Excessive exercise induces cardiac arrhythmia in a young fibromyalgia mouse model

Taiki Nakata; Atsushi Doi; Daisuke Uta; Megumu Yoshimura; Min-Chul Shin

doi:10.1371/journal.pone.0239473

. 2020 Sep 30;15(9):e0239473. doi: 10.1371/journal.pone.0239473

Excessive exercise induces cardiac arrhythmia in a young fibromyalgia mouse model

Taiki Nakata ^1,^2,^#, Atsushi Doi ^2,^3,^*,^#, Daisuke Uta ⁴, Megumu Yoshimura ⁵, Min-Chul Shin ^2,³

Editor: Etsuro Ito⁶

PMCID: PMC7526895 PMID: 32997682

Abstract

Background

Fibromyalgia patients experience cardiovascular complications in addition to musculoskeletal pain. This study aimed to investigate the cardiac effects of a prolonged shallow water gait in a fibromyalgia-induced young mouse model.

Methods

To produce a fibromyalgia mouse model, wild-type mice were administered an intraperitoneal injection of reserpine once a day for three days, and two primary experiments were performed. First, three types of gait tests were performed before and after the reserpine injections as follows: (i) 5 minutes of free gait outside the water, (ii) 1 minute of free gait in shallow warm water, and (iii) 5 minutes of free gait in shallow warm water. Second, electrocardiogram recordings were taken before and after the three gait tests. The average heart rate and heart rate irregularity scores were analyzed.

Results

Exercise-induced cardiac arrhythmia was observed at 1-minute gait in shallow water during the acute stage of induced FM in young mice. Further, both cardiac arrhythmia and a decrease in HR have occurred at 5-minute gait in shallow water at the same mice. However, this phenomenon was not observed in the wild-type mice under any test conditions.

Conclusion

Although a short-term free gait in shallow warm water may be advantageous for increasing the motor activity of FM-model mice, we should be aware of the risk of prolonged and excessive exercise-induced cardiac arrhythmia. For gait exercises in shallow water as a treatment in FM patients. We suggest a gradual increase in exercise duration may be warranted.

Introduction

Fibromyalgia (FM) is an immune system-related intractable disease with higher morbidity rates in women compared with men [1, 2]. The symptoms of FM comprise musculoskeletal pain [3], fatigue [4], sleep disorders, and mood disorders [4]. In general, medications [5, 6], psychotherapy [7], counseling [8], cognitive behavior therapy [9], self-care instructions [10, 11], and rehabilitation or therapeutic exercises [12, 13] are recommended as treatments for FM patients. Underwater walking is a therapeutic exercise that has often been utilized for gait training for patients with painful diseases, such as osteoarthritis [14], rheumatoid arthritis [15], arthroplasty [16], and FM [17–20]. Although the functional activity of antigravity muscles, such as the erector spinae, gluteus maximus, quadriceps, and triceps surae, is required for walking, underwater walking appears to decrease overload on the antigravity muscles.

To investigate effective treatments for FM patients, FM-induced animal models have been made for basic animal research. FM was initially induced by acidic saline injection [21], hydrochloric acid injection [22], or intermittent cold stress [23]. However, these animal models showed comparative recovery within two weeks. Reserpine, an anti-hypertensive medication, has recently been utilized for generating a FM animal model, and it appears to be effective in producing long-term symptoms of FM [24–26]. Reserpine blocks vesicular monoamine transporters. As a result, reserpine-injected FM-induced animals experience a depletion in monoamines in the presynaptic terminals of the central and peripheral synapses [25].

Recently, FM patients have been reported to have higher heart rates (HR) [27], impaired cardiac function [28], and a higher risk of coronary heart disease-related events [29]. Moreover, FM-induced rats were also observed to develop an increase in cardiac sympathetic events [30]. Thus, both FM patients and animals appear to experience cardiovascular complications. However, there have been no studies regarding gait exercise-induced cardiac arrhythmia in animal models.

Our study aimed to investigate whether a prolonged walk in shallow water can be used as a simple, therapeutic exercise in an animal model. Secondly, we aimed to test whether prolonged walking in water results in cardiac arrhythmia in a young FM-induced mouse model.

Material and methods

Animals

Male C57BL/6J mice (5 weeks old, 20 g, n = 39) were purchased from Kyudo, Inc. (Kumamoto, Japan) and housed at a controlled temperature (24 ± 1 ºC) and humidity (55 ± 10%) with a 12-hour light-dark cycle and freely available food and water. The Animal Care Committee of Kumamoto Health Science University approved the animal experiments. Animal experiments were conducted in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals (NIH publications No. 80–23, revised 1996).

Reserpine-injected FM model mice

To induce FM in the mice, an intraperitoneal reserpine injection (1 mL/kg) was administered to wild-type mice once a day for three days [25]. Reserpine was purchased from Nacalai Tesque (No. 30013–81, Kyoto, Japan) and dissolved in 100% glacial acetic acid (1 mg/0.05 mL) (A solution). Distilled water (1 mg/0.95 mL) was subsequently added to the A solution (B solution, 1 mg/1 mL), in which 5% glacial acetic acid was contained in the B solution. Further, the B solution was diluted to a final concentration of 0.5% acetic acid with distilled water (stock solution), which was subcutaneously injected into the mice once a day at a dosage of 10 mL/kg for three days. Therefore, one injected dose would constitute 0.3 ml of reserpine (stock solution) for a mouse with a body weight of 30 g.

Experimental design

Three sets of experiments were performed. First, before and after the reserpine injection, the weight and rectal temperature of the FM-induced mice were recorded, and the free gait inside the cage was videotaped (weight and rectal temperature, n = 6; video recordings of free gait, n = 9). Second, three types of gait tests were performed before and after the reserpine injection (pre-reserpine injection, n = 10; post-reserpine injection, n = 6). The three gait tests were as follows: (i) 5 minutes of free gait out of the water, (ii) 1 minute of free gait in shallow warm water, and (iii) 5 minutes of free gait in shallow warm water. Third, electrocardiogram (ECG) recordings (see below “Evaluation of cardiac function with three gait tests”) were obtained before and after the gait tests. In this set of experiments, ECG was performed for ten FM-induced mice because only ten mice (out of 20) were injected with reserpine. The remaining ten wild-type mice that were not injected with reserpine were used as controls. For the gait tests and the ECG recordings, the mice were first examined in the cage without water. The mice were then placed in cages with shallow water for a 1-minute gait test. After finishing the 1-minute gait test, the mice were carefully wiped with a Kim towel (60001, Nihon Paper Crecia Co, Tokyo, Japan). Two hours after finishing the 1-minute gait test, the mice performed a 5-minute gait test in shallow water. All the gait tests and ECG recordings performed before and after the generation of the FM-induced mice used the same protocol.

