Cooling of cultured water can resist heart failure caused by collagen deposition and necrosis of cardiac bers under chronic heat stress in sturgeon

Chaoyang Zhang Sichuan Agricultural University Miao Zhang Sichuan Agricultural University Zihan Xu Sichuan Agricultural University Datian Li Sichuan Agricultural University Quan Gong Sichuan Academy of Agricultural Sciences Xiaoli Huang Sichuan Agricultural University Jiayun Wu Sichuan Agricultural University Zhi He Sichuan Agricultural University Xiaogang Du Sichuan Agricultural University Defang Chen Sichuan Agricultural University Jun Jiang Sichuan Agricultural University Jun Du Sichuan Agricultural University Shiyong Yang (  yangshiyong@sicau.edu.cn ) Sichuan Agricultural University


Introduction
Global warming and the construction of river dams can cause the water temperature of rivers to increase for extended periods of time which threatens the survival of aquatic organisms [1][2][3][4][5][6]. Chronic heat stress (CHS) occurs when the environmental temperature exceeds the optimal temperature range for sh to survive for a long-term [7][8][9][10]. CHS can directly threaten the physiological stability of freshwater species, affecting their behavior, growth, development, reproduction, immune, digestion, and circulatory system, and can even cause death [11][12][13][14][15][16].
Acipenser baerii is widely distributed in important economic sh in the northern hemisphere, and the caviar produced by adult sh has extremely high nutritional value [17][18][19][20]. Sichuan Province's sturgeon farming production ranks among the top in China, and its normal growth temperature of sturgeon is maintained at 5-20°C all year round (Data form Sichuan Runzhao Fishery Co., Ltd). However, in summer, the water temperature of sturgeon breeding ponds often increases suddenly and lasts for 9-12 days, causing the CHS and a large number of deaths of cultured sturgeons. Mai et al. found that the growth of A. baerii was signi cantly hindered when the feeding water temperature was maintained at 24°C, and the survival rate at 27°C was signi cantly reduced [21]. Our team's CHS experiments show that the survival of A.baerii decreases when the water temperature exceeds 20°C (Yang et al. 2021). In the process of growth and development, the normal maintenance of heart function is essential to ensuring normal growth, development, and reproductive behavior [22]. Heart, the life-sustaining organ of sh, generates pressure to circulate the blood, transport oxygen from sh gills to various tissues, and remove metabolic waste products, thereby supporting active biological processes [23]. Therefore, the normal physiological temperature of the heart is the prerequisite for maintaining the high production e ciency of sturgeon. In recent years, an increasing number of studies have examined the effects of heat stress on sturgeon [6, 14,24]. However, few have examined the effect of CHS on the sturgeon heart and establish the relationship between heart structure and function.
Tissue blood perfusion requires rhythmic and periodic contraction and relaxation of the heart ventricles and complete cardiomyocyte support. Among them, the changes in ECM (extracellular matrix), led by brillar collagen composition, play an important role in affecting the heart movement of sh [25]. Fibrillar collagen is the most common structural bres in the ECM. The brillar collagen forms stiff bres that maintain and support the alignment of myocardial cells by bearing wall stress [26,27]. However, excessive deposition of cardiac collagen will increase the passive stiffness of the chamber wall and reduce the chamber compliance and dilatability of the ventricular cavity, which can have implications for diastolic lling [28]. However, changes in the composition of myocardial collagen in the heart of A. baerii under heat stress and the relationship between the composition of myocardial collagen and changes in cardiac function need to be further studied.
Moreover, pumping blood is considered the main function of the heart, its performance can affect sh behavior, as well as changes in organs, tissues, and cells (intercellular stroma) [15,[29][30][31]. However, adverse and extreme external pressure stress may cause damage to the structure of myocardial cells and cause necrosis [32]. This is particularly concerning because most sh hearts lack coronary circulation and only receive oxygen via diffusion from the venous return. Unlike the spongy layer, which receives venous blood, the compact layer of the sh ventricle has a coronary blood supply from the gill [33]. However, heat stress can cause serious damage to the gill tissue of sh, decrease in oxygen exchange capacity, and severely damage the oxygen supply to the heart [34]. For sh, the thickness and structural integrity of the dense layer of the ventricle are important prerequisites for maintaining the pumping function of the heart [35,36]. This heart organ organization can impose limits on sustaining cardiac function at different temperatures and may lead to myocardial cell necrosis [37]. However, the in uence of A. baerii myocardial necrosis under CHS on cardiac structure and the relationship between myocardial necrosis and changes in cardiac function need further research.
In present study, we systematically investigated the behavior of sturgeon under CHS, as well as the structure, function and molecular regulatory pathway of heart tissue. we applied echocardiographic techniques and transcriptomics analysis to detect changes in cardiac function indicators in sturgeon to further establish the link between heart tissue structure and pumping function under CHS. Meanwhile, the counteracting effect of aquaculture water cooling on CHS was systematically tested. Our research has established a foundation for the connection between heart structure and function in sturgeon under CHS and provides a reference for the protection of wild and farmed cold-water sh.

