Abstract

   Heat stress resulting from global climate change has been demonstrated
   to adversely affect growth, development, and reproduction of marine
   organisms. The Zhikong scallop (Chlamys farreri), an important
   economical mollusk in China, faces increasing risks of summer mortality
   due to the prolonged heat waves. The heart, responsible for
   transporting gas and nutrients, is vital in maintaining homeostasis and
   physiological status in response to environmental changes. In this
   study, the effect of heat stress on the cardiac function of C. farreri
   was investigated during the continuous 30-day heat stress at 27 °C. The
   results showed the heart rate of scallops increased due to stress in
   the initial phase of high temperature exposure, peaking at 12 h, and
   then gradually recovered, indicating an acclimatization at the end of
   the experiment. In addition, the levels of catalase (CAT), superoxide
   dismutase (SOD), and total antioxidant capacity (T-AOC) exhibited an
   initial increase followed by recovery in response to heat stress.
   Furthermore, transcriptome analysis of the heart identified 3541
   differentially expressed genes (DEGs) in response to heat stress.
   Subsequent GO and KEGG enrichment analysis showed that these genes were
   primarily related to signal transduction and oxidative stress, such as
   the phosphatidylinositol signaling system, regulation of actin
   cytoskeleton, MAPK signaling pathway, FoxO signaling pathway, etc. In
   addition, two modules were identified as significant responsive modules
   according to the weighted gene co-expression network analysis (WGCNA).
   The upregulation of key enzymes within the base excision repair and gap
   junction pathways indicated that the heart of C. farreri under heat
   stress enhanced DNA repair and maintained cellular integrity. In
   addition, the variable expression of essential signaling molecules and
   cytoskeletal regulators suggested that the heart of C. farreri
   modulated cardiomyocyte contraction, intracellular signaling, and heart
   rate through complex regulation of phosphorylation and calcium dynamics
   in response to heat stress. Collectively, this study enhances our
   understanding of cardiac function and provides novel evidence for
   unraveling the mechanism underlying the thermal response in mollusks.

   Keywords: Chlamys farreri, heart, antioxidant, transcriptome, heat
   stress

1. Introduction

   The Intergovernmental Panel on Climate Change (IPCC) has declared that
   the world has been warming since the industrial revolution. The
   increasing emission of greenhouse gases and the resulting climate
   change have been identified as the primary drivers of the occurrence of
   extreme high-temperature weather events, presenting a substantial risk
   to marine ecosystems [[40]1,[41]2,[42]3,[43]4,[44]5,[45]6,[46]7].
   Mollusks, as significant components of marine ecosystems, are
   ectothermic organisms that exhibit particular sensitivity to
   fluctuations in temperature [[47]8]. Numerous studies have reported
   that extreme high temperature has adverse effects on the life stages of
   mollusks [[48]9,[49]10,[50]11]. For example, mollusks may produce
   oxygen radicals after exposure to temperature fluctuations, which
   causes changes in the activity of antioxidant enzymes, including
   catalase (CAT), superoxide dismutase (SOD), glutathione S-transferases
   activities (GST), etc. [[51]12,[52]13]. In the study of Patinopecten
   yessoensis and Haliotis discus hannai, when exposed to heat stress, the
   CAT and SOD activities first increased significantly, and then
   gradually recovered to the control level as the stress time increased
   [[53]14]. In the Manila clam Ruditapes philippinarum, it could enhance
   the resistance to marine heat waves by decomposing more energy reserves
   to increase antioxidant capacity and decreasing the energy supply for
   reproduction [[54]15]. The high activity of SODs in the gills of
   Scapharca inaequivalvis and Tapes philippinarum were involved in the
   physiological role in respiration and protection against oxidative
   stress caused by environmental fluctuations [[55]16]. These studies
   indicate that mollusks may possess the capacity to migrate oxidative
   damage resulting from heat stress and acclimate to climate warming.
   However, the focus of the aforementioned studies is predominantly on
   mantle or gill, with limited exploration of the effects on other
   tissues or organs.

