iii). Thus, we used the position of the positive probe aligned to the heart for later experiments.

Fig 1
A, Different ECG waveform with distinct P, R, S, and T waves when probes were at a different location relative to the heart (red dots). We recommend aligning the positive probe to the heart (ii), whereby the ECG waveform is similar to that in humans. B, Microsurgery was conducted to open the pericardium sac and thus eliminate noise and enhance amplitude of the waveforms by about 10-fold. C, Change in heart rate with surgery. bpm indicates beats per minute; WT, wild-type.Quality of electrocardiography (ECG) signals is improved by optimizing location of the probes and microsurgery.

An adult zebrafish is able to survive microsurgery that partially opens the pericardial sac to remove the silver-white membrane [3033]. We found that the operation is also able to boost the ECG signal, as indicated by about 10-fold higher amplitudes of P, QRS, and T waves, accompanied by significantly reduced background noise (Fig 1B). Microsurgery has no significant impact on HR (Fig 1C).

The design of the ECG recording system we used allows an adult fish to be physically constrained without anesthesia for a short time, during which ECG signals with P wave and QRS complex can be documented despite a relatively noisy background caused by movement of the fish body and gills (Fig 2A). The average HR in WT fish at ambient temperature (ie, 24°C) is about 100 beats per minutes (bpm). Because the zebrafish is an ectotherm that adjusts its body temperature according to its environment [26], we built a chamber for temperature control and plotted the change of HR as a function of chamber temperature. We found that the HR could change from 80 bpm at 22°C to 180 bpm at 32°C. Interestingly, we noted a higher beat to beat variability at lower temperature, e.g. between 22°C and 26°C, which warrants further quantification and follow up studies in the future (Fig 2A and 2B). A similar nearly linear HR-temperature relationship was noted when fish were anesthetized (Fig 2B). Because the HR could increase about 10 bpm per 1°C, temperature control was needed to ensure consistent ECG data in adult fish.

Fig 2
A, Representative electrocardiogram (ECG) for a single fish without anesthesia. Chamber temperature was changed from 22°C to 32°C. B, Heart rate significantly increased with increased chamber temperature, which is independent of anesthesia. Inset shows in-house temperature-controlled chamber. C, Representative ECG for a single fish before and after anesthethia. Temperature was controlled at 28°C. Noise caused by gill movement is reduced after 6 minutes of anesthesia. D, Heart rate to the time after anesthesia. E, Schematic of protocol for conducting ECG with the iWorx system. bpm indicates beats per minute.Optimization of temperature and anesthesia for reliable ECG recording in adult zebrafish.

Anesthetization of the fish with tricaine incubation effectively reduced the background noise caused by body and gill movement, which become stable at 6 minutes after anesthesia (Fig 2C). Moreover, tricaine initially increased HR significantly, which peaked at about 90 seconds, likely due to stress response and excessive excitement of sympathetic nerves. Then the HR gradually decreased to the baseline level (Fig 2D). Also, the incidence of HR variation was higher during the first minute of ECG documentation.

Accordingly, we recommend the following protocol for obtaining a reliable ECG signal with an iWorx system: 1) open the pericardial sac and remove the silver-white membrane 7 days before ECG recording; 2) put fish into 0.02% tricaine E3 water for 6 minutes at desired temperature; and 3) one minute after a fish is held on a dented sponge when more reliable ECG signals can be obtained, record ECG signals for additional 2 minutes (Fig 2E).

### 3.2 PP interval more than 1.5 seconds can be used to define an SA episode in zebrafish

Similar to waveforms in both mice and humans, a typical zebrafish ECG waveform consists of P, QRS, and T waves that are comparable to other previously published recordings (Fig 3A) [3436]. The average HR in an adult zebrafish is about 100 bpm at ambient temperature, similar to the rate of 60 to 100 bpm in humans. In contrast, the average HR in mice is 450 to 600 bpm [34], which is significantly higher than that in humans and fish (Fig 3B). In contrast to a relatively stable HR in both humans and mice, HR variation after anesthesia was higher in adult zebrafish, as indicated by the significantly higher variation in either PP intervals or PPi / PPi+1 (ratio of 2 adjacent PP intervals) (Fig 3B–3C). Therefore, the baseline HR variation in WT fish needs to be defined; this is the foundation for modeling related human rhythmic diseases such as SA and SSS. As a control, we included Tg(SCN5A-D1275N) in our study, the first zebrafish model for inherited SSS [37].

Fig 3
A, Electrocardiographic (ECG) waveforms with distinguishable P, QRS, and T waves were similar in zebrafish, mice, and humans. B, Heart rate variation (HRV) was measured by quantifying average PP intervals every 30 beats in a representative fish, mouse, and human. HR in the zebrafish was similar to that in a human but was much higher than that in a mouse. HRV in zebrafish was larger than that in both a mouse and a human. C, HRV was measured by quantifying PPi/PPi+1 (ratio of 2 adjacent PP intervals) in a representative fish, mouse, and human. The ratio in a zebrafish was much larger than that in both a mouse and a human.Heart rate was more variable in zebrafish than in mice and humans under anesthesia.