Fish maintenance
All animal handling procedures were approved by the animal care and use committee of Sichuan Agricultural University in accordance with the animal experiment guidelines under license NO. ZCY-2019202031. 400 healthy A. baerii of similar weights (84.531 ± 2.713 g) and body lengths (31.491 ± 1.746 cm) were purchased from Sichuan Runzhao Fishery Co., Ltd. China. The sh were held in tanks under a 12 h light : dark cycle with an uninterrupted oxygen supply to ensure dissolved oxygen remained above 7.5 mg/L. The water in the tanks was pretreated with UV light and an aeration process, and 20% of the culture water was renewed every day. The pH ranged between 7-8 and ammoniacal nitrogen and nitrite were maintained at 0-0.015 mg/L. The sh were fed with 1% of the sh body weight of commercial feed three times daily. Eventually, sh that were responsive, robust, and healthy were selected for experimentation.

Experimental treatment
According to the study by Yang et al., (in 2021) A. baerii exhibits signi cant increases in fatality and organ damage when the breeding water temperature reaches 28°C [14]. Based on the observations, the sturgeon in this experiment were divided among three groups to simulate normal and CHS conditions: a control group at 20°C (C) and two elevated temperature groups at 24°C (M) and 28°C (H). A total of 180 sh were randomly assigned to the three treatments, with each group consisting of four parallel tanks holding 15 sh each. For the control group, the sturgeon were raised at normal room temperature (20°C) for the duration of the experiment. The CHS groups were also held at room temperature for the rst 14 days. Then, according to the heating scheme (Supplementary table 1 Heart disease activity index (HDAI) evaluation Based on the symptoms the sturgeon in the farm experienced in summer, we established a novel heart disease activity index (HDAI) with reference to the research by Chen et al. The index was designed to evaluate the "heart failure-like" behavior in A. baerii under CHS (Table 1) [38,39]. Similar to the above experimental treatments (Section 2.2). The procedure was designed to assess the degree of damage to the A. baerii after experiment CHS. The monitored variables included feeding rate, swimming speed, imbalance rate, respiratory rate, cardiac color, and percentage of ventricular injury, and all variables were blind scored by two trained researchers. The evaluation experiment included CHS treatment and a recovery period. Feeding rate = total feed intake (g) / total body weight (g) × 0.01 × 100%. To ensure measured swimming speed sturgeon are not subject to mechanical equipment and environmental impact. We used the camera to randomly record the swimming trajectory (curved or straight) and time of the horizontal sturgeon in the treatment tank one hour before feeding. Swimming speed = Swimming distance (cm) / time (s).
Imbalance rate = number of lateral ip / total number. Meanwhile, the sturgeon was recorded for 5 minutes continuously one hour before feeding during the experiment. Six sturgeons in each group were randomly selected for respiration frequency statistics. Respiratory rate (T/min) = number of operculum opening and closing cycles / time (min). Sensory score for the color of the dissected sturgeon heart (n = 3), then the hearts were dissected from sturgeon in each treatment and xed in 4% paraformaldehyde overnight at room temperature. Para n sections were prepared and stained with haematoxylin and eosin (H & E). Sections were photographed under a light microscope (Nikon, Tokyo, Japan). The obtained heart histopathological images were analyzed using Image J 1.35a (National Institutes of Health, USA) image analysis software. Percentage of ventricular injury = ventricular injury area / total cross-sectional area of ventricle × 100%. Scoring system of HDAI for sturgeon can be seen in Table 1 (n ≥ 3).