Body weight

The body weight of the FM-induced mice was measured using a weighing balance (Sefi 1B-1KM, Osaka, Japan). Awake mice were allowed to walk onto the weighing balance. After the mice became stationary, the body weight was recorded from the digital display of the weighing scale once it had stabilized. Body weight was measured for up to six days after the third reserpine injection (n = 6) [31].

Rectal temperature

Rectal temperature was used as a measure of body temperature in the mice (CTM-303, TERUMO, Tokyo, Japan). At first, awake mice were placed on top of the cage (KN-604, Natsume Seisakusyo Co, Tokyo, Japan); the roof of the cage was made of strong mesh-wire. Then, while the mice gripped the mesh-wire with both forelimbs, the examiner picked up their tails. A standard method of rectal temperature measurement, involving the insertion of a small-diameter temperature probe with Vaseline (Vaseline HG, Taiyo Seiyaku Co, Tokyo, Japan) into the anus at a depth >2 cm, was used. The temperature-sensitive probe was connected to equipment that indicated a digital temperature scale. Rectal temperatures were taken for six days after inducing FM in the model mice (n = 6) [31].

Evaluation of locomotive behavior using three types of gait tests

Three types of gait tests were performed. The temperature of the shallow warm water was 40–42 ºC for the 1-minute and 5-minute free gait tests. Warm water was placed inside a plastic cage (KN-601-B, Natume Co. Tokyo, Japan) at a depth of 1 cm. Subsequently, movement during the three gait tests was recorded using a digital camera (100 frames/s, TZ-35, Panasonic, Osaka, Japan) placed above the cage. While tracking the gait of the mice from above, gait distance (cm/1 or 5 minutes), maximum speed (cm/s), and average speed (cm/s) were measured. In the analysis of the gait videos, Avidemux (http://fixounet.free.fr/avidemux/), Any Video Converter (http://www.any-video-converter.com/products/for_video_free/), VirtualDub (http://www.virtualdub.org/), and ImageJ (https://fiji.sc/), which are open-source software programs, were used [37]. The videos were cut with Avidemux and edited with Any Video Converter and VirtualDub. The edited videos were then analyzed with ImageJ [37].

Evaluation of cardiac function using three gait tests

A disposable self-adhesive Ag/AgCl snap dual-electrode (#272S, NORAXON, Scottsdale, AZ, USA) was cut into two pieces and used to detect the ECG signals before and after the gait tests. The mice were kept in the prone position by gently holding the back of their neck. While a mouse was held, one part of the electrode was placed onto the palmar surface of the right forefoot as the positive electrode, and the other part of the electrode was placed onto the plantar surface of the left hind-foot as the negative electrode. Usually, clear ECG signals were recorded for at least 20 seconds, which suggested that 200–250 cycles on average were recorded from one mouse. The ECG signals were amplified using a differential amplifier (model 1700, AM-System, Sequim, WA, USA) and digitized using a digitizer (Axon DigiData 1322, Molecular Device, San Jose, CA, USA). The average HR (HR means numbers of “R” in a minute; see S1D Fig), standard deviation (SD) of the HR, and HR irregularity score were analyzed using DataView 11 (University of St Andrews, Scotland, UK) [32, 33]. The HR irregularity score was determined for each cycle using 50 ECG cycles with the formula (for consecutive cycle length values) stated below:

S n = 100 * A B S (P n - P n - 1) / P n - 1

(Sn = score of the nth cycle, Pn = period of the nth cycle, Pn-1 = period of the cycle preceding the nth cycle, ABS = the absolute value) [34, 35].

Statistical analysis

Experimental data were expressed as average ± SD. Single comparisons were conducted using the Wilcoxon’s signed-rank test and the Mann-Whitney U-test for paired and unpaired groups, respectively. Statistical significance was set at a p-value<0.05. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics [36].

Results

The effects of reserpine injections on body condition and behavior

The three injections of reserpine resulted in a gradual decrease in the weight of the young mice (Fig 1A). However, six days after the reserpine injection, their body weight increased slightly. In addition, the rectal temperature of the FM-induced mice decreased slightly (Fig 1B). Further, regarding the free gait video analysis before and after the reserpine injections, three gait parameters, namely, distance (m) (wild-type: 4.17 ± 0.57 vs. FM: 0.53 ± 0.21, p<0.05, n = 9; Fig 1Ci), maximum speeds (cm/s) (wild-type: 33.1 ± 2.92 vs. FM: 8.94 ± 2.89, p<0.05, n = 9; Fig 1Cii) and average speeds (cm/s) (wild-type: 8.67 ± 1.20 vs. FM: 1.10 ± 0.43, p<0.05, n = 9; Fig 1Ciii), decreased after FM had been induced in the mice.

Free gait facilitation in shallow warm water in wild-type mice and FM-induced model mice

Using a video recorder, differences in the 5-minute free gait were investigated in the wild-type mice both in and out of shallow warm water (Fig 2Ai and 2Aii). Five minutes of free gait in shallow warm water was used to observe enhancement in gait distance covered (m) (out of water: 1.22 ± 0.33 vs. in shallow water: 2.06 ± 0.73, p<0.05; Fig 2Aiii), maximum speed (cm/s) (out of water: 25.46 ± 5.74 vs. in shallow water: 56.65 ± 14.47, p<0.05; Fig 2Aiv), and average speed (cm/s) (out of water: 4.57 ± 1.28 vs. in shallow water: 8.81 ± 2.86, p<0.05; Fig 2Av). Fig 1D shows that a decrease was observed in the gait distance covered and maximum speed by the same mice after the reserpine injections. However, the addition of shallow warm water into the cage improved gait distance (m) (5-minute gait out of water: 0.44 ± 0.48, 1-minute gait in shallow water: 1.43 ± 1.23, p<0.05, 5-minute gait in shallow water: 6.20 ± 0.34; Fig 2Biv) and maximum speed (cm/s) (5-minute gait out of shallow water: 3.66 ± 4.16, 1-minute gait in shallow water: 19.92 ± 4.16, p<0.05, 5-minute gait in shallow water: 31.50 ± 25.43; Fig 2Bv). Although the distance covered (m) among the FM-induced mice remained less than that covered by the wild-type mice (wild-type: 20.58 ± 7.66 vs. FM: 6.20 ± 3.42, p<0.05; Fig 2Ci), the maximum speed (cm/s) in the FM-induced model mice was not statistically different from that in the wild-type mice (wild-type: 56.65 ± 15.25 vs. FM: 31.50 ± 25.43; Fig 2Cii), even when considering the lesser maximum speed attained in the FM-induced mice.