In humans, a PP or RR interval more than 1.6 seconds is considered a candidate episode for SA, and an interval more than 2.0 seconds is widely accepted as a definitive SA episode. Given that HR is 60 to 90 bpm in humans and 100 bpm in unanesthetized adult zebrafish at ambient temperature, or a 1.33-fold difference, we deduced that an abnormal PP or RR interval in fish is 1.2 to 1.6 seconds. We measured both PP and RR intervals in 59 WT fish at 1 year old and found these two parameters are similar. Thus we used the PP interval as the parameter to determine episode for SA. Out of these 59 WT fish, three had episodes of PP intervals more than 1.2 seconds, one of which was more than 1.6 seconds. In contrast, 10 of 16 Tg(SCN5A-D1275N ) fish had episodes of PP intervals of more than 1.2 seconds, seven of which were more than 1.6 seconds (Fig 4A). We manually analyzed ECG signals for two WT fish and three Tg(SCN5A-D1275N) fish that were considered to have SA if 1.2 seconds was used as a cut-off but not if 1.6 seconds was used as a cut-off. Because of the high candidate episode number per minute in WT fish number 1 and 2 and Tg(SCN5A-D1275N) fish number 2, we adopted more than 1.5 seconds as an arbitrary criterion to define an SA episode (Fig 4B and 4C). On the basis of this criterion, 5.1% of WIK WT fish at 1 year old have SA episodes and 50.0% of Tg(SCN5A-D1275N) fish have SA episodes. Consistently, RMSSD, another index that was used to quantify HR variability, was significantly higher in Tg(SCN5A-D1275N) fish than WT fish (Fig 5A and Table 1). The average HR is significantly lower, a finding supporting that bradycardia frequently accompanies SA (Fig 4A).

Fig 4
A, Summary of electrocardiographic (ECG) data from 59 wild-type (WT) and 16 Tg(SCN5A-D1275N) fish at 1 year old. Different cut-offs of PP intervals were used to determine an episode of SA. B, Two wild-type (WT) fish and 3 Tg(SCN5A-D1275N) fish that were considered to have SA when 1.2 seconds was used as cut-off, but not if 1.6 seconds was used as cut-off, were further analyzed by reviewing the raw ECG. We considered WT fish 1 and 2 and Tg fish 2 as SA-positive, and others were not because of low frequency of long PP interval. An PP interval more than 1.5 seconds was recommended as a criterion to define SA in zebrafish. C, Representative SA episodes in a WT fish and a Tg(SCN5A-D1275N) fish. epm indicates episodes per minute.Evidence supports PP interval more than 1.5 seconds as a criterion to define a Sinus Arrest (SA) episode in adult zebrafish.
Fig 5
A, RMSSD in wild-type (WT) fish at different ages and in Tg(SCN5A-D1275N) fish. B, Quantification of P-wave amplitude in WT fish at different ages and Tg(SCN5A-D1275N) fish. C, Quantification of QRS complex amplitude in WT fish at different ages and Tg(SCN5A-D1275N) fish. D, Quantification of QRS/P ratio in WT fish at different ages and Tg(SCN5A-D1275N) fish. RMSSD and QRS/P ratio are higher, while P wave amplitude is lower, in WT-3 year subpopulation than in other younger siblings. RMSSD, P wave amplitude, QRS/P ratio in the Tg(SCN5A-D1275N) fish are higher than those in age-matched WT control. **P < .01 and ***P < .001 (one-way analysis of variance). ns indicates not significant.Root mean square of successive differences (RMSSD) increases, QRS/P ratio increases, and P-wave amplitude reduces during the aging process.
Table 1
Heart rates and SA incidence comparison in WT and Tg(SCN5A-D1275N) transgenic fish.
Fish TypeAgeNAverage HR, bpmPercentage of Fish With SA (No. of Fish)SA frequency epm
WT0.5y34103.5±18.8*5.9(2)**1.8±0.3
1y59103.3±15.7*5.1(3)**2.5±1.2
2y32103.6±14.2*6.3(2)**1.5±0.5
3y884.6±22.525.0(2)3.0±2.0
Total133101.6±18.46.8(9)2.2±1.3
Tg(SCN5A-D1275N)1y1673.8±14.9###50.0(8) ###1.9±1.1
SA, sinus arrest. epm: episode per minute. HR, heart rate. bpm, beat per minute.
*: p<0.05;
**: p<0.01, Compare with 3 ys WT group;
###: p< 0.001, Compare with 1 y WT group.

### 3.3 Percentage of WT fish with episodes of SA (WTSA) significantly increases during the aging process

Because SSS is an aging-associated disorder in humans, we queried whether the percentage of fish with SA episodes increases in aged fish. We found a remarkable increase of SA in fish at 3 years old compared to 0.5–2 years old (Table 1). Consistently, RMSSD was significantly increased along the aging process (Fig 5A). The average HR decreased gradually (Table 1), as did the P-wave amplitude (Fig 5B). In contrast, QRS complex amplitude showed no obvious difference among the age groups (Fig 5C). As a consequence, the QRS/P ratio of WIK fish at 3 years old was larger than that in younger fish (Fig 5D). Because P-wave amplitude depends on the position of the probes in adult zebrafish (See Fig 1A), we recommend the use of QRS/P ratio as a more reliable index. Interestingly, the frequencies of SA episodes within the subpopulation with SA episodes did not differ significantly in WT fish of different ages or in Tg(SCN5A-D1275N) fish (Table 1).

For further investigation, we referred to the subpopulation of all WT fish with SA episodes as WTSA and their siblings without SA episodes as WTNormal. Similarly, we named the subpopulation of Tg(SCN5A-D1275N) fish with SA episodes Tg(SCN5A-D1275N)SA and their siblings without SA episodes Tg(SCN5A-D1275N)Normal. In a comparison of WTSA and WTNormal subgroups, the WTSA subgroup had significantly higher RMSSD (Fig 6A), lower HR (Fig 6B), and higher QRS/P ratio (Fig 6C). Similarly, the Tg(SCN5A-D1275N)SA subgroup had significantly higher RMSSD (Fig 6A) and lower HR (Fig 6B) than the Tg(SCN5A-D1275N)Normal subgroup, but the QRS/P ratio remained unchanged (Fig 6A).

Fig 6
A, Root mean square of successive differences (RMSSD) of wild-type (WT) and Tg(SCN5A-D1275N) fish in SA subgroup is higher than in normal fish. B, Heart rate of WT and Tg(SCN5A-D1275N) fish in SA subgroup is lower than in normal fish. C, QRS/P ratio of WT and Tg(SCN5A-D1275N) fish in SA subgroup is larger than in normal fish. **P < .01 and ***P < .001 (nonparametric tests). bpm indicates beats per minute.Obvious features of Sinus Arrest (SA) exist in SA-like subgroup compared with normal fish.