Myocardial enzyme determination
Myocardial enzyme is a biomarker of myocardial injury used to monitor the degree of myocardial necrosis [40,41]. To further determine whether myocardial cells were necrotic, the content of myocardial enzymes in the plasma was measured. The myocardial enzyme leakage activity indexes for plasma were determined using diagnostic kits produced by Nan Jing Jian Cheng Bioengineering Institute (Nanjing, China) following the manufacturer's instructions. The blood from each sample was centrifuged at 3500 g 10 min at 4°C. The diagnostic kits used in this experiment measured LDH (lactate dehydrogenase) (A020-2-2), AST (aspartate aminotransferase) (C010-2-1), and CK (creatine kinase) (A032-1-1). Assessments were performed so that the operators were 'blinded' to eliminate the potential for operator bias. Assessments were conducted in conditions temperature corresponding to the treatment temperature of the sturgeon for < 5 min, allowing echocardiographic recordings of at least three cardiac cycles. The water environment during the detection process and the indoor temperature during the detection are the corresponding experimental treatment temperatures.

Echocardiography and histopathological examination
With the help of the color-Doppler mode, sampling windows of pulsed-wave Doppler were positioned at the atrioventricular valve (AV), bulboventricular valve (BV), abdominal aorta (AA), and between the two valves. Clear images were captured and the Cardiac Software Package (CHISON, China) was used to analyze the relevant parameters. The pulsed-wave Doppler velocity measurement was taken from a position as parallel to the blood ow as possible. The angle was adjusted if necessary, but adjustments were limited to a maximum of 15 degrees.
Basic parameters, such as body weight (BW), carcass weight (CW), full length (FL), and body length (BL) were measured promptly after echocardiographic examinations. Ratios of ventricular and heart mass to BW or CW were chosen to assess changes in cardiac morphology. Dissected tissues were immediately xed in 10% neutral-buffered formalin for histopathologic examination. The samples were trimmed, dehydrated, and embedded in para n wax before sectioning at 4 µm for H & E and Masson trichrome staining. Images were recorded on a light microscope (Nikon, Tokyo, Japan). The degrees of change in brillar collagen content, atrophy, in ltration, necrosis, hyperplasia of fat, and blurred epicardial border in the heart were evaluated according to Huang et al [45]. Every change was assessed with a score (S) ranging from 0 to 6 depending on the degree of change: (0) unchanged, (2) mild change, (4) moderate change, and (6) severe change (diffuse lesion).
The obtained histopathological images were analyzed using Image J 1.35a (National Institutes of Health, USA) image analysis software. The basic calculation was conducted as follows: ventricle stratum spongiosum myocardial density (%) = pixels (area of myocardium) / pixels (total area of ventricle) ×100%; brillar collagen content in the myocardium of the stratum spongiosum = pixels (area of myocardial collagen) / pixels (total area of ventricle stratum spongiosum) ×100%.