Cardiac effects of free gait in shallow warm water in wild-type mice

To determine the effects of the gait tests on cardiac function, ECGs were analyzed before and after the tests in the wild-type mice (Fig 3Ai and 3Aii). Under conditions of 5 minutes of free gait in and out of shallow warm water, no significant change was observed in the average HR (beats/min) (before and after the 5-minute gait out of water: 646.82 ± 18.98 and 667.38 ± 17.66; Fig 3Bi) (before and after the 5-minute gait in shallow water: 689.08 ± 20.19 and 661.95 ± 24.00; Fig 3Bii), SD of the HR (beats/min) (before and after the 5-minute gait out of water: 18.98 ± 16.37 and 17.66 ± 14.84; Fig 3Biii) (before and after the 5-minute gait in shallow water: 20.19 ±12.94 and 24.00 ± 11.37; Fig 3Biv), and HR irregularity score (before and after the 5-minute gait out of water: 2.81 ± 1.85 and 2.51 ± 1.88; Fig 3Bv) (before and after the 5-minute gait in shallow water: 3.34 ±2.10 and 2.92 ± 1.79; Fig 3Bvi). Further, the HR irregularity score statistically correlated with the SD (Fig 3C). Therefore, only the HR irregularity scores were reported (without the SD) in the results as an evaluation of HR rhythm.

Cardiac effects of free gait in shallow warm water in FM-induced model mice

The ECG findings were assessed both before and after gait training for the FM-induced mice (Fig 4Ai and 4Aii). However, no significant change was observed in the average HR (beats/min) before and after gait training (before and after the 5-minute gait out of water: 701.14 ± 57.52 and 713.46 ± 45.61; Fig 4Bi) (before and after the 1-minute gait in shallow water: 686.70 ± 52.53 and 681.45 ± 55.58; Fig 4Bii). Although no increase was observed after the 5-minute free gait test out of the water in the HR irregularity score (before and after the 5-minute gait out of water: 2.95 ± 1.59 vs. after 5-minute gait out of water: 3.33 ± 2.41; Fig 4Biii), a significant increase was observed after the 1-minute free gait test in the shallow warm water (before and after the 1-minute gait in shallow water: 1.88 ± 0.77 and 5.57 ± 4.46, p<0.05; Fig 4Biv).

Cardiac effects of long-term free gait in shallow warm water in the FM-induced model mice

The final part of the experiment comprised an investigation of the effects of 5 minutes of free gait in shallow warm water in terms of cardiac function (Fig 5Ai). As shown in Fig 5Aii, after 5 minutes of free gait in shallow warm water, the HR of the FM-induced mice seemingly fluctuated. The HR (beats/min) significantly decreased after 5 minutes of free gait in the shallow warm water (before and after 5-minute gait in shallow water: 688.83 ± 56.55 and 622.92 ± 119.13, p<0.05; Fig 5Bi). Furthermore, a significant increase was similarly observed in the HR irregularity scores (before and after 5-minute gait in shallow water: 2.50 ± 1.71 and 12.17 ± 8.09, p<0.05; Fig 5Bii). The average HR (beats/min) after 5 minutes of free gait in shallow warm water was higher than after 1 minute of free gait in shallow warm water (after 5-minute gait in shallow water in wild mice: 671.85 ± 76.17, vs. after 5-minute gait out of water in FM model mice: 713.46 ± 45.61 vs. after 1-minute gait in shallow water in FM model mice: 681.45 ± 55.58 vs. after the 5-minute gait in shallow water in FM model mice: 622.92 ± 119.13, p<0.05; Fig 5Ci). The HR irregularity score was also higher after 5 minutes of free gait in shallow warm water than after 1 minute of free gait in shallow warm water (after 5-minute gait in shallow water in wild mice: 3.27 ± 2.22 vs. after 5-minute gait out of water in FM model mice: 3.33 ± 2.41 vs. after 1-minute gait in shallow water in FM model mice: 5.57 ± 4.46 vs. after 5-minute gait in shallow water in FM model mice: 12.17 ± 8.09, p<0.05; Fig 5Cii).

Fig 5 — (A) An example of ECG raw data involving FM-induced model mice. (Left panel of Ai) Before 5 minutes of free gait in shallow warm water. (Right panel of Ai) After 5 minutes of free gait in shallow warm water. (Aii) A line graph illustrating the change in HR before and after 5 minutes of free gait in shallow warm water. Bar and line graphs comparing the average HR before and after 5 minutes of free gait in shallow warm water. (Bii) Bar and line graphs are comparing the IS of the HR before and after 5 minutes of free gait in shallow warm water. (C) Bar and line graphs are comparing both HR (Ci) and the IS of the HR (Cii) under the four experimental conditions. After 5 minutes of free gait in shallow water in wild-type mice (gait in water [5 min]). After 5 minutes of free gait out of water in FM-induced model mice (gait out of water-gait [5 min]). After 1 minute of free gait in shallow warm water in FM-induced model mice (gait in water [1 min]). After 5 minutes of free gait in shallow warm water in FM-induced model mice (gait in water [5 min]). ECG, electrocardiogram; FM, fibromyalgia; HR, heart rate; IS, irregularity score.

Discussion

This study reported that exercise-induced cardiac arrhythmia was observed at 1-minute gait in shallow water during the acute stage of induced FM in young mice. Both cardiac arrhythmia and a decrease in HR have occurred at 5-minute gait in shallow water at the same mice. However, this phenomenon was not observed in any wild-type mice under any test conditions. Herein, we discuss the proposed mechanisms of prolonged shallow water exercise-induced cardiac arrhythmia in reserpine-injected FM-induced mice, the implication of free gait exercise in shallow warm water, and the clinical applications of the outcomes.

Mechanisms of prolonged exercise-induced cardiac arrhythmia in reserpine-injected FM-induced model mice

Neuromodulators control cardiac function [37], and neuromodulator imbalances may cause cardiac arrhythmias [38]. For instance, reserpine appears to deplete monoamines, such as noradrenaline, serotonin, and dopamine, in the central and peripheral nervous systems [25]. Therefore, excessive exercise-induced cardiac arrhythmia in the FM-induced mice may be due to arrhythmia derived from the peripheral region [37], the central nervous system [37], or the neuromuscular region [39]. First, the release of neuromodulators, especially noradrenaline and acetylcholine, regulates cardiac function [37, 38]. Therefore, such exercise may have over-stressed the FM-induced mice and caused the cardiac arrhythmia due to a depletion in the level of monoamines needed for cardiac function. Second, it has been reported that monoamines released in the rostral ventrolateral medulla (of the medulla oblongata) play a crucial role in the control of autonomic function, such as in respiratory and cardiac regulation [37, 40–42]. Therefore, monoamine depletion in the medulla might equally result in cardiac arrhythmia. Third, monoaminergic neurotransmission in the cortical motor area also might be downregulated [37]. Fourth, the neuromuscular region may be indirectly involved. The FM model mice showed a temporary decrease in weight (Fig 1A), which may have been due to muscle atrophy because of pain and reduced movement [39]. Acetylcholine is only released at the neuromuscular junction, as it is not a monoamine [43]. However, skeletal muscles are controlled by motor neurons in the ventral horn of the spinal cord. It is reported that noradrenaline, one of the monoamines, excites motor neurons [44], suggesting that changes in monoamine release in the ventral horn of the spinal cord may modulate the excitability of the motor neurons, which may downregulate the acetylcholine release in neuromuscular junction [44]. Further, FM patients are often afflicted with fatigue syndrome [4]. Muscle atrophy and fatigue may also be the cause for the decrease in body weight of FM-induced mice after reserpine injection that was observed in this study (Fig 1A). Therefore, excessive gait might overload the heart muscles during the acute stage in FM mice.

