Effect of exercise preconditioning on myocardial content of Sphingosine1-phosphate and its mechanism in rats after exhaustive exercise

Xinnuan Wei; Weiyuan Yang; Junxiang Zhou; Luoyuan Cao; Wenxing Jiang

doi:10.1371/journal.pone.0340313

. 2026 Jan 7;21(1):e0340313. doi: 10.1371/journal.pone.0340313

Effect of exercise preconditioning on myocardial content of Sphingosine1-phosphate and its mechanism in rats after exhaustive exercise

Xinnuan Wei ¹, Weiyuan Yang ^1,^*, Junxiang Zhou ¹, Luoyuan Cao ², Wenxing Jiang ¹

Editor: Amirreza Khalaji³

PMCID: PMC12779141 PMID: 41499553

Abstract

Objective

This study aimed to investigate the effects of exercise preconditioning on rat myocardial Sphingosine1-phosphate(S1P) content and its potential mechanisms of heart protection.

Methods

A rat model of exercise preconditioning followed by exhaustive exercise was established. Rats were randomized to four groups: control (C), exercise preconditioning (EP), EP plus the S1PR1-selective antagonist W146 (EP + W146), and EP plus the MEK1/2 inhibitor PD98059 (EP + PD98059). Following a final exhaustive swim, comparisons across groups revealed that EP attenuated myocardial injury and apoptosis, an effect which was abolished by both W146 and PD98059.

Results

1. Exercise preconditioning (EP) significantly attenuated exhaustive exercise-induced myocardial injury and apoptosis (P < 0.001); 2. EP significantly elevated myocardial S1P levels (P = 0.002), and S1PR1-selective antagonist (W146) abolished this cardioprotective effect (P = 0.016 for apoptosis); 3. Most importantly, MAPK pathway inhibition (PD98059) abrogated the protective effect of EP, as evidenced by significantly increased apoptosis (P = 0.002), despite unaltered S1P levels.

Conclusion

In summary, beyond confirming S1P elevation with exercise preconditioning, our findings propose the S1P→MAPK signaling axis as a novel mechanistic pathway warranting future validation.

Introduction

Exercise preconditioning, characterized by repeated brief intermittent high-intensity exercise, enhances the heart’s tolerance to prolonged ischemic hypoxia and is considered effective in reducing myocardial ischemic injury. Mechanisms include improvements in calcium handling and upregulation of endogenous antioxidant enzymes [1,2]. Sphingosine1-phosphate (S1P) has been shown to protect cardiomyocytes during ischemic preconditioning and postischemic conditioning [3], potentially through mechanisms involving upregulation of endogenous antioxidant enzymes and improved calcium handling [4]. Previous reports indicate that endurance exercise may upregulates sphingosine kinase 1 (SPHK1) activity, enhancing S1P synthesis. S1P binding to receptors (e.g., S1PR1/S1PR2) activates downstream MAPKs (ERK1/2 and p38), which suppress pro-apoptotic proteins, promote mitophagy, and bolster antioxidant defenses [5]. Therefore, this experiment aims to investigate whether exercise preconditioning exerts myocardial protective effects via activation of the MAPK signaling pathway by increasing S1P content, using an animal model of exercise preconditioning and exhaustive exercise.

Materials and methods

1.1. Primary reagents and kits

S1P, the S1PR1-selective antagonist W146, and the MEK1/2 inhibitor PD98059 were obtained from GlpBio Corporation (USA). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit and the Annexin V-FITC/PI apoptosis detection kit were purchased from Roche (Germany).The S1P ELISA kit (Catalog No: UCEG013Ge) was acquired from Wuhan Yunclone (China).

1.2. Experimental animals and ethics statement

1.2.1. Experimental animals.

Twenty-two healthy male Sprague-Dawley (SD) rats, weighing between 200 and 220 g, were housed under routine conditions with 5 animals per cage. They were provided with ad libitum access to water and standard rodent diet, maintained at a room temperature of 20−22°C with a relative humidity of 45%−50% and a 12-hour light-dark cycle. The SD rats and their feed were supplied by Wu’s Experimental Animal Center, Fujian Province, under license SCXK (Hong’s) 2018−0006. All procedures involving experimental animals were conducted in accordance with the Guiding Opinion on the Ethical Treatment of Experimental Animals issued by the Ministry of Science and Technology of the People’s Republic of China [6].

1.2.2. Methods of sacrifice.

At the conclusion of the experiment, all rats were humanely euthanized using carbon dioxide (CO2) inhalation, in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). The CO2 euthanasia process was conducted as follows: (1)Animals were placed in a well-ventilated euthanasia chamber. (2)CO2 was gradually introduced into the chamber at a flow rate of 3–70% of the chamber volume per minute to avoid rapid asphyxiation. (3)Euthanasia was confirmed by monitoring physiological signs (cessation of breathing, absence of heartbeat, and corneal reflex) for a minimum of 5 minutes post-loss of consciousness. (4)Secondary verification of death was performed by cervical dislocation.This method was selected for its rapid onset of unconsciousness, minimizing distress, and its compliance with institutional ethical guidelines. The euthanasia procedure was performed by trained personnel with appropriate certification in animal handling and euthanasia techniques.

1.2.3. Efforts to alleviate suffering.

To ensure the welfare of animals and minimize suffering throughout the study, the following measures were implemented:(1)Experimental Design & 3R Principles: Reduction: The sample size was optimized through statistical power calculations to minimize animal numbers while maintaining scientific validity. Refinement: Exercise protocols were designed to gradually acclimate animals to swimming (1-week acclimation period with 15-minute daily weightless swimming sessions) to reduce stress associated with forced exercise.Rationale for Exercise Model: The swimming protocol was selected as a non-invasive, whole-body exercise model with established scientific validity for studying physiological adaptations. (2)During Experimentation:Progressive exercise intensity: The tail-loaded swimming exercise (3% body weight) was introduced only after animals demonstrated physical adaptation during the acclimation phase.Real-Time monitoring: Trained observers continuously monitored animals during the exhaustive swimming session to identify signs of distress (e.g., dysregulated swimming actions, prolonged submersion). The “exhaustion endpoint” was defined by the inability to maintain buoyancy for >5 seconds, at which point the animal was immediately removed from the water to prevent drowning-related suffering.Environmental enrichment: Rats were housed in groups in environmentally enriched cages (including bedding material, chew toys, and hiding spaces) to promote natural behaviors and reduce stress.(3) Post-Exercise & Post-Sacrifice Care: Immediately after the exhaustive swimming session, animals were gently dried with towels and placed in a warm recovery area (maintained at 28–30°C) to prevent hypothermia.Animals were monitored for signs of pain or distress post-exercise; analgesics (e.g., buprenorphine) were administered as needed under veterinary supervision.Euthanasia was performed under deep anesthesia (using pentobarbital) when necessary to ensure painless transition.