Echocardiographic image data analysis
Clear echocardiographic images were obtained and o ine analyses were performed. With reference to the analysis process established by Fang et al., [42], the measurement results were analyzed as follows. The epicardial border was outlined using the Cardiac Function Measurement Package (CHISON, China) and the end-diastolic (E-d) and end-systolic (E-s) ventricular areas (A) were calculated (Fig. 1). The diameter of the AV valve annulus during systole (D) was also recorded. In addition, ventricular lling (atrioventricular ow) and ejection ow (BV ow) were also measured ( Fig. 1). Corresponding parameters such as peak velocity, mean velocity, and velocity time integral (VTI) can be automatically calculated when using the continuous wave Doppler mode. The calculated formulas-cross-sectional area (CSA) was used to evaluate stroke volume (SV, µL), cardiac output (CO = SV*HR), and pulsatility index (PI = ventricular blood ow peak systole / end-diastolic trough). SV was calculated as CSA × VTI. De ning CSA as πD 2 / 4, the VTI of the BV valve was measured using pulsed Doppler. The pulsed Doppler image was used to estimate ventricular ejection time (VET) by measuring the time of ventricle ejection from start to nish (Fig. 1).
RNA-seq 2.7.1 RNA extraction, library preparation and illumina sequencing The heart in sturgeon in different treatment group were selected for RNA-seq, each group including three replicates. Total RNA was extracted from the tissue using TRIzol® Reagent according the manufacturer's instructions(Invitrogen). Then RNA quality was determined using 2100 Bioanalyser (Agilent) and quanti ed using the ND-2000 (NanoDrop Technologies

Statistical analysis
Continuous variables were expressed as mean ± standard deviation (SD). Categorical variables were expressed as counts and percentages. Data were analyzed using SPSS version 27. Differences between groups were assessed with one-way ANOVAs followed by post hoc analyses with Dunnett's tests. Differences between the means were considered signi cant or extremely signi cant at the 95% or 99% con dence levels (P < 0.05 or P < 0.01), respectively.

Results
Systemic symptoms of heart failure in A. baerii caused by CHS A. baerii developed relatively obvious heart failure symptoms after CHS treatment. Starting from day 1 to 19, the HDAI scores of the sh in group H were signi cantly (P < 0.05 or P < 0.01) higher than those of the control group ( Fig. 2A). Notably, the cardiac HDAI in group H abruptly peaked from day 4 to day 12, during which time it remained at a high level. Speci cally, there were signi cant increases in feeding rate, swimming speed, imbalance rate, respiratory rate, cardiac color, and percentage of ventricular injury scores relative to the control group for most of the days between 4-12 (Fig. 2C). Fish in the heat-stressed group showed a signi cant recovery trend from day 18 to 19 (P < 0.05). The survival rates of the group H was signi cant lower than the control group (Fig. 2B). The survival rate dropped signi cantly from day 5 and continued to decrease until the end of the heat stress period. During the recovery period, there was a clear recovery trend in the M group, while the survival rate of the H group continued to decline (Fig. 2B). Generally, sturgeon in the heat stress groups showed systemic cardiopathic symptoms. In the control group, almost no deaths occurred during the experiment, no obvious pathological symptoms were observed, and the HDAI scores remained relatively stable without signi cant change.
Signi cant irregular ventricular shape and heart atrophy in A. baerii caused by CHS Immediately after the CHS and following recovery, we performed further measurements of morphological indicators of heart condition and basic parameters of the health of A. baerii. Severe abnormal shapes, darkening of the ventricle, and atrophy of the heart increased with the intensity of CHS, but were partially relieved after the recovery period (Fig. 3A). Speci cally, the ventricle darkening and ventricle atrophy were the most obvious with increasing CHS intensity. Next, the basic parameters were measured. All morphological indicators of the heart showed a decreasing trend with increasing intensity of CHS. The indexes related to heart weight showed signi cant reductions in group H, but returned to normal levels after the recovery period. Meanwhile, there was a tendency for the ventricles to atrophy under CHS (Table  2). However, BW, CW, and BL were not signi cantly affected in the experimental groups (Table 2).