Implications of free gait in shallow warm water as a form of exercise for FM-induced mice

In the investigation of the free gait of FM-induced mice in shallow warm water, consideration was given to the following: a short-duration free gait, cardiovascular issues, the use of inexpensive equipment, and the simplest and easiest method of assessment. While a free gait in shallow warm water enabled an easy assessment of distance covered and average and maximum speeds, the risk of cardiac arrhythmia remained, especially during the long-duration 5-minute exercise of the FM-induced model mice in the acute stage of this disease. While a treadmill that assesses passive gait in the FM-induced model mice may be inaccessible and expensive, the cheaper treadmill alternative from the pet shop cannot precisely control the speed. On the contrary, short-term free gait sessions in shallow warm water were found to be an inexpensive and easy way for assessing the FM-induced mice. Further, the method used in this study allowed us to perform both the exercise and its evaluation of multiple mice in one cage at the same time.

Study limitations and clinical applications

This study had several limitations. First, HR was measured for only 20 seconds. If the HR had been continuously monitored, the relationship between exercise load, gait time, and cardiac arrhythmia could have been more clearly determined in the FM-induced mice. Second, the study had a critical experimental limitation. Although cardiac abnormalities were detected in ECG recordings acquired after the gait test, owing to the difficulty in performing ECG recordings during the gait tests, we could not determine the starting point of the abnormality nor if it began during the gait test. Third, we could not precisely analyze the ECG recordings, such as QRST wave analysis, acquired from the mice since the QRST wave is unclear in the ECG signals from the mice (S1D Fig). Therefore, we also could not specify which regions of the heart were normal or abnormal.

While there is a study showing that FM patients have similar cardiovascular responses to submaximal exercise as healthy control subjects [45], other new studies reported that FM patients had higher HR [27] and impaired cardiac function [28]. Further, they seem to have a higher risk of coronary heart disease-related events [29] and delayed HR recovery after a treadmill graded gait exercise [46]. However, it could not be clear how these cardiovascular problems in FM patients relate to the exercise-induced cardiac arrhythmia and decrease of HR in FM model mice. Further, we do not know how cardiovascular problems aggravate disease progression. Inevitably, previous studies reported that moderate aerobic exercise reduces both musculoskeletal pain and autonomic dysfunction for FM patients [17, 47–49]. Therefore, medical staff should be aware of the following three matters. First, FM patients have both musculoskeletal pain and cardiovascular risks. Second, the load and the time spent performing therapeutic exercises for FM patients should be precisely arranged, with initially low and short duration; otherwise, unexpected cardiovascular events may occur during exercise in these patients. Third, when FM patients perform therapeutic exercises, ECG or HR monitoring should be considered.

Conclusion

This study reported a prolonged and excessive exercise-induced cardiac abnormality involving a decrease in the HR and the occurrence of cardiac arrhythmia in the acute stage of FM-induced mice. Although a short-term free gait in shallow warm water may be advantageous for increasing the motor activity of FM-model mice, our data indicates it can be associated with changes in heart rate and even arrhythmias. Physiotherapists and other health professionals should be aware of these potential risks when considering strenuous exercise as a treatment in FM patients. We suggest a gradual increase in exercise duration may be warranted.

Supporting information

S1 Fig. Experimental protocols and flow charts of the experiments.

(A) The experimental protocol to produce FM-induced mice. (B) A flow chart of the first experiment to measure body weight and rectal temperature, and video recordings of free gait inside the cage. (C) A flow chart of the second experiment with video recordings before and after the three types of gait tests. (D) Raw data from ECG recordings taken to evaluate cardiac function. R means a negative peak of wave in the ECG. (E) A flow chart of the third experiment for ECG recordings before and after the three types of gait tests. ECG, electrocardiogram; FM, fibromyalgia.

(TIF)

Click here for additional data file.^{(545.6KB, tif)}

Acknowledgments

Data Availability

All relevant data are within the manuscript.

Funding Statement

Kumamoto Health Science University fellowship grant (No. 2018-C-11) and a Grant-in-Aid for Scientific Research (C), JSPS to A.D. (19K11383).

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PLoS One. doi: 10.1371/journal.pone.0239473.r001

Decision Letter 0

Etsuro Ito

25 Jun 2020

PONE-D-20-16502

A long and excessive exercise-induced abnormal cardiac arrhythmia in young model mice with acute stage fibromyalgia

PLOS ONE

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The papers described that a prolonged and excessive exercise induced cardiac abnormality involving a decrease in heart rate and the occurrence of cardiac arrhythmia during the acute stage of induced fibromyalgia (FM) in model mice. This phenomenon was not observed in any of the wild-type mice under any of the test conditions.

This research is potentially interesting, however, following points should be addressed.

1. Has this paper been proofread by native speakers?

2. In the abstract, “Results: the cardiac” should be corrected as “Results: The cardiac”.

3. In the text, references 16-19 are missing from the text.

4. “In general, medications (Ferreira-Dos-Santos et al., 2018; Sarmento et al., 2019),”

A quote of above references is mistaken. Please use numeric references.

5. For the instruments or reagent, model number, company name, city, province (if USA), country should be included.

For example,

camera (100 frames/sec, TZ-35, Panasonic, Japan)

digitizer (Axon DigiData 1322, USA)

6. How the mice were fixed a position for measurements of the ECG signals?

7. Authors utilized following formula, Sn = 100*ABS (Pn – Pn-1)/Pn-1, for analysis of fluctuation of the ECG signals.

Is it a standard way to observe the variation of the ECG signals?

8. Mice generally do not like walking or swimming in the water; even depth is pretty shallow.

Why did you come up with that idea?

9. What is the purpose of measuring rectal temperature in FM animals, as there is nothing in the discussion of rectal temperature?

Was there any previous study on temperature changes?

10. Have you already tried walking in a shallow pool as a stimulus or exercise for FM mice?

If so, what is the effect?

11. The authors described four mechanisms caused by monoamine depletion as a mechanism of excessive exercise-induced cardiac arrhythmias in FM-induced model mice injected with reserpine.