1.2.4. Ethics statement.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of NingDe Municipal Hospital of Ningde Normal University (Approval No.: IACUC-20200310) on March 21, 2020. All procedures were performed in strict accordance with the National Standard of China (GB/T 35892-2018) and followed the ARRIVE guidelines 2.0 as well as the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1.3. Experimental design, grouping, and animal models

The sample size of n = 5–6 per group was determined for this exploratory study. As this research was designed to investigate novel mechanistic insights into the S1P/MAPK pathway, a formal a priori power calculation was not feasible. The sample size was selected based on common practices in preliminary physiological research and practical feasibility, with the goal of identifying robust effect trends to inform future hypothesis-driven investigations. The rats were acclimatized for 1 week and subjected to daily 15-minute sessions of weightless swimming exercise. After this adaptation period, rats were randomized into intervention and control groups using a computer-generated randomization list. The random sequence was generated using computer-generated block randomization (block size 4–6) via SAS 9.4, stratified by body weight and baseline measurements. Allocation concealment was ensured using sequentially numbered, sealed, opaque envelopes prepared by a statistician not involved in the experiment. Envelopes were opened only after the rat was enrolled and baseline data recorded. An independent researcher, unrelated to animal housing or treatment, performed the allocation. Randomization occurred immediately before intervention initiation. And 22 rats were randomly divided into the following groups: normal control (group C, n = 5), exercise-preconditioning (EP group, n = 5), EP with the S1PR1-selective antagonist W146 (EP + W146, n = 6), and EP with the MEK1/2 inhibitor PD98059 (EP + PD98059, n = 6).The group C was maintained under routine conditions for 3 weeks. The remaining groups underwent intermittent swimming with a tail-loaded weight equivalent to 3% of their body mass to establish long-term exercise-preconditioned animal models, following the method described by Margonato et al [7]. This involved daily sessions of non-weight-bearing swimming for 15 minutes, followed by tail-loaded swimming in a pool measuring 40 cm deep and 50 cm in diameter at a temperature of 37 ± 2°C. The exercise duration gradually increased from 30 minutes per day in week 1, 1 hour per day in week2, and 2 hours per day in week 3, conducted 6 days per week. The swimming intensity (e.g., swim speed or resistance) does not progressively increased along with the duration. The EP + W146 group received an intraperitoneal injection of W146 (20 mg/kg) 10 min prior to the commencement of each exercise session. The EP + PD98059 group also received PD98059 (100 mg/kg) via identical timing and route (i.p., 10 min before exercise). All inhibitors were given before every exercise session, with doses adjusted according to body weight. All reagents were prepared in normal saline as the vehicle.The preconditioning regimen for each treatment group was identical to the exercise protocol. Three weeks after the experiment commenced, rats in the groupC, EP group, EP + W146 group, and EP + PD98059 group underwent a single bout of exhaustive swimming exercise with a tail-loaded weight equivalent to 3% of their body mass. Exhaustion was determined by evident swimming fatigue, extremely slow paddling speed, continuous sinking, and an inability to persist in swimming.

1.4. Euthanasia and tissue collection

Following euthanasia, which was performed thirty minutes post-modeling as detailed in section 1.2.2, tissue and blood samples were collected for subsequent analysis. Immediately thereafter, blood was drawn via cardiac puncture, and the hearts were rapidly excised, weighed, and rinsed with sterile saline. A portion of the myocardial tissue was snap-frozen for biochemical assays, while another portion was fixed for histological examination.

1.5. Biochemical and histological analyses

1.5.1. Quantification of myocardial S1P.

Myocardial S1P levels were quantified using a competitive ELISA approach. Tissue samples (~100 mg) were homogenized in ice-cold phosphate-buffered saline (1:10, w/v) using a mechanical homogenizer. The homogenate was centrifuged at 3,000 × g for 10 min at 4°C, and the supernatant was collected for analysis. S1P concentrations were determined using a commercial ELISA kit according to the manufacturer’s protocol, with absorbance measurements performed on a BioTek microplate reader (Bolton Company, USA). The assay detection range was 12.35–1000 ng/mL, with a sensitivity of ≤4.73 ng/mL. Total protein concentration in the supernatant was measured using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, USA). Final S1P levels were normalized to total protein content and expressed as ng/mg protein.

1.5.2. Histopathological analysis.

Myocardial tissues were fixed in 10% neutral buffered formalin for 24 hours, routinely processed through a graded ethanol series and xylene, and embedded in paraffin. Sections (4–5 µm thick) were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) following standard procedures. Specifically, nuclei were stained with hematoxylin, differentiated in acid alcohol, and blued in tap water. Cytoplasmic counterstaining was performed with eosin. Finally, sections were dehydrated, cleared in xylene, and mounted. All sections were examined under a light microscope (NIKON ECLIPSE E100, Japan) at 400 × magnification by two experienced pathologists blinded to the experimental groups to assess histological features such as cellular morphology and tissue architecture.

1.5.3. TUNEL assay.

Apoptosis in myocardial tissues was detected using the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, performed according to the manufacturer’s protocol. Nuclei of apoptotic cardiomyocytes were stained brown, while non-apoptotic nuclei appeared blue. For each group, three randomly selected sections were analyzed. From each section, three non-overlapping fields were captured at 200 × magnification. The apoptotic index was calculated as the percentage of TUNEL-positive nuclei relative to the total number of nuclei in the field. To ensure specificity, positive controls (tissue sections treated with DNase) and negative controls (sections incubated without terminal deoxynucleotidyl transferase enzyme) were included in each assay batch. Automated image analysis with predefined thresholds for nuclear size and staining intensity was used for quantification, and results were verified independently by two blinded observers.

1.5.4. Western blot for p-MEK.

The expression of p-MEK was evaluated by Western blot analysis. Protein samples were separated by SDS-PAGE, transferred onto PVDF membranes, and probed with specific primary and secondary antibodies. p-MEK levels were normalized to the loading control (e.g., GAPDH) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

1.6. Blind analysis protocol

To minimize bias in data acquisition and analysis, an assessor-blinded study design was implemented throughout the experimental procedures. Personnel responsible for conducting biochemical assays (ELISA, Western blot), performing histopathological evaluations, and scoring TUNEL assays were unaware of the group assignments until all quantitative analyses were completed.