To exclude the possibility that the increased incidence of SA in the WTSA subpopulation during the aging process was specific to the WIK line, we checked TU and NIHGR fish, 2 other WT lines that are maintained in our laboratory. The incidence of SA among different WT fish lines was comparable at the same ages (Table 2). Similarly, the HRs were comparable among different lines at the same ages.

Table 2
SA incidence in different WT strains.
WT strainAgeNAverage HR, bpmPercentage of Fish With SA (No. of Fish)SA frequency epm
Wik1y59103.3±15.75.1(3)2.5±1.2
Tu1y35103.3±12.200
Wik3y884.6±22.525.0(2)3.0±2.0
NIHGI3y2381.2±16.721.7(5)2.0±3.0
SA, sinus arrest. epm: episode per minute. HR, heart rate. bpm, beat per minute.

### 3.4 WTSA fish manifest defective response to atropine, which is different from that in Tg(SCN5A-D1275N)SA fish

Given that the increased RMSSD and reduced HR could be a consequence of the presence of SA arrest episodes, additional evidence is needed to prove sinus node dysfunction in WTSA. Because chronotropic incompetence is often part of sinus node dysfunction, we assessed whether WTSA fish manifested this disability. We tested HR response to atropine, an inhibitor of parasympathetic nerves, by directly applying the solution to the gill. Application of 2 μg/g atropine solution effectively increased HR, and application of 4 μg/g or higher concentration maximized HR (Fig 7A). The maximum HR was reached in 5 minutes and maintained for about 60 minutes (Fig 7B). Therefore, we administered 4 μg/g atropine and measured HR changes at 5 minutes after application. The baseline HR of WTSA fish was lower than that in WTNormal fish (Fig 7C), and atropine administration did not increase HR in WTSA fish to the degree it did in WTNormal fish, as indicated by the significant difference in HR between the WTSA and WTNormal groups (Fig 7C). In contrast, atropine administration increased HR to the maximal level in the Tg(SCN5A-D1275N)SA fish, although its baseline HR is lower than in Tg(SCN5A-D1275N)Normal fish (Fig 7D). Together, these results indicate sinus node dysfunction in the WTSA fish, supported by the evidence that reduced atropine response occurred in the WTSA but not the Tg(SCN5A-D1275N)SA fish.

Fig 7
A, Quantification of heart rate in response to different concentrations of atropine (one-way analysis of variance). B, Heart rate response to atropine (4 μg/g) over time (one-way analysis of variance). C, Quantification of heart rate increases in response to atropine (4 μg/g) in WT fish. Heart rate in WTSA subgroup did not reach maximum, as in WTNormal siblings. D, Quantification of heart rate increases in response to atropine (4 μg/g) in Tg(SCN5A-D1275N) fish. Heart rate in fish with SA episodes reached maximal in their siblings. **P < .01 and ***P < .001 (non-parametric tests). ns indicates not significant.Chronotropic incompetence in subpopulation of Wild-Type (WT) fish with Sinus Arrest (SA) but not Tg(SCN5A-D1275N) fish.

### 3.5 WTSA fish manifest minor cardiac dysfunction

We determined whether WTSA fish hearts manifest any abnormal cardiac structural or functional changes. On high-frequency echocardiography on 9 WTSA and 10 WTNormal fish, there were no significant changes in ejection fraction (EF), end-diastolic volume (EDV)/body weight, end-diastolic volume (EDV)/body weight, ratio between the early (E) ventricular filling velocity and late (A) ventricular filling velocity (E/A ratio), or myocardium performance index (MPI) (Fig 8), but a significantly reduced E wave amplitude and A wave amplitude in WTSA fish suggested minor cardiac dysfunction (Fig 8F and 8G).

Fig 8
A, Representative B mode echocardiogram of a zebrafish heart at end-diastole (top left) and end-systole (top right) and pulsed-wave Doppler images for quantification of E wave, A wave, IVCT, ET, and IVRT. B-G, Quantification of cardiac function indices between WTNormal and WTSA siblings. *P < .05 (Student t test). A indicates peak velocity of late ventricular filling; BW, body weight; E, peak velocity of early ventricular filling; EDV, end-diastolic volume; EF, ejection fraction; ESV, end-systolic volume; ET, ejection time; IVCT, isovolumic contraction time; IVRT, isovolumic relaxation time; LAX, long axis; MPI, myocardial performance index; ns, not significant; SAX, short axis; WTSA, wild-type fish with SA; WTNormal, wild-type fish without SA.Cardiac function remains unchanged between wild-type fish with (WTSA) and without (WTNormal) sinus arrest.

## 4. Discussion

### 4.1 Reliable ECG signals can be obtained from adult zebrafish

Compared with existing zebrafish ECG systems that use needle probes invasively inserted into the zebrafish dermis to capture the signal [20, 33, 38, 39], here, we deployed non-invasive electrodes that were applied to the surface of the heart using the commercially available iWORX ECG recording system. We mainly focus on P waves and QRS waves that are sufficient to define SSS. Notably, our methods have not been optimized to reliably monitor T wave. We determined the best position of these probes for generating ECG patterns that resemble those from humans and found that opening the pericardial sac is effective for enhancing the signal and reducing background noise. High-resolution, low-noise single-channel ECG signals can be reliably obtained from more than 90% of fish. A unique application of the ECG recording system we used here does also allow us to document ECG from awake fish, because it is possible to physically constrain the fish and prevent body movement for a short time. Nevertheless, we still prefer to anesthetize the zebrafish to obtain more stable and clearer ECG signals.