Fibrillar collagen level increased and cardiomyocyte necrosis caused structural damage to the heart
To observe the histopathological changes of A. baerii heart failure, heart sections were stained following the protocols of H & E and Masson trichrome staining. Masson's trichrome staining showed that the changes were extremely pronounced in the compact layer in the heat-stressed group (Fig. 3A). The most obvious changes were the decrease in the thickness of the compact layer and the dramatic increase in the content of brillar collagen (Fig. 3A, B and E). Meanwhile, CHS also caused the density of the spongy layer to signi cantly decrease and the brillar collagen content to signi cantly increase (Fig. 3C, D). All indices showed different degrees of recovery after the recovery period (Fig. 3).
Sections were systematically examined, revealing that the most obvious lesions were mainly concentrated in the ventricular tissue. Speci cally, H & E staining showed massive necrosis of cardiomyocytes in the compact layer of the ventricle in group H, exhibiting symptoms of myocardial atrophy and in ammatory cell in ltration (Fig. 4A c, f, g). The histopathological score of the compact layer also signi cantly increased (P < 0.05) (Fig. 4A h). CHS also resulted in signi cant increases in epicardial fat and increased blurring of the boundary between the compact layer and the epicardium compared to the control (Fig. 4A g). To further con rm the presence of necrosis in the cardiomyocytes we tested the myocardial enzyme leakage levels in the plasma. In general, the levels of myocardial enzymes exhibited an upward trend under CHS, but the increase in CK was most obvious (Fig. 4D) (P < 0.05).

The regulatory mechanism of cardiac brosis and myocardial necrosis under CHS
To study the speci c role of cardiac brosis and myocardial necrosis in the heart of A. baerii, we selected the group C, H and R-H heart for transcriptome sequencing. Transcriptomics analysis showed that brous collagen deposition increased after CHS. It is manifested as an increase in the production of collagen bers (COL1A, COL2A, COL4A and COL6A) and a down-regulation of the expression of genes that regulate the degradation of collagen bers (COLase 3 and COLase 4). In the necroptosis regulatory network, down-regulated of the Caspase 8, leading to inhibition of apoptotic pathways in cardiomyocytes and shifting to necrosis. The present studies have shown that mitochondrial ssion may be an important inducement of cardiomyocyte necrosis, accompanied by the signi cant up-regulation of DNM1L and Hsp 90 after CHS (Fig. 6L and M). However, timely cooling can alleviate the process of cardiac brosis and cardiomyocyte necrosis to a certain extent.

Blocked heart activity under severe CHS
To further investigate the association between changes in cardiac tissue structure and cardiac function, echocardiography combined with B-mode imaging and Doppler imaging were used. In the current study, both the ventricular end-diastolic area (VEDA) and ventricular end-systolic area (VESA) showed increasing trends in CHS conditions, with signi cant differences in the VESA (P < 0.05). After the recovery period, the M group remained unchanged, while the VEDA and VESA continued to rise in the H group (P < 0.05) ( Fig. 7A and B). CHS resulted in a signi cant reduction in atrioventricular valve diameter, which did not return to normal levels after the recovery period (Fig. 7C). Meanwhile, a marked HR increase was observed in the H group compared to control group, but after recovery the HR was no longer elevated (Fig. 7D).
CHS caused decrease in heart ejection effectiveness Impaired cardiac activity may directly change blood ow parameters. Therefore, ultrasound Doppler analysis was used to analyze the changes in cardiac blood ow velocity. After CHS, there was no signi cant change in AA blood ow velocity in group M, but group H showed a signi cant increase (Fig. 8). The blood ow velocities from the BA and AV were signi cantly reduced in the recovered sh from group H compared to the control sh (Fig. 8B, C) (P < 0.05). Except group M and H, no signi cant differences in AA blood ow velocity were detected among groups.
In addition, indicators of cardiac function were measured using the Cardiac Function Measurement Package. With increasing CHS intensity, SV, CO, and VET showed signi cant and sequential decreases in the M and H groups compared to the control group (P < 0.05) (Fig. 7F, G and H). However, the opposite trend was true for the PI of group H (Fig. 7E). Considering the trends in HR, which suggested decreased myocardial contractility and increased cardiac afterload, the changes could be characterized as those of a typical high-resistance and low-output heart failure. During the recovery period, almost all indicators tended to return to normal levels (group C), in particular, SV, CO, and VET were signi cantly increased in the R-H group (P < 0.05) (Fig. 7F, G and H). However, the SV and CO levels did not return to normal levels in group H.