1.7. Statistical analysis

The concentration of S1P was designated as the single primary endpoint. All statistical analyses were performed using SPSS (version 23.0, IBM, Armonk, NY, USA) and GraphPad Prism (version 10.0, GraphPad Software, San Diego, CA, USA). The normality of all continuous variables was confirmed using the Shapiro-Wilk test. Consequently, data are presented as mean ± standard deviation (SD). For the comparison of the primary endpoint (S1P) and other continuous variables between two groups, the independent two-tailed Student’s t-test was employed. For comparisons across more than two groups, one-way analysis of variance (ANOVA) was applied, followed by Tukey’s post-hoc test for multiple comparisons. The assumption of homogeneity of variances for ANOVA was verified using Levene’s test. For all key comparisons, effect sizes (Cohen’s d for t-tests and η² for ANOVA) along with their 95% confidence intervals (95% CI) are reported. All tests were two-sided, with statistical significance set at P < 0.05.

Results

2.1. Exercise preconditioning attenuates myocardial injury induced by exhaustive exercise in rats

As shown in Fig 1, exercise preconditioning (EP) provided significant protection against exhaustive exercise-induced myocardial injury. Cardiac hypertrophy was not a contributing factor, as indicated by the comparable heart weights between group C and the EP group (P = 0.388; Fig 1A and Table 1). Instead, the cardioprotective effects of EP were evidenced at the tissue and cellular levels. Histological analysis of H&E-stained sections demonstrated markedly improved myocardial preservation in the EP group, characterized by reduced cardiomyocyte rupture and interstitial changes (Fig 1B). Correspondingly, TUNEL assay revealed that EP significantly suppressed exhaustive exercise-induced cardiomyocyte apoptosis (P < 0.001; Fig 1C and 1D and Table 2).

Table 1. Comparison of heart weight between group C and the EP group.

Variable & (Group)	n	Mean ± SD	Statistical Model	P Value	Effect Size	95% CI
The Weight of Heart (g)			t-test	0.388	Cohen’s d = 0.58	[−0.88 2.04]
Group C	5	1.21 ± 0.04
EP Group	5	1.18 ± 0.06

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Table 2. Comparison of the myocardial apoptotic index between group C and the EP group.

Variable & (Group)	n	Mean ± SD	Statistical Model	P Value	Effect Size	95% CI
Apoptotic Index(%)			t-test	<0.001	Cohen’s d = 6.33	[2.75,9.90]
Group C	5	66.20 ± 12.77
EP Group	5	4.80 ± 5.03

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2.2. Exercise preconditioning mediates cardioprotection during exhaustive exercise in rats via enhanced sphingosine-1-phosphate (S1P) generation

2.2.1. Exercise preconditioning upregulates myocardial S1P content in rats subjected to exhaustive exercise.

Following exhaustive exercise, the myocardial S1P content in the EP group was significantly higher than in group C (P = 0.002; Fig 2A and Table 3), with a large effect size (Cohen’s d = 2.79, 95% CI [−4.25, −1.34]).

Table 3. Myocardial S1P concentration in group C and EP-group rats after exhaustive exercise.

Variable & (Group)	n	Mean ± SD	Statistical Model	P Value	Effect Size	95% CI
S1P(ng/mg)			t-test	0.002	Cohen’s d = 2.79	[−4.25, −1.34]
Group C	5	1.19. ± 0.62
EP Group	5	1.36 ± 0.62

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2.2.2. S1P attenuates exhaustive exercise-induced eyocardial injury in rats.

Administration of the S1PR1-selective antagonist W146 significantly abrogated the cardioprotective effects of exercise preconditioning. The heart weight of rats in the EP + W146 group was significantly higher than in the EP group (P = 0.008; Fig 2B and Table 4). Consistent with this, H&E-stained sections revealed more prominent cardiomyocyte rupture and interstitial edema in the EP + W146 group compared to the EP group (Fig 2C). Furthermore, TUNEL assay demonstrated a significant increase in exhaustive exercise-induced cardiomyocyte apoptosis in the EP + W146 group (P = 0.016; Fig 2D, 2E and Table 5).

Table 4. Comparison of heart weight between the EP and EP + W146 groups.

Variable	n	Mean ± SD	Statistical Model	P Value	Effect Size	95% CI
Weight of heart (g)			t-test	0.008	Cohen’s d = 2.03	[−0.19, −0.04]
EP Group	5	1.18 ± 0.06
EP + W146 Group	6	1.30 ± 0.05

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Table 5. Comparison of the myocardial apoptotic index between the EP and EP + W146 groups.

Variable & (Group)	n	Mean ± SD	Statistical Model	P Value	Effect Size	95% CI
Apoptotic Index (%)	5		t- test	0.016	Cohen’s d = 1.80	[−3.24, −0.18]
EP Group		4.80 ± 5.03
EP + W146 Group	6	35.30 ± 22.32

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2.3. S1P protects against exhaustive exercise-induced myocardial injury in rats via the MAPK pathway

2.3.1. MEK1/2 inhibition abrogates S1P-mediated exercise preconditioning.

Administration of the MEK1/2 inhibitor PD98059 revealed a critical role for the MAPK pathway. A one-way ANOVA identified a significant main effect of group on p-MEK expression (F(3,8) = 113.74, P < 0.0001, η² = 0.977), as shown in Fig 3A. Post-hoc testing confirmed that the exhaustive exercise-induced increase in p-MEK observed in the EP group was significantly abrogated in the EP + PD98059 group (P < 0.05). In contrast, myocardial S1P content after exhaustive exercise did not differ significantly between these two groups (P = 0.73; Fig 3B and Table 6).