To reduce experimental variation of the HR, we studied the effect of temperature and noted a nearly linear relationship: an increment in temperature of 1°C results in an increment of about 10 bpm in zebrafish. As a consequence, HR at 32°C could be double in the same fish at 22°C. Therefore, strictly controlled temperature is essential for obtaining reliable ECGs in adult zebrafish [35, 36]. Because a zebrafish facility is typically operated at about 28.5°C and the room temperature when conducting ECG is usually about 24°C, we recommend a 1-hour acclimation step before conducting ECG, which could improve data consistency. Of note, because our in-house temperature-controlled chamber might not be ideal, a better temperature-controlled chamber, preferably as a commercially available product, is needed. We did not consider the contribution of evaporation on the body surface to body temperature, which can be considerable when a fish is out of water. An alternative solution is to document ECG signals when a fish is kept within the water, as enabled by ECG jacket technology [40].

Prompted by previous studies that showed significant effects of anesthesia on cardiac function and rhythm [41, 42], we determined the dynamics using the iWorx system. We decided on a 6-minute sedation protocol that ensures deep sleep, as indicated by the effective halt of gills and body movement. Our series of optimization protocols enabled us to obtain reliable ECG signals with consistent results.

### 4.2 Zebrafish can be used to model aging-associated SSS

On the basis of our studies of abnormal heart beating in WT and Tg(SCN5A-D1275N) fish, we recommend an PP interval of more than 1.5 seconds as a standard to define an SA episode in adult zebrafish. The use of this arbitrary standard enabled us to quantify SA with software and may make future large-scale analysis of ECG data possible. Given the dramatic effect of body temperature on HR, the use of an absolute PP interval as a criterion must be restricted to a fixed temperature. The criterion needs to be adjusted when ECG is done at temperatures significantly different from 24°C. Future studies are warranted to determine whether the relative index (ie, the ratio of PP intervals between the 2 neighboring intervals) is a better index than the absolute PP interval to define SA in adult zebrafish. Because the P wave is somewhat variable in a zebrafish ECG, extra cautions need to be taken when an adult fish manifests atrioventricular block that could disengage P waves and R waves.

The establishment of a reliable ECG platform and of a criterion to define an SA episode in an adult zebrafish enabled us to uncover aging-associated SSS in this animal. The percentage of WT fish with SA episodes can be as high as 25% when fish are 3 years old. This subpopulation of WT fish with SA episodes did have other SSS phenotypic traits that are shared with the Tg(SCN5A-D1275N) model, a known transgenic fish model for inherited SSS. Bradycardia was found in both models, a finding supporting sinus node dysfunction. However, aging-associated SSS has important differences from the Tg(SCN5A-D1275N) model. First, this subpopulation of WT fish has reduced P amplitude and increased QRS/P. Second, this subpopulation of WT fish, but not the Tg(SCN5A-D1275N) model, had compromised HR responses to atropine. The data suggested that parasympathetic nerve control of the sinus node is damaged in aging-associated SSS, but not in the transgenic model. These phenotypic differences must reflect different molecular natures of these two SSS models. Similar to aged humans with SSS, fibrosis in the sinus node contributes to the pathogenesis [2, 8], which might explain the increased QRS/P ratio [43]. In contrast, SCN5A is more prominently expressed in the peri-node region, which results in SSS via a different mechanism [44].

### 4.3 A small subpopulation of young WT fish has SA episodes

A notable finding of the study is the identification of a small population of young WT fish that have episodes of SA, when we used an PP interval of more than 1.5 seconds as a standard. Even after we reduce the influence of many confounding factors such as temperature and anesthesia, we still noted a higher variation of HR in adult zebrafish. It is plausible that, compared with mammals, fish lack a certain functional mechanism that ensures stringent control of automacity of heart beating. Whether genetic heterogeneity in the fish WT lines contributes to this phenomenon remains to be clarified. The higher basic incidence in WIK fish than in TU fish tends to support this idea. Possibly, WIK fish have more SSS-related sequence variants than TU fish.

In summary, our data established aging-associated SSS and SA models in adult zebrafish, laying a foundation for discovering underlying genetic factors. A large number of zebrafish mutants have already been generated with either mutagenesis screening or genome editing technology, some of which might affect heart rhythm. The identification of a small subpopulation of WT fish with SA episodes speaks to extra caution when interpreting phenotypes from these mutants. Sufficient numbers of age-matched controls are needed to confidently conclude whether the affected gene is a new genetic factor for SA or SSS.

## Acknowledgements

This work was supported by grants from National Institutes of Health (NIH) R01-HL107304, R01-HL081753, R01-GM63904 and the Mayo Foundation to XX; the Ted and Loretta Rogers Cardiovascular Career Development Award Honoring Hugh C. Smith from Mayo Clinic (FP00093430) to YD, and National Science Foundation (NSF) #1652818 and NIH R41-OD024874 to HC. We thank Beninio Gores and Kashia B. Stragey for managing the zebrafish facility; Dr. Diane Fatkin, Victor Chang Cardiac Research Institute, Australia, for sharing the Tg(SCN5A-D1275N) fish.

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25 Feb 2020

PONE-D-20-03005

Aging-Associated Sinus Arrest and Sick Sinus Syndrome in Adult Zebrafish

PLOS ONE

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5. Review Comments to the Author

Reviewer #1: Aging-Associated Sinus Arrest and Sick Sinus Syndrome in Adult Zebrafish

In this manuscript the authors aim to define Zebrafish as a new model for aging-associated Sick Sinus Syndrome. They first describe an optimization procedure for measuring ECG signals in anesthetized Zebrafish; they define a time threshold of PP interval for the determination of Sinus arrest episodes in Zebrafish; they evaluate the quantity of SA episodes during the aging process and they show a higher incidence in old fishes; they finally show a difference in the response to Atropine in young and old fishes.

Here are some points to take in consideration:

Major points:

INTRODUCTION

Lines 43-48: please give more details about the sinoatrial node (SAN), intrinsic heart rate and the autonomic nervous system.