What do you think about other mechanisms besides monoamine depletion for excessive exercise-induced cardiac arrhythmia, as the potential mechanism of fibromyalgia symptoms is not yet known?

12. In the Fig. 1A, “(Days after 3td reserpine injection)” should be corrected as “(Days after 3rd reserpine injection)”.

13. In the Fig. 2A, why did the bodyweight of FM mice decrease right after first shot of the reserpine?

Then, are there any changes in the behavior of FM model animals in day and night time?

14. In the reference list, following journals should be corrected.

43. Kanda Y. Investigation of the freely available easy-to-use software {’EZR’} for medical statistics. Bone Marrow Transplant. 2013;48: 452–458. should be corrected as

43. Kanda Y. Investigation of the freely available easy-to-use software ’EZR’ for medical statistics. Bone Marrow Transplant. 2013;48: 452–458.

49. Beech DJ. Actions of neurotransmitters and other messengers on Ca2+ channels and K+ channels in smooth muscle cells. Pharmacol Ther. 1997/01/01. 1997;73: 91–119. doi:10.1016/s0163-7258(97)87271-3, should be corrected as

Beech DJ. Actions of neurotransmitters and other messengers on Ca2+ channels and K+ channels in smooth muscle cells. Pharmacol Ther. 1997/01/01. 1997;73: 91–119. doi:10.1016/s0163-7258(97)87271-3.

Reviewer #2: The authors use chronic reserpine treatment for 1 week to induce cardiac arrhythmias, that manifest as irregular HR upon brief 1 minute walking in warm water, and that become exacerbated (with reduced mean HR) when walking in warm water is prolonged (5 mins). As reserpine dosing has been previously shown to induce fibromyalgia (FM) symptoms in mice, and as human FM patients have cardiac abnormalities, this new finding adds an additional aspect to this FM model that may have utility in researching mechanisms or treatments for FM. In particular, the approach is readily accessible with minimal costs, which can broaden the utility of this model.

The data is clear in the Figures, and appropriately presented and analyzed. The experiments appear robust and well designed. The Figures are very clear.

A key point therefore is to look further into the ECG pattern and describe (and show) what the abnormalities are that cause the irregularity. I think the data should already be there.

There are many areas in the text that need clarification. The English is good, but the authrors just need to be more concise with details, rationale and conclusion. I have many suggestions below (sorry) but I hope it helps a bit and improves the paper.

Abstract

Background is wrong. Study doesn’t aim to test a treatment. Its investigating CVS effects of the reserpine-FM model. Methods: Last sentence unclear, seems a conclusion, not a method, but then seems to contradict result? Just leave out

Ethics – anaesthesia not mentioned.

Data – Some misunderstanding I think. Data stated as not available but no real reasons given? Says all data in ms, but individual values not in ms?

Title “Abnormal” cardiac arrhythmia. By definition, arrhythmias are abnormal. Is this needed? Delete? And does long and excessive refers to exercise, not arrhythmia? I suggest something like “Identification of exercise induced cardiac arrhythmia in fibromyalgia model mice”

Intro.

- Ref 14 describes that the reserpine model has been described quite thoroughly as a model of FM. So this needs to be sated and the features described in the Introduction. SO the rationale is then to evaluate exercise to reduce cardiac abnormalities associated with this model? Then one needs to 1st characterize these cardiac abnormalities (or other features of FM such as allodynia), then test exercise against these control features. And the study rationale made clearer. Its unclear whether cardiac abnormalities found before.

L92. Suggests reserpine used to reduce FM symptoms (line 92), but you are using to produce FM symptoms. Rewrite to clarify.

L10 9. Is anything known of cardiac symptoms in FM mice? Has this been seen before? This is an important point, as the paper may be the first to identify this so should be clarified in the introduction. Why is it stated a “possibility of cardiovascular complications”.

And treadmills are available for mice – cheaply in pet shops - delete this.

Methods.

L130. Reserpine 1ml/kg. What dose in mg/kg? And 1ml/kg is about 0.02 ml in a 20g mice. Check this. Can you achieve such low volumes accurately? What type of needle?

How did you do “out of the water” and “shallow water”. Were the mice taken from home cages to the plastic cage with warm water? Was the out of water the same procedure (ie taken from home cage to plastic cage) but without the water. Was every mice given the three tests? Was it wiped to clear any residual scent between tests? How did the gait software work – via some digital tracking?

Were these animals anesthetized to place the electrodes and for rectal temperature? Please give details

ECG. Seems nice. How was the period measured? (eg between peak of QRS complex). many cycles used to obtain a mean irregularity score?

In general, Figure 1 describes the experiments and cohorts well. Use this when your going through the animal numbers.

Results.

Figures are great – very clearly show methods protocol. Results clearly described

Was the irregularities related to the distance travelled? For instance, they were seen with 1 min warm water but not 5 mins out of water. Was the distance and speed (ie exertion) greater in 1 min warm water?

The mice lost a lot of weight. Figures say 7 days after reserpine, but weight graphs only go out to 5 or 6 days after reserpine?

What causes the irregularity? Is it missed or delayed beats? Can the authors look at the ECG and analyze what the specific irregularity(ies) is/are?. Does this relate to human FM abnormalities?

Discussion

What the paper shows is that it extends the reserpine FM model to show that cardiovascular abnormalities occur with 1 and 5 min warm water exercise, being an increase in irregular rhythm and, for 5 mins, a decrease in HR. This should be stated clearly in the context of whether FM models have seen cardiovascular parameters, how it relates to human FM and what may be the nature and cause of the irregularity.

Is this interpreted as an exercise effect or a stress-effect, or some combination?

Some discussion of what irregularity means. Is this abnormalities in specific regions or the QRST waves (Q-T delay for example) or is ECG shape normal?

P280-281 RVM “monomaines play a crucial role” – in what? How precisely? This sounds interesting and maybe relevant

285-290. It seems an ischemic effect postulated due to poor blood flow regulation. Cardiac blood flow largely autoregulation isn’t it, rather than Noradrenalin? I don’t see how neuromuscular effects or cardiac blood flow could be involved. Most arhhythmas due to deficits in cellular control of excitability, so 1st need to identify the nature of the “irregularity” then start looking towards possible effects on cardiac channels or they posttranslational regulation

4.2 makes an excellent point of this is a simple design with cheap, accessible methods. I think a mouse exercise wheel is also cheap from a pet shop, and one can set for one direction and count revolutions over time.

Conclusion. Even 1 min warm water gait had increased irregularity, so cant conclude this is safe and “beneficial”, although I appreciate it did increase movement (due to fear or stress?). So conclusion should rewrite.