Fig 3 — (A) Expression of p-MEK protein. Representative Western blots and densitometric analysis are shown. Data are presented as mean ± SD (n = 3 per group). One-way ANOVA revealed a significant main effect of group (F(3,8) = 113.74, P < 0.0001, η² = 0.98). Post-hoc Tukey’s test showed that the p-MEK level in the EP group was significantly higher than that in the EP + PD98059 group (^*P < 0.05), ^**P < 0.001 vs. other relevant groups. (B) Myocardial S1P content. S1P was quantified by ELISA. Data are expressed as mean ± SD (n = 5−6 per group). An independent samples Student’s t-test showed no significant difference between the EP and EP + PD98059 groups (P = 0.73, Cohen’s d = 0.04, 95% CI [−1.42, 1.50]).(C) Heart weights of rats. Data are expressed as mean ± SD (n = 5−6 per group). An independent samples Student’s t-test showed no significant difference between the EP and EP + PD98059 groups (P = 0.849, Cohen’s d = 0.12, 95% CI [−1.25, 1.49]). (D) Representative H&E-stained sections of rat myocardium (400×). The EP + PD98059 group exhibits increased cardiomyocyte rupture and interstitial changes compared to the EP group. Scale bar: 25 µm. (E & F) Cardiomyocyte apoptosis assessed by TUNEL staining. Data are expressed as mean ± SD (n = 5−6 per group). An independent samples Student’s t-test indicated a significantly higher apoptosis index in the EP + PD98059 group than in the EP group (P = 0.002, Cohen’s d = 2.58,[−4.43, −0.73]). Scale bar: 25 µm.

Table 6. Comparison of myocardial S1P concentration between the EP and EP + PD98059 groups.

Variable & (Group)	n	Mean ± SD	Statistical Model	P Value	Effect Size	95% CI
S1P(ng/mg)			t-test	0.732	Cohen’s d = 0.04	[−1.42, 1.50]
EP Group	5	1.37 ± 0.62
EP + PD98059 Group	6	1.35 ± 0.83

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2.3.2. Role of MEK1/2 signaling in exercise preconditioning-induced cardioprotection.

Inhibition of MEK1/2 with PD98059 had no significant effect on heart weight compared to the EP group alone (P = 0.849; Fig 3C, Table 7). However, it exacerbated exhaustive exercise-induced myocardial injury, as evidenced by more severe cardiomyocyte rupture and interstitial changes (Fig 3D) and a significant increase in cardiomyocyte apoptosis (P = 0.002; Fig 3E, 3F, Table 8).

Table 7. Comparison of heart weight between the EP and EP + PD98059 groups.

Variable	n	Mean ± SD	Statistical Model	P Value	Effect Size	95% CI
Weight of heart (g)			t-test	0.849	Cohen’s d = 0.12	[−1.25, 1.49]
EP Group	5	1.18 ± 0.06
EP + PD98059 Group	6	1.17 ± 0.08

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Table 8. Comparison of the myocardial apoptotic index between the EP and EP + PD98059 groups.

Variable & (Group)	n	Median [IQR]	Statistical Model	P Value	Effect Size	95% CI
Apoptotic Index(%)			t- test	0.002	Cohen’s d = 2.58	[−4.43, −0.73]
EP Group	5	4.80 ± 5.03
EP + PD98059 Group	6	30.01 ± 12.33

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Discussion

Exercise preconditioning, characterized by repeated transient intermittent high-intensity exercise, enhances the heart’s resilience against prolonged ischemic hypoxia and is considered effective in mitigating myocardial ischemic damage [8]. Several studies have demonstrated that exercise preconditioning can alleviate myocardial damage in rats subjected to exhaustive exercise, evident as reduced myocardial hypertrophy, myocardial rupture, and myocardial interstitial hyperplasia [9,10]. In this experiment, HE staining revealed significantly less myocardial rupture and interstitial hyperplasia in rats subjected to exercise preconditioning compared to those without preconditioning. However, there was no significant difference in heart weight between the exercise-preconditioned group and the control group, suggesting a potential balance between myocardial apoptosis, hypertrophy, and interstitial hyperplasia in the control group. Additionally, exercise preconditioning may exert myocardial protective effects by reducing cardiomyocyte free radical release [9] and up-regulating LC3 protein via other pathways [11].

Studies have demonstrated the protective role of S1P for cardiomyocytes in ischemic preconditioning and postischemic conditioning [3]. The findings of this experiment indicate that myocardial S1P content in exhausted rats subjected to exercise preconditioning is significantly higher compared to the control group, consistent with Morrell MBG et al.‘s findings [12]. The mechanism by which exercise influences S1PR1 protein content involves central actions of IL-6 in the hypothalamus [13], increased plasma S1P content through exercise, leading to elevated S1P levels in skeletal muscle [14], and exercise-induced release of vascular endothelial cell-derived S1P [15,16].

The results further revealed that inhibition of S1PR1 with W146 significantly increased the myocardial apoptotic index following exercise preconditioning, suggesting that the S1P/S1PR1 axis is critical for conferring anti-apoptotic protection.. Addition of the S1PR1-selective antagonist significantly increased heart weight, myocyte rupture, and interstitial changes in exhausted rats, collectively indicating that S1P protects myocardium in exhausted rats. Its mechanisms may involve promoting the biological effects of muscle through enhanced expression of S1PRs in skeletal muscle, such as excitation-contraction coupling, activation of satellite cells, and protection via improved mitochondrial functions that enhance skeletal muscle cell adaptability to exercise training [14–17].

Additionally, the results of this study indicate that there was no significant difference in S1P content in exhausted rats between the exercise-preconditioned + the MEK1/2 inhibitor PD98059 groups, suggesting that PD98059 do not markedly reduce S1P levels in this context. Western blot analysis showed significantly weaker p-MEK expression in the EP + PD98059 group compared to the exercise-preconditioned group. HE staining indicated increased myocyte rupture and interstitial changes in the EP + PD98059, suggesting greater myocardial damage compared to the non-MEK1/2 inhibitor group. And studies show that PD98059 exacerbates myocardial injury in exercise-precondition by blocking survival signals, inhibiting endogenous protectants and aggravating oxidative stress, underscoring the indispensable role of MAPK in adaptive cardioprotection,which can explain why apoptosis increases [18,19]. These findings suggest that S1P likely plays a role in myocardial protection through MAPK signaling pathways independent of affecting S1P content.

A key question in S1P-MAPK biology is whether their relationship constitutes a bidirectional feedback loop or can exhibit a dominant directional flow in specific physiological contexts. Although prior work in endurance adaptation has described a robust S1P-MAPK positive feedback loop [5,16], our findings in an exercise preconditioning model provide evidence for the latter. The demonstration that MAPK inhibition completely abolishes protection while leaving S1P levels unchanged allows us to disentangle this interplay, positioning MAPK activation as an essential downstream event contingent upon S1P signaling. This proposes a refined, S1P→MAPK hierarchical model that complements the existing concept of a feedback loop and offers a more precise mechanistic framework for exercise-induced cardioprotection.