Before introducing the index for diagnosis of SSS, the possible causes of SSS should be mentioned , like fibrosis, remodeling of the SAN and ion channels dysfunction.

Lines 84-85: the authors didn’t mention these SSS phenotypic traits before, but they just mentioned PP interval. It’s not clear if they are just present in the Zebrafish model or how they relate to humans. Please explain these indexes and add citations.

METHODS

Line 99: at which pH?

Line 106: is this the time for induction of anesthesia? How long does this anesthesia last?

RESULTS

Lines 172-178: the authors state they optimized the recordings by finding the best position for the ECG electrodes. This investigation was already done by other authors (Crowcombe 2016; Zhao 2019). In this previous work the T wave was positive, while in this work it’s not clear. It would be nice if the authors could identify more efficiently the T wave and explain in which way their findings represent an improvement.

Line 197: Why do you have an increase in HR after injection of anesthesia? Is it just due to stress induced by the injection? Please comment.

Line 203: in the methods they say they leave the fish for 3 minutes in the E3 water with Tricaine; here it seems they leave them for 6 minutes. It’s confusing. It would be better if in the methods they could explain better the protocol they use and how Tricaine works.

Line 204: the recordings are performed 7 minutes after induction of anesthesia. Is this sufficient to reach the steady state after the stress associated with the injection procedure? From the figure it seems that after 6 minutes HR is still decreasing. And, again, for how long does the anesthesia last?

Lines 212-213: are they comparing HR in normal conditions or under anesthesia? From the manuscripts it’s not clear.

Line 244: is it the P-wave amplitude a safe parameters to consider? As the authors mentioned previously, a movement in the positioning of the electrodes could change the amplitude of the waves; this could influence the results.

Lines 250-251: it’s not clear if they now call WTSA the 3 year old-fishes of or all the WT fishes with SA.

Lines 254-255: The higher RMSSD and the lower HR could just be a consequence of the presence of SA arrest episodes that are included in the calculation of the means of these parameters.

How do the authors consider regulation by the autonomic system?

Lines 265-266: Please calculate intrinsic heart rates of 1 year-old and 3 year-old fishes. And then it would be possible to use this parameter as a starting point to check responses to atropine. Without this we don’t know the reference value for the comparison.

Lines 277-278: they state that these data indicate a sinus node dysfunction in WTSA fishes supported by the fact that response to Atropine is different from the one found in the model for SSS. This is controversial.

Line 497: this legend title is confusing. It gives a negative connotation of the use of anesthesia but throughout the work anesthesia is used because it gives better ECGs.

Figure 1 B: it is not clear which wave is the T wave. And in any case the 2 ECGs look quite different from the one they define as the correct one in Figure 1A.

Figure 2 A: between 22°C and 26°C there seems to be a higher beat to beat variability. Why? The authors don’t mention anything about it.

Figure 2 B: please comment the high difference between heart rate in presence or absence of Tricaine.

Figure 2C: in the methods the authors say that they anesthetize the mice for 3 minutes and then move to the chamber for recordings. How did they do the recordings after 1.5 minutes? Is the induction of anesthesia enough? Or probably they did the measurements 1.5 minutes after the 3 minutes of incubation with Tricaine. It’s not completely clear.

Figure 3: it is not clear whether they are comparing Zebrafish under anesthesia to human without. In this case it would make no sense. If it isn’t like this, please specify it.

I would also prefer to see a comparison of ECGs with some previous published recordings in Zebrafish.

Figure 5A: the variability among 3-year old fishes it’s really high. Increasing of the number of experiments would help.

DISCUSSION

Lines 295-297: the authors consider the recordings from awake fishes. They should explain why at the end they chose to record under anesthesia. From the results it seems that it is because of lower noise. But on the other hand a measure without anesthesia could have some advantages. Please discuss .

Line 334: It’s not possible to conclude from their results if it is a bradycardia due to intrinsic problems or to autonomic imbalance.

Line 349: the high HR variation could just be due to body temperature decrease during experiments.

Minor points:

Line 60: “the most prevalent genes involved in congenital SSS”.

Line 88: The authors write “Methods and Methods”. They probably mean “Materials and Methods”.

Line 167: mean ± SD.

Line 214: Figure 3B-C.

Reviewer #2: In their manuscript, Yen et al. present a thorough and carefully carried out analysis on the heart rhythm of adult zebrafish. The authors first optimized the protocol for the measurement of electrocardiogram using the commercially available iWork system. They obtained stronger and more consistent ECG signals by opening the pericardial sac and controlling temperature of the local environment. Using this improved protocol, the authors examined the heart rate of wild type fish and Tg(SCN5A-D1275N), an established zebrafish model for inherited sick sinus syndrome model. Using a criterion of PP interval > 1.5 seconds, approximately half of the Tg(SCN5A-D1275N) fish are defined as having SA episodes. More interestingly, the authors noted an increased percentage of wild type fish with SA episodes as they age. Furthermore, using the anticholinergic drug atropine, the authors demonstrate different sick sinus symptom-related traits between Tg(SCN5A-D1275N) and wild type aged fish. The authors conclude that zebrafish might serve as an alternative model for the study of aging-associated sick sinus syndrome. Overall, the manuscript is clearly written. The data are of high-quality and the improved protocol for ECG recording will help establish zebrafish as an animal model for the study of sinus dysfuntion.

1. It is surprising to note that the heart rate of adult zebrafish is quite variable based on the authors’ measurement. Could the authors comment on how their data compared to previous reports? It would also be very helpful if the authors could comment on what might contribute to the irregular heartbeat detected in their recording. The traces shown in Fig.2A illustrate that the heart rate is more irregular when recorded at lower temperatures (22 -26oC v.s. 30-32oC). Would the temperature or the surgery contribute to the high variability in heart rate?