Discussion
CHS caused by the increase of water temperature in aquaculture ponds in summer may lead to heart failure of sturgeon and affect the development of sturgeon industry. However, how CHS affects the changes in sturgeon's heart structure and function is still unclear and its effect on sh production signi cance for aquaculture need to be further studied. This study explored the changes in the structure and function of the sturgeon's heart under CHS, and tried to establish the connection. Combined with histopathology, echocardiographic detection technology and transcriptomics analysis, it was found that CHS caused A. baerii's ventricular ber collagen content to increase and myocardial necrosis, which caused A. baerii's cardiac dysfunction and decreased pumping effectiveness. After the recovery period, although most results showed that the heart function of the heat-stressed group had a tendency to recover. However, there were still injuries in group H that could not eliminate the symptoms. It indicates that the sturgeon heart has a certain ability to regulate itself, which may be accomplished through the regulation of ber collagen content and the proliferation of myocardial cells [35,46,47].
The present study identi ed the proliferation of myocardial interstitial myocardial collagen and myocardial necrosis as important factors leading to the cardiac imbalance and heart failure. Myocardial brosis is an important causes of cardiac dysfunction in A. baerii. Myocardial brosis is a pathological reaction that causes an imbalance in the collagen ratio due to increased deposition of collagen in the myocardial interstitium, which may induce a variety of cardiovascular diseases [26,48]. Studies have shown that increased ventricular stiffness and decreased cardiac function have been observed in Atlantic cod (Gadus morhua) at warm acclimation [31]. Meanwhile, in zebra sh, where there is signi cantly less thick collagen bres in the hearts of sh acclimated to 20°C compared with those acclimated to 28°C [46]. We guess that the zebra sh myocardial collagen bers will show the opposite trend after warm acclimation. Fibrous collagen deposition increases ventricular stiffness. Similarly, after CHS, the proportion of A. baerii's ventricular ber collagen increased signi cantly in group H. Fibrous collagen increases the passive stiffness of the chamber wall, and excessive myocardial brosis will reduce chamber compliance and dilatability, which may have a signi cant impact on ventricular diastolic lling [28]. We believe that it is caused by delayed or hindered degradation of collagen bers and over-synthesis to maintain part of the heart function. Therefore, we observed that the VEDA of the ventricle remains unchanged but the VESA increases signi cantly. Meanwhile, CO and SV in the CHS group were signi cantly reduced and implying decreased pumping effectiveness. Such changes are common, and permanent, in the hearts of patients suffering from cardiac hypertension, dilated cardiomyopathy or chronic congestive heart failure, and collagen ber increased greatly contribute to the associated diastolic dysfunction and eventual heart failure [49][50][51][52]. Considering the importance of the ventricles to the whole heart, this may be an important factor in inducing heart failure in sturgeon. Study found that the coldinduced increase in salmon heart collagen deposition was reversed after experiencing chronic warming [23,35]. Therefore, the signi cant decrease in collagen content of A. baerii's ventricular bers during the recovery period can be attributed to the degradation of collagen.
Furthermore, cardiac myocytes necrosis, which is one of the main pathological changes in the hearts of A. baerii under CHS, can cause cardiac atrophy and dysfunction [32,53]. Study found that the H 2 O 2 consumption capacity of cardiac mitochondria in rainbow trout decreased with increasing temperature, which may increase the risk of oxidative stress and cause cardiomyocyte necrosis [54]. In present study, histopathology and myocardial enzyme levels con rmed the presence of necrosis of myocardial cells. And transcriptomics studies have found that cardiac mitochondrial damage may mediate myocardial cell necrosis. Experiment found that the delayed delivery of ion channel ux action potentials caused by the collapse of cardiac myocytes may seriously affect pumping in sh hearts. [55]. However, as an increase in physiological temperature increases HR in sh [16,56], this suggests that the heart is pumping less blood per beat at a faster rate. Therefore, we have observed an increasing trend of the AA ow velocity under chronic heat stress (group H) in sturgeon, but the BV and AV ow do not change signi cantly. It explains the signi cant increase in A. baerii heart rate and the signi cant decrease in SV after CHS (group H). Meanwhile, damage to myocardial structure will cause insu cient energy supply, abnormal ventricular contraction (increased ventricular volume at the end-systolic area), and lead to a decrease in stroke volume and pumping e ciency [57]. Therefore, cardiac function in A. baerii under heat stress is imply a reduction in myocardial contractility and an augmentation of cardiac afterload, matching the typical characteristics of high-resistance and low-output heart failure.
Like most cold-water sh sh, the sturgeon's living space and life activities are under threat and being compressed by rising water temperatures, which is apparent in the Yangtze River Basin [4,6]. In addition, sturgeon grow slowly and usually take 7-8 years to reach sexual maturity [20]. Cardiac dysfunction is undoubtedly an important risk factor that could hinder the growth and development of sturgeon, potentially increasing their age at maturation. Due to the inability of their hearts to quickly respond to high water temperatures, most sturgeon could exhibit "cardiac syndrome" (like S. salar) in the high water temperature environment in summer [58,59]. According to the results of the echocardiography and microstructure observations after the recovery period, it was found that the sturgeon's heart was unable to completely return to normal working levels. Current evidence suggests that the upper limits of temperature tolerance in sh are primarily determined by the points at which their hearts fail to maintain tissue perfusion and adequate oxygenation [60,61]. Disorders of heart function may affect sh swimming performance, predator avoidance, foraging behavior, and alter a species geographic distribution [62, 63], leading to the local extinction of sh species [4]. Therefore, avoiding high water temperature sturgeon farming and seeking effective cooling methods may be important directions for sturgeon farming in the future. In addition, behavioral observations of juvenile sturgeon should be performed regularly during the conservation and breeding of endangered sturgeon species, and antistress prevention products should be added appropriately to improve heart health.