Previous studies have reported that activation of the S1P/S1PR1/STAT3 signaling pathway may promote inflammatory damage and fibrosis of the cardiac valves [20]; and the activation of S1P receptors plays a crucial role in inhibiting the activation of Caspase-3, a key executor of apoptosis, by stimulating the AKT pathway [21]. And the studies suggests that eNOS-knockout mice lose S1P-mediated cardioprotection [22]; endothelial S1PR1 regulates pressure overload-induced cardiac remodelling through AKT-eNOS pathway [23]. Whether exercise preconditioning influences S1P synthesis through these pathways awaits further experimental validation.

In summary, while our findings confirm established roles of exercise preconditioning and S1P, they propose a novel hierarchical relationship by suggesting that the MAPK pathway is a necessary downstream component of S1P-mediated protection. This proposed S1P→MAPK axis represents a key mechanistic insight from our work, and future work is warranted to fully validate this signaling hierarchy.

Supporting information

S1 File. WB-raw-images.

Contains the complete set of original, uncropped, and unprocessed Western blot images from which the data presented in this study were derived. The lanes containing the molecular weight markers are present. The corresponding molecular weight calibration reference for these markers is provided in S2 File.

(ZIP)

pone.0340313.s001.zip^{(10.3MB, zip)}

S2 File. Molecular weight marker reference.

Provides a reference image showing the precise alignment of the molecular weight markers (in kDa) with their corresponding bands in the original Western blot images contained in S1 File. This allows for independent verification of protein sizes presented in the main figures.

(PDF)

pone.0340313.s002.pdf^{(4.1MB, pdf)}

S3 File. Raw H&E source images.

Contains all original, uncropped microscope images supporting the histology results in Fig 1B, Fig 2C, and Fig 3D, provided as a ZIP archive (S3_File RAW HE.zip). The archive includes the following files, with their specific correspondences detailed below: S3 File C.jpg: The 40x field for the Control group panel in Fig 1B. S3 File EP 1.jpg: The 40x field for the EP group panel in Fig 1B. S3 File EP 2.jpg: The 40x field for the EP group panel in Fig 2C. S3 File EP 3.jpg: The 40x field for the EP group panel in Fig 3D. S3 File W.jpg: The 40x field for the EP + W146 group panel in Fig 2C. S3 File P.jpg: The 40x field for the EP + PD98059 group panel in Fig 3D. S3 File EP Source.jpg: The low-magnification (3x) source image from which the three EP group 40x fields (EP_1, EP_2, EP_3) were cropped. S3 File EP Guide.tif: An annotated guide image. The locations of the cropped 40x fields are outlined and labeled as EP_1 (for Fig 1B), EP_2 (for Fig 2C), and EP_3 (for Fig 3D) within this source image.

(ZIP)

pone.0340313.s003.zip^{(34.3MB, zip)}

S4 File. Supplementary representative histological images (H&E staining).

This file contains additional, representative H&E-stained images from the study that were not featured in the main figures but provide further context and demonstrate the consistency of observations within each experimental group. The images cover all four groups: Control (C), EP group (EP), EP + W146 (W), and EP + PD98059(P). Both low-magnification (3x) overviews and high-magnification (40x) detail views are included where available. These supplementary images support the robustness and generalizability of the histological findings presented in the manuscript.

(ZIP)

pone.0340313.s004.zip^{(27.7MB, zip)}

Data Availability

All data generated or analysed during this study are available in the Figshare repository: [Dataset for “Effect of Exercise Preconditioning on Myocardial Content of Sphingosine1-phosphate and its Mechanism in Rats after Exhaustive Exercise “] (DOI: [https://doi.org/10.6084/m9.figshare.30473615]).

Funding Statement

This work was supported by the General Program of Fujian Provincial Natural Science Foundation, China (Grant No. 2024J01941) to Xinnuan Wei.

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PLoS One. doi: 10.1371/journal.pone.0340313.r001

Decision Letter 0

Amirreza Khalaji

16 Jul 2025

Dear Dr. Yang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: N/A

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

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: The authors are presenting a rodent study in which the want to describe the effect of Exercise Preconditioning on Myocardial Content of S1P and its Mechanism after Exhaustive Exercise. Unfortunately, the data presented is not comprehensive enough to seriously underline the authors' conclusions. There is a clear need for improvement both in the methodology and in the presentation and interpretation of the data. Furthermore, the knowledge gap to be filled here is very small. There is some data on S1P in exercise and training that should have been discussed in more detail here. Overall, the idea is a good one, but significant improvements are needed before publication. My suggestions are as follows:

Abstract:

- Typos in S1P

- Methods described in great detail in relation to the rest.

- The results are difficult to understand. The enumeration (1.,2.,3.) seems more like a bullet point list. Where possible, the wording should also be chosen so that results are compared with the control group. Otherwise this is very misleading.

Methods:

- The headings should be more clearly separated from the text. Reading would be easier if the headings were also visually marked (bold, italics, etc.).

- W146 is not just an “S1P receptor blocker”, but a specific antagonist of S1PR1. This should be made clear, as S1P can mediate its effects via the other S1PRs

- The description of PD98059 as a MAPK blocker is also not ideal. It is a MAPKK inhibitor (MEK1/2)

- It remains unclear in which solvent the substances were administered

- Were there any differences in the duration of the final “single bout of exhaustive swimming exercise” in the test animals?

- The preparation and the amount of myocardial tissue to be prepared for the S1P determination is not mentioned

Results:

- The authors refer to tables in several places. However, these cannot be found in the manuscript.

- The designation “group C” for the controls is confusing, especially as the figures show “Control”.

- Fig. 1A: A reduction in heart weights cannot be seen from the graph alone. Even with the caveat that this is not a significant change, the authors should not describe these heart weights of the groups as different.

- When describing and presenting data, one should always compare the treatment with the controls. This alternate presentation (e.g. Results to Fig. 1B) is misleading. Here it would be “were decreased in EP group...”

- Fig. 1B: Can the histological effect also be quantified?

- Fig. 1D: 75% apoptosis in the myocardium after a single swim seems almost implausibly high to me. Does this match the literature? Is there any way to explain this? The animals should hardly be viable after this training.

- Fig. 2A. It is unclear how this unit comes about. The amount of S1P in the myocardium cannot be given as ng/ml without further information and explanations.

- Fig. 2A: Are there also differences in plasma S1P?

- Fig. 2C: Is there a possibility of quantification? A change in cardiomyocyte rupture, as described by the authors, can hardly be detected.