2. The ECG analysis identified a subpopulation of wild type and Tg(SCN5A-D1275N) fish with SA episodes. Given the irregular heartbeat observed in wild type and Tg(SCN5A-D1275N) fish, it would be helpful for the authors to clarify whether WTSA and Tg(SCN3A-D1275N)SA fish consistently expressing SA episodes. Also, have the authors re-examined WTnormal and Tg(SCN3A-D1275N)normal fish? Do some of them exhibit SA when re-examined?

3. The authors showed that atropine treatment increases the heart rate of Tg(SCN3A-D1275N)SA to the level of Tg(SCN3A-D1275N)normal fish, but the heart rate of atropine-treated WTSA fish is still slower than atropine-treated WTnormal. The authors conclude that there is a reduced atropine response occurred in the WTSA but not the Tg(SCN3A-D1275N)SA fish. However, atropine treatment appears to increase the heart rate in both WTSA and WTnormal fish (Fig.7C). Please clarify.

1. Figure 4A, percentage of 3yr-old wild type fish with long PP interval should be 62.5% for PP>1.2 s, 25% for PP> 1.5 S and 25% for PP>1.6 s.

2. Page 15, line 286, it should be Figure 8 F and G.

3. Table 2 is missing.

**********

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31 Mar 2020

Reviewer #1: Aging-Associated Sinus Arrest and Sick Sinus Syndrome in Adult Zebrafish

In this manuscript the authors aim to define Zebrafish as a new model for aging-associated Sick Sinus Syndrome. They first describe an optimization procedure for measuring ECG signals in anesthetized Zebrafish; they define a time threshold of PP interval for the determination of Sinus arrest episodes in Zebrafish; they evaluate the quantity of SA episodes during the aging process and they show a higher incidence in old fishes; they finally show a difference in the response to Atropine in young and old fishes.

Here are some points to take in consideration:

Major points:

INTRODUCTION

Lines 43-48: please give more details about the sinoatrial node (SAN), intrinsic heart rate and the autonomic nervous system.

Thanks to your suggestion, we made the following changes (new lines 44-49): “Heart rhythm is primarily initiated by the automatic beating of pacemaker cells located in the sinoatrial node (SAN), a specialized area in the upper right chamber of a mammalian heart. The initial innate electrical potential transmits from the SAN to the atrioventricular node (AVN) and finally passes to the His-Purkinje system. This well-controlled rhythmic contraction is modulated both positively by sympathetic nerves and negatively by parasympathetic nerves [1]”.

Before introducing the index for diagnosis of SSS, the possible causes of SSS should be mentioned, like fibrosis, remodeling of the SAN and ion channels dysfunction.

Thanks to your suggestion, we added the following sentences to mention the possible causes of SSS (new lines 51-55): “The causes of SSS can be divided into both intrinsic and extrinsic factors that disrupt the SAN function. Intrinsic causes include age-related degenerative fibrosis, ion channel dysfunction, and remodeling of the SAN. Extrinsic factors include the use of certain pharmacologic agents, metabolic disturbances, and autonomic dysfunctions that exacerbate SSS”.

Lines 84-85: the authors didn’t mention these SSS phenotypic traits before, but they just mentioned PP interval. It’s not clear if they are just present in the Zebrafish model or how they relate to humans. Please explain these indexes and add citations.

We now add sentences to mention these phenotypic traits. We also include related citations to show these indices are presented in both Zebrafish and humans (new lines: 59-61, and 91-92)

METHODS

Line 99: at which pH?

pH 7.0. It is now added to the text (new line: 107).

Line 106: is this the time for induction of anesthesia? How long does this anesthesia last?

Yes, this is the time (3 minutes) we used to induce anesthesia before we start to operate microsurgery for opening the pericardium sac. The anesthesia usually lasted for additional 1-2 minutes, which is sufficient for us to complete the surgery. After the surgery, fish were returned to tank water for recovery. We now clarified this protocol in the text (new lines 114-115, 118-119)

RESULTS

Lines 172-178: the authors state they optimized the recordings by finding the best position for the ECG electrodes. This investigation was already done by other authors (Crowcombe 2016; Zhao 2019). In this previous work the T wave was positive, while in this work it’s not clear. It would be nice if the authors could identify more efficiently the T wave and explain in which way their findings represent an improvement.

Other published work mostly used probes that were invasively inserted into the zebrafish dermis to capture the signal. In contrast, we deployed non-invasive electrodes that were applied to the surface of the heart using the iWORX ECG recording system. In this manuscript, we mainly focus on P waves and QRS waves that are sufficient to define SSS. Our methods have not been optimized to reliably monitor T wave. Thanks to your suggestion, we added these two references, and described the limitation of our methods in the discussion section (new lines 307-312).

Line 197: Why do you have an increase in HR after injection of anesthesia? Is it just due to stress induced by the injection? Please comment.

This is an interesting discovery that we do not have full explanation. Our speculation is that the increased HR by tricaine is a consequence of either stress response or excessive excitement of sympathetic nerves. Notably, we didn’t inject tricaine to induce anesthesia. We meant “incubation” by “administration”. To avoid confusion, we now use the word “incubation”. We added our speculation to the text (new lines 207).

Line 203: in the methods they say they leave the fish for 3 minutes in the E3 water with Tricaine; here it seems they leave them for 6 minutes. It’s confusing. It would be better if in the methods they could explain better the protocol they use and how Tricaine works.

We used different anesthesia times of tricaine in different experiments. For microsurgery, we used 3 minutes of tricaine incubation. For ECG and Echo, fish need to be more deeply anesthetized; thus, we used a longer anesthesia time (6 minutes). We clarified these points in the method section (lines 114-115, 133).

Line 204: the recordings are performed 7 minutes after induction of anesthesia. Is this sufficient to reach the steady state after the stress associated with the injection procedure? From the figure it seems that after 6 minutes HR is still decreasing. And, again, for how long does the anesthesia last?