**********

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Sep 30;15(9):e0239473. doi: 10.1371/journal.pone.0239473.r002

Author response to Decision Letter 0

18 Aug 2020

PLOS ONE's suggestion

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Answer for PLOS ONE's suggestion

We have revised the manuscript and figures in accordance with the PLOS ONE style templates.

Additional Editor Comments:

When submitting your revision, we need you to address these additional requirements.

The two independent reviewers basically like your manuscript, and so please revise it according to their comments,

Answer for additional Editor Comments:

We have uploaded our figure files to the PACE digital diagnostic tool.

Reviewer's Responses to Questions

Question #1:

Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Response:

We thank the reviewers for this feedback.

Question #2:

Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Response:

We thank the reviewers for this feedback.

Question #3:

Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: No

Response:

We have included all data underlying the findings in our manuscript to ensure the data is fully available.

Question #4:

Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

Response:

The manuscript has been reviewed and revised by Editage.

Question #5:

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Response:

We thank the reviewers for their peer review.

Reviewer’s Comments to the Author

Reviewer #1:

The papers described that a prolonged and excessive exercise induced cardiac abnormality involving a decrease in heart rate and the occurrence of cardiac arrhythmia during the acute stage of induced fibromyalgia (FM) in model mice. This phenomenon was not observed in any of the wild-type mice under any of the test conditions.　This research is potentially interesting, however, following points should be addressed.

Comment 1: Has this paper been proofread by native speakers?

Response: The manuscript has been reviewed and revised by Editage.

Comment 2: In the abstract, “Results: the cardiac” should be corrected as “Results: The cardiac”.

Response: We have revised the results in the abstract (line 46).

Comment 3: In the text, references 16-19 are missing from the text.

Response: We have ensured that references 16–19 are appropriately cited within the text (line 61) and at the end of the manuscript (Lines 493-499). Furthermore, a couple of references have already been omitted.

Comment 4: “In general, medications (Ferreira-Dos-Santos et al., 2018; Sarmento et al., 2019),”

A quote of above references is mistaken. Please use numeric references.

Response: We have ensured that all the references are listed at the end of manuscript and are numbered in the order in which they appear in the text. We have also ensured that all in-text citations are listed as numbers within square brackets in accordance with PLOS ONE’s formatting requirements.

Comment 5: For the instruments or reagent, model number, company name, city, province (if USA), country should be included.

For example,

camera (100 frames/sec, TZ-35, Panasonic, Japan)

digitizer (Axon DigiData 1322, USA)

Response: We have included the model number, company name, city, and country for all commercial sources of instruments and reagents (lines 105,147,148, 152, 164, 176).

Comment 6: How the mice were fixed a position for measurements of the ECG signals?

Response: We have added a description to clarify that the mice were gently held in the prone position by the examiner (Line 176).

Comment 7: Authors utilized following formula, Sn = 100*ABS (Pn – Pn-1)/Pn-1, for analysis of fluctuation of the ECG signals.

Is it a standard way to observe the variation of the ECG signals?

Response: The irregularity score (IS) is often used to evaluate respiratory rhythm. However, the IS might not be standard in the analysis of ECG signals. In this study, since the heartbeat of the mice was fast, we decided to evaluate the fluctuation of the heartbeat. Therefore, in this study, we utilized the IS of the respiratory rhythm to evaluate the regularity of the heartbeat. Further, a recent study used a similar analysis method to evaluate the R-R interval (Karey E, Pan S, Morris AN, Bruun DA, Lein, PJ, Chen, C. The use of percent change in RR interval for data exclusion in analyzing 24-h time domain heart rate variability in rodents. Front. Physiol. 2019;10:693)

Comment 8: Mice generally do not like walking or swimming in the water; even depth is pretty shallow. Why did you come up with that idea?

Response 8: We have included a description in the Introduction section to clarify that we used underwater walking as a method to decrease overload on the antigravity muscles (line 64).

Comment 9: What is the purpose of measuring rectal temperature in FM animals, as there is nothing in the discussion of rectal temperature?

Was there any previous study on temperature changes?

Response: We initially thought that reserpine related monoamines downregulation may change the rectal temperature since the body temperature is controlled by the autonomic function as a phenomenon of the monoamines downregulation. However, the changing of the temperature was temporary, and we could not scientifically clear the reason about the temporal change of the temperature. Further, we have included a description in the Material and Methods section that clarifies that measuring rectal temperature was used as method to measure body temperature in the mice. We have also included a reference to a prior study that used this method (reference 31). The change in the rectal temperature was negligible and thus, we did not expand further on the rectal temperature of the mice in the Discussion section.

Comment 10: Have you already tried walking in a shallow pool as a stimulus or exercise for FM mice? If so, what is the effect?

Response: We have performed these experiments; however, as it is currently under peer-review we cannot describe the results in great detail. Underwater walking does not appear to have an effect on the pain threshold.

Comment 11: The authors described four mechanisms caused by monoamine depletion as a mechanism of excessive exercise-induced cardiac arrhythmias in FM-induced model mice injected with reserpine.

What do you think about other mechanisms besides monoamine depletion for excessive exercise-induced cardiac arrhythmia, as the potential mechanism of fibromyalgia symptoms is not yet known?

Response: The Discussion has been revised to include further detail about how fatigue and muscle atrophy may be related to a decrease in weight in fibromyalgia patients (line 412). It is possible that excessive gait may overload the heart muscles, and not skeletal muscles, in acute stage FM mice.

Comment 12: In the Fig. 1A, “(Days after 3td reserpine injection)” should be corrected as “(Days after 3rd reserpine injection)”.

Response: We have revised this title accordingly in Supplemental Figure 1A (S1 FigureA).

Comment 13: In the Fig. 2A, why did the bodyweight of FM mice decrease right after first shot of the reserpine?

Then, are there any changes in the behavior of FM model animals in day and night time?

Response: Although Nagakura et al. reported a decrease in weight [31], they do not describe a mechanism underlying the weight decrease. Skeletal muscle atrophy should not appear in reserpine injected FM-induced model mice. However, it is still currently unclear why the reserpine injection decreases their weight.

After the reserpine injection, our data show that the locomotive activity of FM model animals is inhibited both during the day time and night time.

Comment 14: In the reference list, following journals should be corrected.

43. Kanda Y. Investigation of the freely available easy-to-use software {’EZR’} for medical statistics. Bone Marrow Transplant. 2013;48: 452–458.

Response: We have revised a new reference 36, and omitted original reference 49.

(line 615).

Reviewer #2:

The authors use chronic reserpine treatment for 1 week to induce cardiac arrhythmias, that manifest as irregular HR upon brief 1 minute walking in warm water, and that become exacerbated (with reduced mean HR) when walking in warm water is prolonged (5 minutes). As reserpine dosing has been previously shown to induce fibromyalgia (FM) symptoms in mice, and as human FM patients have cardiac abnormalities, this new finding adds an additional aspect to this FM model that may have utility in researching mechanisms or treatments for FM. In particular, the approach is readily accessible with minimal costs, which can broaden the utility of this model.