- Fig. 2E: Was the inhibition of other S1PR also tested? S1PR3 in particular also seems to be of interest here

- Fig. 3A. A sectioned Western blot image is not sufficient to support the statement “the expression of p-MEK in the myocardium of rats... was significantly reduced...”. Quantification with statistically significant differences is required here.

- Fig. 3B/C: It is not possible to speak of differences here. The text should be revised.

Discussion

- The authors do offer a number of references to justify the finding mechanism. However, this is not sufficient. It is possible that the increased formation of S1P via SphK1 also plays a role here. This has already been shown for other cells that were also exposed to a certain amount of stress (Benkhoff et al., Circulation. 2025).

Reviewer #2: Strengths

The experimental design is logical and relevant to the study question.

The use of both S1P receptor and MAPK pathway inhibitors helps dissect the mechanisms involved.

The figures support the text well and show clear experimental differences.

Ethical approval and animal welfare guidelines were followed.

Weaknesses and Suggestions for Improvement

1. Sample Size & Randomization: Clarify how rats were randomized into groups. Indicate whether investigators were blinded during analysis.

2. Data Transparency: Include numerical data (mean ± SD) within the main text or supplementary tables. For instance, actual S1P concentrations in each group. Figures are provided, although raw data (e.g., actual ELISA values, full apoptotic counts) could further strengthen transparency.

3. Statistics: Indicate whether post hoc tests were used. Also, include effect sizes or confidence intervals where possible. The manuscript lacks a statement on statistical power or sample size calculation. Exact p-values (rather than simply “P < 0.05”) would improve transparency.

4. Discussion: While generally well-referenced, the discussion would benefit from addressing alternative pathways (e.g., eNOS or STAT3) as mentioned briefly at the end. Consider whether MAPK activation is a downstream result or a parallel pathway influenced by S1P.

5. Figure Legends: Improve clarity. Example: Figure 2 – instead of “showed increase,” write “was significantly higher than the EP group (P < 0.05).”

6. Grammar & Language: Minor editing needed for readability and fluency. A few examples:

Replace “cardiomyocyte rupture and interstitial changes were increased” with “cardiomyocyte rupture and interstitial edema were more prominent.”

“This study was carried out in accordance with the principles of the Basel Declaration.” → "The study adhered to the Basel Declaration on animal research ethics."

"An observable dysregulation of swimming actions..." → Consider simplifying to "evident swimming fatigue."

Use active voice more often. Avoid redundant phrases like “compared to the EP group, the EP+W146 group showed…” repetitively.

Figures and Tables

Figure Quality: Acceptable but can be improved by adding scale bars, clearer labels (e.g., “n = 6/group”), and including actual values in bar charts.

Table Clarity: The tables referenced (Table 1–8) ??? are not embedded or visualized in the version reviewed. If these exist separately, ensure they are submitted as part of the supplemental material.

Reviewer #3: Thank you to the authors for their comprehensive and well-designed study on exercise preconditioning and its effects on myocardial S1P content in rats. The study is timely, and it provides valuable insights into the potential mechanisms underlying exercise-induced myocardial protection. Below are my specific comments aimed at improving clarity, experimental rigor, and presentation of results.

Introduction

Introduction is written summarized and well. The only point that could be mentioned is the relationship of S1P and MAPK in the literature, regardless of preconditioning. Before writing " Previous reports indicate that the MAPK signaling pathway may attenuate cardiac fibrosis, apoptosis, oxidative stress injury, and inflammatory reactions [5]." You can bring a bridge from s1p to MAPK and their importance in preconditioning… .

Methodological Recommendations:

1. Clarification of Exercise Protocol:

o The manuscript states that the exercise duration gradually increased from 30 minutes/day to 2 hours/day. It would be helpful to specify how the intensity of the swimming exercise changed during this period. For example, was the swimming intensity (e.g., swim speed or resistance) progressively increased along with the duration? Detailing the intensity would improve the reproducibility of the exercise protocol.

2. Control Group and Randomization:

o While the animals were randomly assigned to groups, it is essential to explicitly mention whether the experimenters were blinded to the group assignments and the outcome measures (e.g., apoptosis index, S1P levels). Blinding helps reduce potential biases during outcome assessments.

3. Sample Size Justification:

o While the sample sizes for each group are mentioned (e.g., n=5 or n=6), the manuscript would benefit from a clear justification of how these sample sizes were determined. Were power analyses conducted to ensure that the study had enough statistical power to detect differences between groups? If not, this could be a limitation to address in the revised manuscript.

4. S1P Measurement:

o The manuscript mentions using an ELISA kit to measure myocardial S1P content. Did you validate the S1P ELISA kit against other methods, such as mass spectrometry, to ensure its accuracy? This validation would strengthen the reliability of the S1P data.

5. Inhibition Timing and Dosing:

o The manuscript describes the use of the S1P receptor blocker W146 and MAPK inhibitor PD98059, but the exact timing of administration relative to the exercise sessions could be more clearly defined. Were these inhibitors given before every exercise session or just once a day? Was the dose adjusted over time based on the animals' body weight? These details are critical for reproducibility and understanding the effectiveness of the treatments.

6. Method of Exhaustion Induction:

o The criterion for exhaustion (e.g., observable dysregulation of swimming actions, slow paddling speed, continuous sinking) is subjective. While this is commonly used in exercise studies, addressing this issue in limitation would enhance the clarity of the findings.

Results and Data Presentation:

1. Figures

o It would be informative to provide the exact data or statistical analysis in the figure legend to allow the reader to understand how significant the difference is between groups.

o In Figure 3E, apoptosis is increased in the EP + PD98059 group. A discussion about the mechanisms contributing to this finding (i.e., how MAPK inhibition could increase myocardial damage despite exercise preconditioning) would be helpful to provide a more comprehensive interpretation of the data.

Reviewer #4: Comment 1: The tables are not visible in the main text. Please include them at the end of the manuscript instead of submitting them as a supplementary file.

Comment 2: While the study suggests MAPK signaling is involved in S1P-mediated protection, the molecular mechanism remains speculative. Consider discussing alternative or complementary signaling pathways such as AKT or STAT3, which were briefly mentioned but not tested.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

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PLoS One. 2026 Jan 7;21(1):e0340313. doi: 10.1371/journal.pone.0340313.r002

Author response to Decision Letter 1

10 Sep 2025

We have made correction according to the Reviewer’s comments.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0340313.s006.docx^{(23.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0340313.r003

Decision Letter 1

Amirreza Khalaji

29 Sep 2025

Dear Dr. Yang,

Please submit your revised manuscript by Nov 13 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Amirreza Khalaji

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #5: (No Response)

Reviewer #6: All comments have been addressed

Reviewer #7: All comments have been addressed

Reviewer #8: (No Response)

**********

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

Reviewer #3: Yes

Reviewer #5: No

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #3: Yes

Reviewer #5: No

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

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

The PLOS Data policy

Reviewer #3: Yes

Reviewer #5: No

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: No

**********

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

Reviewer #3: Yes

Reviewer #5: No

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

Reviewer #3: Dear authors,

Thank you for applying the recommendations. The current version of the manuscript is clearer and more rigorous.