Conclusions
In conclusion, we have con rmed that timely cooling of the culture water can alleviate the heart failure of A. baerii caused by CHS. CHS can cause A. baerii to show obvious "heart failure" characteristics, and cardiac function examination reveal a reduction in myocardial contractility and an augmentation of cardiac afterload, matching the typical characteristics of high-resistance and low-output heart failure characteristics. The rate of A. baerii 's cardiac brosis and myocardial necrosis was relieved after the culture water was cooled. Therefore, the present study shows that maintaining normal growth water temperature and timely cooling during cold-water sh farming are important measures for healthy farming.  The HDAI (A) and survival rate % (B) of A. baerii in different temperature groups at different time points.

Declarations
HDAI component parts, including: feeding rate; swimming speed; imbalance rate; respiratory rate; cardiac color; and percentage of ventricular injury (C). Survival curves were estimated by the Kaplan-Meier method, and signi cance analysis was performed by the log-rank test. P < 0.05 (*) and P < 0.01 (**) indicate that groups are signi cant or extremely signi cant different from the control group.   DEGs of brous collagen regulation-axis in A. baerii. Mapping the brous collagen regulation-axis network in the heart of A. baerii based on transcriptome data. A-I: the column map showed the expression level of reads of regulate collagen deposition genes in the transcriptome (n = 3, data were presented as means ± standard deviation). * or *** represents a signi cant or highly signi cant difference (P < 0.05 or P < 0. 001) between two groups.

Supplementary Files
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