Indeed, the heart rate continued to reduce after 6 minutes of anesthesia, but not significantly. Comparing to earlier time points, we considered that fish reach relatively steady state after 6 minutes of anesthesia. Indeed, more reliable ECG signals can be recorded. The anesthesia usually lasts additional 2 minutes that enables us to obtain reliable ECG recording (see Figure 2E). We added these details to the text (new lines 216-217).

Lines 212-213: are they comparing HR in normal conditions or under anesthesia? From the manuscripts it’s not clear.

Sorry for the confusion. We meant under anesthesia. We added this premise accordingly (new line 226).

Line 244: is it the P-wave amplitude a safe parameters to consider? As the authors mentioned previously, a movement in the positioning of the electrodes could change the amplitude of the waves; this could influence the results.

We agree. We deleted statement that the P-wave amplitude alone can serve as “an index indicating a decline in sinus node function” and used only the QRS/P ratio instead as a more reliable index (new lines 257-261).

Lines 250-251: it’s not clear if they now call WTSA the 3 year old-fishes of or all the WT fishes with SA.

Sorry for the confusion. WTSA refers to all the wild-type fish with SA (new lines 264-265).

Lines 254-255: The higher RMSSD and the lower HR could just be a consequence of the presence of SA arrest episodes that are included in the calculation of the means of these parameters.

How do the authors consider regulation by the autonomic system?

We agree that there is a possibility that these two indices could be simply a consequence of the presence of SA arrest episodes. Indeed, this was the reason that prompted us to seek additional evidences by checking the autonomic system in section 3.4., which lead us to identify reduced response to atropine. We added this rationale to the text (new line 2791-281).

Lines 265-266: Please calculate intrinsic heart rates of 1 year-old and 3 year-old fishes. And then it would be possible to use this parameter as a starting point to check responses to atropine. Without this we don’t know the reference value for the comparison.

As a pilot study, we have tested different doses of atropine to identify the maximal dose that completely remove the influence of parasympathetic nerve system. We then detected uniquely reduced response in WTSA that is different from WTnormal and Tg(SCN5)SA, which strongly supports a defective SAN in WTSA. In the literature, it appears acceptable to define autonomic dysfunction in SSS by only manipulating parasympathetic nerves system (Vavetsi S et al. Europace. 2008). We agree that IHR is an interesting index to define in adult zebrafish. However, we plan to measure it in our future studies.

Vavetsi S, Nikolaou N, Tsarouhas K, Lymperopoulos G, Kouzanidis I, Kafantaris I, Antonakopoulos A, Tsitsimpikou C, Kandylas J.Consecutive administration of atropine and isoproterenol for the evaluation of asymptomatic sinus bradycardia. Europace. 2008;10(10):1176-81. doi: 10.1093/europace/eun211. Epub 2008/08/13. PubMed PMID: 18701603.

Lines 277-278: they state that these data indicate a sinus node dysfunction in WTSA fishes supported by the fact that response to Atropine is different from the one found in the model for SSS. This is controversial.

Because reduced atropine response only occurs in WTSA fish, we reasoned that sinus node dysfunction in these fish is likely characterized with aberrant parasympathetic control. By contrast, Tg(SCN5)SA fish does not manifest this phenotypic trait, suggesting a different SSS mechanism. We clarified this statement further in our discussion section (new lines 359-363).

Line 497: this legend title is confusing. It gives a negative connotation of the use of anesthesia but throughout the work anesthesia is used because it gives better ECGs.

Thanks for your suggestion. We agree and now change the legend title to “Optimization of temperature and anesthesia for reliable ECG recording in adult zebrafish” (new lines 551-552).

Figure 1 B: it is not clear which wave is the T wave. And in any case the 2 ECGs look quite different from the one they define as the correct one in Figure 1A.

We acknowledge that our current protocol is mainly opted to study P and QRS waves, but have not been optimized to reliably define the T-wave. We added this point to the text (new line 311-312).

Figure 2 A: between 22°C and 26°C there seems to be a higher beat to beat variability. Why? The authors don’t mention anything about it.

Figure 2 B: please comment the high difference between heart rate in presence or absence of Tricaine.

Thanks for pointing out this potentially highly interesting phenomenon. We now described this observation in the text and indicated that it is warranted for further quantification and further study in the future (new lines 202-204).

We now comment the high difference between heart rate in presence or absence of tricaine (new lines 209-211).

Figure 2C: in the methods the authors say that they anesthetize the mice for 3 minutes and then move to the chamber for recordings. How did they do the recordings after 1.5 minutes? Is the induction of anesthesia enough? Or probably they did the measurements 1.5 minutes after the 3 minutes of incubation with Tricaine. It’s not completely clear.

Sorry for the confusion. The times indicated in this Figure are anesthesia times that we used before we move the fish to the chamber for ECG documentation.

Indeed, 1.5 minutes seems not sufficient, as indicated in Figure 2C, and we recommend 6 minutes of anesthesia, as shown in Figure 2E.

Figure 3: it is not clear whether they are comparing Zebrafish under anesthesia to human without. In this case it would make no sense. If it isn’t like this, please specify it.

In this case, we intended compare ECG from fish, mouse and human under anesthesia. We replaced the previous ECG image of an awaken patient with a new ECG image of a patient under anesthesia. We now specify this fact in the text (new lines 560-561).

I would also prefer to see a comparison of ECGs with some previous published recordings in Zebrafish.

We added references for several previous published recordings, which stated similar HR (new lines 221-222).

Figure 5A: the variability among 3-year old fishes it’s really high. Increasing of the number of experiments would help.

We agree that the RMSSD index in 3-year old WT-wik fish is high. Unfortunately, we don’t have more WT-wik fish for this experiment. Nevertheless, the statistic power is sufficient for us to reach our conclusion.