Best wishes

Reviewer #5: The manuscript investigates whether exercise preconditioning (EP) mitigates exhaustive-exercise–induced myocardial injury in rats and explores a mechanistic pathway involving sphingosine-1-phosphate (S1P) and MAPK signaling across four groups (Control, EP, EP+W146, EP+PD98059), with outcomes including heart weight, histology, apoptosis, S1P levels, and p-MEK. This is a revised submission; however, substantive issues remain in methodological reporting (e.g., blinding, euthanasia parameters, randomization, sample-size justification), statistical transparency (tests, assumptions, multiple-comparison control, effect sizes/CIs), and internal consistency between text, tables, and figure legends. Several contradictions (e.g., non-significant results described as decreases) and formatting/artifact problems persist, affecting interpretability and reproducibility. In its current form, the study requires major revision to meet PLOS ONE’s rigor and reporting standards.

Major comments

1. Results describe a decrease “not statistically significant,” and the table shows no difference (e.g., p≈0.95), yet the Figure 3 legend asserts a decrease. All sections must agree and use neutral wording that reflects the statistics.

2. Results indicate no significant difference (P>0.05), but the figure legend states “Cardiac weight was increased.” Legend phrasing should mirror the statistical outcome.

3. Table content contains a merged/duplicated line and mixes mean±SD with median(IQR) for the same endpoint. Clean the table and use one summary metric aligned with the applied test.

4. CO₂ flow is reported as “3–70% chamber volume per minute,” and elsewhere euthanasia is described under deep anesthesia “when necessary.” Specify one guideline-concordant method with exact parameters and apply it consistently.

5. Blinding is stated despite visibly different procedures. Specify who was blinded, how (coding/masking), and which outcomes were assessed blinded (e.g., coded histology/apoptosis); otherwise revise to assessor-blind.

6. Power is reported using a regression effect size (f²=0.15) for a four-group design; achieved n≈5–6/group is small for histology/apoptosis. Provide an ANOVA/GLM-appropriate calculation (primary outcome, effect size, α, power) or state that the study is exploratory.

7. P-values are reported without naming the tests, assumption checks, multiple-comparison handling, effect sizes, or 95% CIs. Add a Statistics subsection with software, tests/models per endpoint, diagnostics, and corrections; report estimates with CIs.

8. Clarify whether heart weight is absolute or normalized (and how), define apoptosis index quantification (fields, thresholds, blinding), provide S1P kit/catalog details, and include n/group in figure panels.

9. Randomization is stated without method/timing (sequence generation, concealment, independent allocator). Provide concrete implementation steps.

Minor comments

10. Figure legends—units and n/group: Add units (e.g., S1P ng/mL, °C) and n/group in each legend; ensure legend wording reflects the actual statistical outcome (e.g., “no significant difference” where applicable).

11. Data availability / ethics alignment. Ensure the Data Availability and Ethics statements match Methods with precise institutional approval details and access to raw per-animal data (and original images where applicable).

Reviewer #6: (No Response)

Reviewer #7: Thank you for the opportunity to review; the manuscript has been carefully revised—no further changes are required.

Reviewer #8: Dear Authors

Thank you for your submission and for the substantial effort invested in a valuable. Yor revision sounds good and almost complete, but there are minor things to care. The comments that follow are offered in a constructive spirit to help sharpen the manuscript’s clarity and maximize its usefulness to researchers.

My comments:

-Reconcile the direction of S1P change (EP vs Control) across text, tables, and figures; ensure the narrative matches the numeric values and final conclusions.

-Consolidate to one Statistics section (remove legacy one-way ANOVA text if not used).

-Name a single primary endpoint (used for power) and map each outcome (heart weight, S1P, apoptosis, p-MEK) to: -distribution check (e.g., Shapiro–Wilk),-summary measure (mean±SD or median[IQR]), exact test used, post-hoc method for the 4-group design, multiple-comparison control (e.g., Tukey, Dunn, Holm/Benjamini–Hochberg), effect sizes and 95% CIs.

-Resolve software(version) inconsistencies (e.g., SPSS 17 vs newer; Prism) and state the final versions only.

-Use W146 = S1PR1-selective antagonist; PD98059 = MEK1/2 (MAPKK) inhibitor.

Remove any remaining “MAPK blocker” wording in text, tables, and figure legends.

-Report myocardial S1P normalized to tissue mass (ng/g) or protein (ng/mg); if retaining ng/mL homogenate, provide a clear calculation pipeline (homogenization volumes, protein assay) so others can reproduce it.

Add operational details, who generated or kept the randomization list, how allocation was concealed, who assigned animals, who was blinded for TUNEL scoring and Western densitometry, and when blinding was lifted.

Reconcile inconsistent euthanasia, and anesthesia descriptions; ensure the Methods reflect the actual procedure and are consistent across manuscript and submission materials.

-Include representative images with scale bars.

-Describe field selection, counting thresholds, automation, and positive-negative controls to address potential TUNEL over-calling.

-Remove tracked-changes artifacts, duplications; correct any duplicated or corrupted table entries.

-Ensure each legend lists n/group, exact test, post-hoc method, and exact P-value criteria.

- Add explicit statements on: subjective exhaustion criterion; small sample size; single sex; ELISA vs LC-MS/MS for S1P; lack of functional cardiac readouts (e.g., echo, infarct size); and inhibitor specificity caveats.

-Specify exact timing of W146 and PD98059 (relative to each preconditioning session and to the exhaustive swim), vehicle, dose per kg, and whether dosing changed over time.

-State clearly what is novel (S1P→MAPK inhibition within an exercise-preconditioning model) versus confirmatory, and situate findings against the most relevant prior EP/S1P cardioprotection literature.

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PLoS One. 2026 Jan 7;21(1):e0340313. doi: 10.1371/journal.pone.0340313.r004

Author response to Decision Letter 2

29 Oct 2025

We are deeply grateful to Dr. Amirreza Khalaji and the Reviewers for their time and insightful comments on our manuscript. We have carefully considered all feedback and have thoroughly revised the manuscript to address each point raised.