The data is clear in the Figures, and appropriately presented and analyzed. The experiments appear robust and well designed. The Figures are very clear.

However the integration of the results with the current literature in the conclusions, and the rationale and introduction, and the way the methods is described needs significant work. The results do not seem to be testing or reporting any FM treatment approach (as stated, for example, in abstract) and the fact that the reserpine treated mouse has been previously validated as a FM model is not mentioned (in fact, it is stated reserpine is a treatment?). What has been shown is that this model induces cardiac abnormalities, and this should be the focus of Intro and Discussion. A key point therefore is to look further into the ECG pattern and describe (and show) what the abnormalities are that cause the irregularity. I think the data should already be there. There are many areas in the text that need clarification. The English is good, but the authors just need to be more concise with details, rationale and conclusion. I have many suggestions below (sorry) but I hope it helps a bit and improves the paper.

Comment 1: Abstract

Background is wrong. Study doesn’t aim to test a treatment. It’s investigating CVS effects of the reserpine-FM model.

Response: The Abstract has been revised to clarify that our aim was to investigate the cardiac effect of a prolonged shallow water gait in an FM-induced mouse model (line 37).

Comment 2: Methods: Last sentence unclear, seems a conclusion, not a method, but then seems to contradict result? Just leave out

Response: We have removed the last sentence of the Methods described in the Abstract in order to improve clarity (line 45).

Comment 3: Ethics – anesthesia not mentioned.

Response: We have not mentioned anesthesia in the Ethics section since we did not use anesthesia for mice when injecting reserpine or during ECGs.

Comment 4: Data – Some misunderstanding I think. Data stated as not available but no real reasons given? Says all data in manuscript, but individual values not in manuscript?

Response: We have revised this and included all experimental data in the manuscript (lines 214, 232, 238, 271, 293, 307, 311, 340, 352).

Comment 5: Title “Abnormal” cardiac arrhythmia. By definition, arrhythmias are abnormal. Is this needed? Delete? And does long and excessive refers to exercise, not arrhythmia? I suggest something like “Identification of exercise induced cardiac arrhythmia in fibromyalgia model mice”

Response: We thank the reviewer for this suggestion of the title. We have deleted “a long” and “abnormal” from the title to remove the redundancy (line 1). The new title is “Excessive exercise induces cardiac arrhythmia in a young fibromyalgia mouse model”.

Comment 6: Introduction

- Ref 14 describes that the reserpine model has been described quite thoroughly as a model of FM. So this needs to be stated and the features are described in the Introduction. Thus, the rationale is then to evaluate exercise to reduce cardiac abnormalities associated with this model? Then one needs to 1st characterize these cardiac abnormalities (or other features of FM such as allodynia), then test exercise against these control features. And the study rationale made clearer. It’s unclear whether cardiac abnormalities found before.

Response: We have revised the Introduction to describe the features of reserpine model. We have also described the cardiac abnormalities associated with this model and clarified the rationale of using exercise to reduce these cardiac abnormalities (starting on line 58).

Comment 7: Original line 92 suggests reserpine used to reduce FM symptoms (line 92), but you are using to produce FM symptoms. Rewrite to clarify.

Response: We have edited this to clarify that we used reserpine to produce FM symptoms (line 74).

Comment 8: Original line109

Is anything known of cardiac symptoms in FM mice? Has this been seen before? This is an important point, as the paper may be the first to identify this so should be clarified in the introduction. Why is it stated a “possibility of cardiovascular complications”.

Response: In a search conducted on the search engine PubMed using the keywords “fibromyalgia” and “cardiac arrhythmia” (on July 10, 2020), we found 26 reports. However, when we used the keywords “fibromyalgia”, “cardiac arrhythmia”, and “animal”, “rat”, or “mouse”, there were no studies found. Therefore, we have edited the Introduction to highlight that there are no known studies that evaluate exercise-induced cardiac arrhythmia in an FM-induced mouse model (line 86). We have also clarified our description of the cardiovascular complications in the Introduction by removing the word “possibility” (line 84).

Comment 9: And treadmills are available for mice – cheaply in pet shops – delete this.

Response: We have removed the sentence about treadmills in the Introduction.

Comment 10: Methods.

Original line 130.

Reserpine 1ml/kg. What dose in mg/kg? And 1ml/kg is about 0.02 ml in a 20g mice. Check this. Can you achieve such low volumes accurately? What type of needle?

Response: We have revised the description of the dosage to 10 mL/kg and clarified that this would be 0.3 mL of reserpine for a mouse with a body weight of 30 g (line 111).

Comment 11: How did you do “out of the water” and “shallow water”. Were the mice taken from home cages to the plastic cage with warm water? Was the out of water the same procedure (ie taken from home cage to plastic cage) but without the water. Was every mice given the three tests? Was it wiped to clear any residual scent between tests?

Response: We used different cages for breeding and the experiments. This included cages with shallow water and cages without water. We have revised our description of the gait test to clarify the method used with the cages (starting at line 128).

Comment 12: How did the gait software work – via some digital tracking?

Response: We have revised our description of the gait software (line 167).

Comment 13: Were these animals anesthetized to place the electrodes and for rectal temperature? Please give details

Response: No, the mice were not anesthetized before we conducted the ECG and measured the rectal temperature. Thus, we have excluded this from our description of measurement of the rectal temperature (line 147).

Comment 14: The measurements of ECG recordings seem nice. How was the period measured? (eg between peak of QRS complex). How many cycles used to obtain a mean irregularity score?

Response: We have revised our description of our measurements of ECG recordings in the Material and Methods section (line 170). We detected ECG signals before and after the gait tests, and the ECG signals were recorded for at least 20 seconds (200–250 cycles were recorded on average for each mouse). The HR irregularity score was determined using 50 ECG cycles.

Comment 15: In general, Figure 1 describes the experiments and cohorts well. Use this when you are going through the animal numbers.

Response: We have changed Fig. 1 to Supplemental Fig. 1.

Comment 16: Results.

Figures are great – very clearly show methods protocol. Results clearly described

Response: We thank the reviewer for this feedback.

Comment 17: Was the irregularities related to the distance travelled? For instance, they were seen with 1 min warm water but not 5 min out of water. Was the distance and speed (ie exertion) greater in 1 min warm water?

Response: We believe that the HR irregularity may relate to the amount of activity, such as gait distance. We think that excessive activity might overload the cardiac muscles and induce cardiac arrhythmia.

We have revised Fig. 2B to show the increase in both gait distance and speed in an individual mouse.

Comment 18: The mice lost a lot of weight. Figures say 7 days after reserpine, but weight graphs only go out to 5 or 6 days after reserpine?