DISCUSSION

Lines 295-297: the authors consider the recordings from awake fishes. They should explain why at the end they chose to record under anesthesia. From the results it seems that it is because of lower noise. But on the other hand a measure without anesthesia could have some advantages. Please discuss.

Thanks for this great suggestion. We now discuss the underlying rational in more detail (new lines 318-319): anesthesia affects the heart rate, but to obtain stable and clear ECG signals, zebrafish need to be fully anesthetized.

Line 334: It’s not possible to conclude from their results if it is a bradycardia due to intrinsic problems or to autonomic imbalance.

Agree. Our current data cannot completely discern these two factors. However, our atropine experiment did unveil a contribution from autonomic imbalance.

Line 349: the high HR variation could just be due to body temperature decrease during experiments.

Indeed, our manuscript aims to alert the readers on the huge impacts of temperature on ECG in adult zebrafish. We agree that a better temperature-controlled chamber is needed to further reduce HR variation. We added this point to the text and changed “eliminate” to “reduce the influence of“(new lines 371-372).

Minor points:

Line 60: “the most prevalent genes involved in congenital SSS”.

We changed to “two well-established causative genes for SSS” (new lines 67-68).

Line 88: The authors write “Methods and Methods”. They probably mean “Materials and Methods”.

Sorry for the typo. We corrected it to Materials and Methods (new line 96).

Line 167: mean ± SD.

Thank you for the correction. We edited it accordingly (new line 178).

Line 214: Figure 3B-C.

Thank you for the correction. We edited it accordingly (new lines 227).

Reviewer #2: In their manuscript, Yen et al. present a thorough and carefully carried out analysis on the heart rhythm of adult zebrafish. The authors first optimized the protocol for the measurement of electrocardiogram using the commercially available iWork system. They obtained stronger and more consistent ECG signals by opening the pericardial sac and controlling temperature of the local environment. Using this improved protocol, the authors examined the heart rate of wild type fish and Tg(SCN5A-D1275N), an established zebrafish model for inherited sick sinus syndrome model. Using a criterion of PP interval > 1.5 seconds, approximately half of the Tg(SCN5A-D1275N) fish are defined as having SA episodes. More interestingly, the authors noted an increased percentage of wild type fish with SA episodes as they age. Furthermore, using the anticholinergic drug atropine, the authors demonstrate different sick sinus symptom-related traits between Tg(SCN5A-D1275N) and wild type aged fish. The authors conclude that zebrafish might serve as an alternative model for the study of aging-associated sick sinus syndrome. Overall, the manuscript is clearly written. The data are of high-quality and the improved protocol for ECG recording will help establish zebrafish as an animal model for the study of sinus dysfuntion.

1. It is surprising to note that the heart rate of adult zebrafish is quite variable based on the authors’ measurement. Could the authors comment on how their data compared to previous reports? It would also be very helpful if the authors could comment on what might contribute to the irregular heartbeat detected in their recording. The traces shown in Fig.2A illustrate that the heart rate is more irregular when recorded at lower temperatures (22 -26oC v.s. 30-32oC). Would the temperature or the surgery contribute to the high variability in heart rate?

Indeed, a major discovery of this manuscript is to report high variation of HR in an adult zebrafish. The phenomenon was not emphasized in previously published papers, many of which might deem the phenomenon as a confounding factor. Here, we provided strong data to demonstrate that this variation is not random, needs to be considered, and is associated with aging. Our data prompts future studies to uncover underlying mechanisms (new lines 381-384).

Thanks for pointing out that the heart rate is more irregular when recorded at lower temperatures. We now described this observation in the text (lines 202-204), and indicated that this phenomenon shall be quantified and further studied as a future direction.

2. The ECG analysis identified a subpopulation of wild type and Tg(SCN5A-D1275N) fish with SA episodes. Given the irregular heartbeat observed in wild type and Tg(SCN5A-D1275N) fish, it would be helpful for the authors to clarify whether WTSA and Tg(SCN3A-D1275N)SA fish consistently expressing SA episodes. Also, have the authors re-examined WTnormal and Tg(SCN3A-D1275N)normal fish? Do some of them exhibit SA when re-examined?

Yes, we have re-examined WTSA and Tg(SCN3A-D1275N)SA fish at different times, and we did find that they manifest SA episodes consistently. It is highly possible that some WTnormal might have SA episodes, if the sensitivity is increased by monitoring ECG with a longer time frame.

3. The authors showed that atropine treatment increases the heart rate of Tg(SCN3A-D1275N)SA to the level of Tg(SCN3A-D1275N)normal fish, but the heart rate of atropine-treated WTSA fish is still slower than atropine-treated WTnormal. The authors conclude that there is a reduced atropine response occurred in the WTSA but not the Tg(SCN3A-D1275N)SA fish. However, atropine treatment appears to increase the heart rate in both WTSA and WTnormal fish (Fig.7C). Please clarify.

Yes, atropine treatment increased heart rates in both WTSA and WTnormal fish, as well as Tg(SCN3A-D1275N)SA and Tg(SCN3A-D1275N)normal fish. However, the rate of their increase is significantly different, suggesting defective autonomic response only occurs in WTSA fish.

1. Figure 4A, percentage of 3yr-old wild type fish with long PP interval should be 62.5% for PP>1.2 s, 25% for PP> 1.5 S and 25% for PP>1.6 s.

Thank you very much for pointing out these errors. We now correct them accordingly.

2. Page 15, line 286, it should be Figure 8 F and G.

Thank you very much. We now correct the typo (new line 303).

3. Table 2 is missing.

We now add back Table 2.

16 Apr 2020

Aging-Associated Sinus Arrest and Sick Sinus Syndrome in Adult Zebrafish

PONE-D-20-03005R1

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17 Apr 2020

PONE-D-20-03005R1

Aging-Associated Sinus Arrest and Sick Sinus Syndrome in Adult Zebrafish

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