Key improvements include:

Explicitly framing the study as exploratory and clarifying the novel S1P→MAPK signaling hypothesis versus confirmatory aspects in the Discussion.

Adding a detailed “Statistical Analysis” subsection and ensuring comprehensive reporting of tests, effect sizes, and confidence intervals throughout.

Enhancing methodological transparency regarding heart weight, apoptosis quantification, S1P normalization, and sample size indication in figures.

Consulting a biostatistician to ensure the rigor and appropriateness of all statistical approaches.

We believe these revisions have significantly strengthened the manuscript and hope it now meets the journal’s standards for publication. Thank you again for the opportunity to improve our work.

PLoS One. doi: 10.1371/journal.pone.0340313.r005

Decision Letter 2

Amirreza Khalaji

11 Nov 2025

Dear Dr. Weiyuan Yang,

Please submit your revised manuscript by Dec 26 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewer #5: The revision does not provide proper, point-by-point answers to the reviewers’ comments; many substantive issues remain unaddressed. For example, a required Declarations section is still missing (ethics approval/consent, data availability with an accessible link, funding, competing interests, and author contributions).

Reviewer #8: Dear authors,

I checked all revision files, unfortunately I can't find point by point response letter or section from authors.

please upload it again.

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PLoS One. 2026 Jan 7;21(1):e0340313. doi: 10.1371/journal.pone.0340313.r006

Author response to Decision Letter 3

12 Nov 2025

Reviewer #5: Thank you for your feedback and for bringing this to our attention. We sincerely apologize for the oversight in our previous submission. The point-by-point response to the reviewers' comments was inadvertently omitted during the upload process. We have now uploaded the "Response to Reviewers" document to the submission portal. This document provides a detailed, point-by-point account of all changes made in response to the reviewers' valuable comments. Regarding the Declarations section, we confirm that all required statements, including the ethics approval and consent which are detailed in section 1.2.4 "Ethics Statement" of the manuscript, are fully included. The complete Data Availability statement with the accessible link is also present within the main manuscript file.

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Attachment

Submitted filename: Response to Reviewers_updated.doc

pone.0340313.s008.doc^{(113KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0340313.r007

Decision Letter 3

Amirreza Khalaji

18 Dec 2025

Effect of Exercise Preconditioning on Myocardial Content of Sphingosine1-phosphate and its Mechanism in Rats after Exhaustive Exercise

PONE-D-25-14601R3

Dear Dr. Weiyuan Yang,

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Amirreza Khalaji

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PLOS One

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PLoS One. doi: 10.1371/journal.pone.0340313.r008

Acceptance letter

Amirreza Khalaji

PONE-D-25-14601R3

PLOS One

Dear Dr. Yang,

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. WB-raw-images.

(ZIP)

pone.0340313.s001.zip^{(10.3MB, zip)}

S2 File. Molecular weight marker reference.

(PDF)

pone.0340313.s002.pdf^{(4.1MB, pdf)}

S3 File. Raw H&E source images.

(ZIP)

pone.0340313.s003.zip^{(34.3MB, zip)}

S4 File. Supplementary representative histological images (H&E staining).

(ZIP)

pone.0340313.s004.zip^{(27.7MB, zip)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0340313.s006.docx^{(23.8KB, docx)}

Attachment

Submitted filename: Response to Reviewers_updated.doc

pone.0340313.s008.doc^{(113KB, doc)}

Data Availability Statement

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PERMALINK

Effect of exercise preconditioning on myocardial content of Sphingosine1-phosphate and its mechanism in rats after exhaustive exercise

Xinnuan Wei

Weiyuan Yang

Junxiang Zhou

Luoyuan Cao

Wenxing Jiang

Roles

Abstract

Objective

Methods

Results

Conclusion

Introduction

Materials and methods

1.1. Primary reagents and kits

1.2. Experimental animals and ethics statement

1.2.1. Experimental animals.

1.2.2. Methods of sacrifice.

1.2.3. Efforts to alleviate suffering.

1.2.4. Ethics statement.

1.3. Experimental design, grouping, and animal models

1.4. Euthanasia and tissue collection

1.5. Biochemical and histological analyses

1.5.1. Quantification of myocardial S1P.

1.5.2. Histopathological analysis.

1.5.3. TUNEL assay.

1.5.4. Western blot for p-MEK.

1.6. Blind analysis protocol

1.7. Statistical analysis

Results

2.1. Exercise preconditioning attenuates myocardial injury induced by exhaustive exercise in rats

Fig 1. Exercise preconditioning attenuates myocardial injury induced by exhaustive exercise in rats.

Table 1. Comparison of heart weight between group C and the EP group.

Table 2. Comparison of the myocardial apoptotic index between group C and the EP group.

2.2. Exercise preconditioning mediates cardioprotection during exhaustive exercise in rats via enhanced sphingosine-1-phosphate (S1P) generation

2.2.1. Exercise preconditioning upregulates myocardial S1P content in rats subjected to exhaustive exercise.

Table 3. Myocardial S1P concentration in group C and EP-group rats after exhaustive exercise.

2.2.2. S1P attenuates exhaustive exercise-induced eyocardial injury in rats.

Table 4. Comparison of heart weight between the EP and EP + W146 groups.

Table 5. Comparison of the myocardial apoptotic index between the EP and EP + W146 groups.

2.3. S1P protects against exhaustive exercise-induced myocardial injury in rats via the MAPK pathway

2.3.1. MEK1/2 inhibition abrogates S1P-mediated exercise preconditioning.

Fig 3. S1P exerts a protective effect in the rat myocardium against exhaustive exercise via the MAPK signaling pathway.

Table 6. Comparison of myocardial S1P concentration between the EP and EP + PD98059 groups.

2.3.2. Role of MEK1/2 signaling in exercise preconditioning-induced cardioprotection.

Table 7. Comparison of heart weight between the EP and EP + PD98059 groups.

Table 8. Comparison of the myocardial apoptotic index between the EP and EP + PD98059 groups.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Amirreza Khalaji

Roles

Author response to Decision Letter 1

Decision Letter 1

Amirreza Khalaji

Roles

Author response to Decision Letter 2

Decision Letter 2

Amirreza Khalaji

Roles

Author response to Decision Letter 3

Decision Letter 3

Amirreza Khalaji

Roles

Acceptance letter

Amirreza Khalaji

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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