Response: We have revised “seven days” to “five and six days” in new S1 Figure A.

Comment 19: What causes the irregularity? Is it missed or delayed beats? Can the authors look at the ECG and analyze what the specific irregularity (ies) is/are? Does this relate to human FM abnormalities?

Response: We think that the ECG irregularity observed in the mice has two patterns. The first pattern is a delayed or skipped ECG, meaning the distance between two ECG cycles is long. The second pattern is a doublet-like ECG, meaning the distance between the two ECG cycles is short. These two components are often mixed. We think that these ECG abnormalities patterns are related to human ECG abnormalities.

Comment 20: Discussion

What the paper shows is that it extends the reserpine FM model to show that cardiovascular abnormalities occur with 1 and 5 min warm water exercise, being an increase in irregular rhythm and, for 5 minutes, a decrease in HR. This should be stated clearly in the context of whether FM models have seen cardiovascular parameters, how it relates to human FM and what may be the nature and cause of the irregularity. Is this interpreted as an exercise effect or a stress-effect, or some combination?

Response:

We clearly put results in content of both results and the beginning of discussion, which reviewer asked to us (starting at line 305, 336 and 377). However, we also explained that it could not be clear how these cardiovascular problems in FM patients relate to the exercise-induced cardiac arrhythmia and decrease of HR in FM model mice. Further, we wrote unknown about how cardiovascular problems aggravate disease progression. (starting at line 452). We are thinking both irregular rhythm and a decrease in HR caused by 5 minutes of warm water exercise mainly originated as a result of the exercise effect. However, we also cannot perfectly exclude the possibility of the exercise-induced stress effect.

Comment 21: Some discussion of what irregularity means. Is this abnormalities in specific regions or the QRST waves (Q-T delay for example) or is ECG shape normal?

Response: Our ECG analysis was based on both the numbers and regularity of detected “R” in the ECG (line 187, see S1 Fig D). We could not precisely analyze the QRST waves, such as the Q-T delay, since the QRST wave was unclear in the ECG signals from the mice. Therefore, we cannot say which parts of the heart are normal or abnormal. We have revised our description of the ECG signals to include this information (line 443).

Comment 22: Original line 280-281

RVM “monomaines play a crucial role” – in what? How precisely? This sounds interesting and maybe relevant

Response: We thank the reviewer for this pertinent comment. Monoamines have been reported to play a crucial role in the control of autonomic function, including respiratory and cardiac regulation. We have added this additional information with supporting references (line 399; references 37, 40–42).

Comment 23: Original line 285-290.

It seems an ischemic effect postulated due to poor blood flow regulation. Cardiac blood flow largely autoregulation isn’t it, rather than Noradrenalin? I don’t see how neuromuscular effects or cardiac blood flow could be involved. Most arrhythmia due to deficits in cellular control of excitability, so 1st need to identify the nature of the “irregularity” then start looking towards possible effects on cardiac channels or they posttranslational regulation

Response: We agree that cardiac blood flow is largely a result of autoregulation. Therefore, we have revised our Discussion section to address this (line 407).

Comment 24: 4.2 makes an excellent point of this is a simple design with cheap, accessible methods. I think a mouse exercise wheel is also cheap from a pet shop, and one can set for one direction and count revolutions over time.

Response: We have revised our Discussion section to discuss how treadmills are also an inexpensive and accessible method, but precise control of the speed may be a limitation (line 426).

Comment 25: Conclusion. Even 1 min warm water gait had increased irregularity, so cannot conclude this is safe and “beneficial”, although I appreciate it did increase movement (due to fear or stress?). So conclusion should rewrite.

Response: We have revised our Conclusion to address this point (starting on line 466). We have clarified that although a short-term free gait test in shallow warm water may be advantageous for increasing the activity of FM-induced mice, we should be aware of the risk of prolonged and excessive exercise-induced cardiac arrhythmia. For gait exercises in shallow water with FM-induced mice, there should be a gradual increase in duration.

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PLoS One. doi: 10.1371/journal.pone.0239473.r003

Decision Letter 1

Etsuro Ito

8 Sep 2020

Excessive exercise induces cardiac arrhythmia in a young fibromyalgia mouse model

PONE-D-20-16502R1

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A minor comment. In the conclusion (repeated in abstract and paper), I feel you could conclude beyond just this mouse model to provide some suggestion for human FM patients. As written the two concluding sentences also seem pretty much the same - about being careful not to induce arrhythmias in the mice. I would suggest a rewrite like following:

"Conclusion: Although a short-term free gait in shallow warm water may be advantageous for increasing the motor activity of FM-model mice, our data indicates it can be associated with changes in heart rate and even arrhythmias. Physiotherapists and other health professionals should be aware of these potential risks when considering strenuous exercise as a treatment in FM patients. We suggest a gradual increase in exercise duration may be warranted"

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PLoS One. doi: 10.1371/journal.pone.0239473.r004

Acceptance letter

Etsuro Ito

15 Sep 2020

PONE-D-20-16502R1

Excessive exercise induces cardiac arrhythmia in a young fibromyalgia mouse model

Dear Dr. Doi:

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PERMALINK

Excessive exercise induces cardiac arrhythmia in a young fibromyalgia mouse model

Taiki Nakata

Atsushi Doi

Daisuke Uta

Megumu Yoshimura

Min-Chul Shin

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Material and methods

Animals

Reserpine-injected FM model mice

Experimental design

Body weight

Rectal temperature

Evaluation of locomotive behavior using three types of gait tests

Evaluation of cardiac function using three gait tests

Statistical analysis

Results

The effects of reserpine injections on body condition and behavior

Fig 1. The effects of reserpine injections on body condition and behavior.

Free gait facilitation in shallow warm water in wild-type mice and FM-induced model mice

Fig 2. Free gait facilitation in shallow warm water in wild-type mice and FM-induced model mice.

Cardiac effects of free gait in shallow warm water in wild-type mice

Fig 3. Cardiac effects of free gait in shallow warm water in wild-type mice.

Cardiac effects of free gait in shallow warm water in FM-induced model mice

Fig 4. Cardiac effects of free gait in shallow warm water in the FM-induced model mice.

Cardiac effects of long-term free gait in shallow warm water in the FM-induced model mice

Fig 5. Cardiac effects of long-term free gait in shallow warm water in the FM-induced model mice.

Discussion

Mechanisms of prolonged exercise-induced cardiac arrhythmia in reserpine-injected FM-induced model mice

Implications of free gait in shallow warm water as a form of exercise for FM-induced mice

Study limitations and clinical applications

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Etsuro Ito

Roles

Author response to Decision Letter 0

Decision Letter 1

Etsuro Ito

Roles

Acceptance letter

Etsuro Ito

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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