   The heart, being a vital component of the circulatory system, is
   responsible for facilitating gases exchange and transporting substances
   externally, thereby playing crucial roles in physiology responses of
   organisms to fluctuations in environmental temperature
   [[56]15,[57]16,[58]17,[59]18]. In vertebrates, many studies have
   reported that heart tissue played a crucial functional role in adapting
   to various external stresses [[60]19,[61]20,[62]21]. Mammalian hearts
   dynamically adjusted heart rate via the autonomic nervous system,
   ensuring a seamless transition from resting states to intense physical
   exertion, thereby accommodating diverse physiological demands [[63]19].
   The regulation of blood pressure is identified as a crucial component
   of the cardiovascular response to stress. Mammalian hearts are noted
   for their capacity to adjust myocardial contractility, thereby
   maintaining hemodynamic stability during periods of duress [[64]20].
   Morrison and colleagues emphasized the significance of the heart in
   regulating vascular tone changes, ensuring preferential blood flow to
   critical organs such as the brain and muscles in the face of adversity
   [[65]21]. These results highlight the diverse functions of the
   vertebrate heart in mitigating physiological challenges, encompassing
   the regulation of heart rate, maintenance of blood pressure, and
   adjustments of blood distribution, ultimately contributing to the
   preservation of organismal homeostasis in adverse conditions. In
   invertebrates, alterations in cardiac function are also responding to
   environmental changes. For example, the heart rate of mollusks
   exhibited varying degrees of impact when subjected to high temperature,
   with instances of cardiac arrest observed at extremely elevated
   temperatures [[66]22,[67]23,[68]24,[69]25]. In addition, the heart rate
   of scallop Argopecten irradians showed an initial increase followed by
   a decrease in response to heat stress, which was similar with activity
   of the antioxidant enzymes CAT and SOD. Further dynamic transcriptome
   analysis of scallop hearts revealed an active defense mechanism against
   heat stress during the acute stage (<24 h), including energy supply,
   correction of misfolded proteins, and enhanced signal transduction
   [[70]26]. These findings collectively demonstrate the significant role
   of the heart in facilitating effective adaptation to heat stress.
   Nevertheless, research on the molecular regulatory mechanisms of the
   heart in marine invertebrates is currently limited, hindering the
   understanding of how mollusks adapt to global warming.

   Zhikong scallop (Chlamys farreri) is an epibenthic and semi-sessile
   mollusk, which is naturally distributed along the coasts of northern
   China, Korea, Japan, and Eastern Russia [[71]27]. C. farreri is an
   economically important mollusk in China owing to its large adductor and
   rich nutrition, which makes a major contribution to aquaculture in
   China [[72]28]. In recent years, heat stress caused by ocean warming
   and marine heat waves has brought serious challenges to the growth and
   reproduction of scallops, even leading to summer mortality under severe
   conditions [[73]29]. The objective of this study was to investigate the
   role of the heart in scallops responding to heat stress and potential
   regulated mechanisms. Firstly, the changes in heart rate and heart
   amplitude of scallops under heat stress were examined. Subsequently,
   the dynamic transcriptome response of scallop hearts to heat stress was
   analyzed. At the same time, the weighted gene co-expression network was
   constructed to identify the responsive modules and hub genes.
   Furthermore, the antioxidant activity of scallop hearts was measured to
   validate their functional role in coping with heat stress. The results
   will provide new insights into the function of the scallop heart in
   response to heat stress and contribute to understanding the potential
   adaption mechanism of scallops to global warming.

2. Materials and Methods

2.1. Ethics Statement

   The experiments in this study were conducted according to the
   guidelines and regulations established by the Institutional Animal Care
   and Use Committee (IACUC) of Ocean University of China.

2.2. Sample Collection and Heat Stress Experiment

   Healthy Zhikong scallops (shell height: 54.95 ± 4.91 mm) were obtained
   from the scallop farming area in Qingdao (Shandong Province, China) and
   transported alive to the laboratory. Scallops were cultured in plastic
   tanks with filtered and aerated seawater (salinity 30.0 psu; pH 8.0;
   temperature 20.0 °C) for 7 days of acclimation. The seawater was
   replaced once per day, and scallops were fed daily with Nitzschia
   closterium (1.0 × 10^5 cells/scallop). After a week of acclimation, a
   total of 180 scallops were randomly divided into three groups and
   exposed directly to the seawater at a temperature of 27 °C, which is
   the upper temperature during summer in the culture area of C. farreri.
   The heart rates of nine randomly selected scallops were detected at
   nine time points (0 h, 3 h, 6 h, 12 h, 24 h, 3 d, 6 d, 15 d, and 30 d)
   during heat stress. And the heart tissues of nine scallops were
   dissected and frozen in liquid nitrogen at the same time points, and
   they were divided into three groups, with three individuals in each
   group forming one sample for subsequent RNA extraction and biochemical
   assay. Among them, the individuals sampled at 20 °C were set as the
   control group (0 h).

2.3. Heart Performance Measurement

   Heart performances of scallops were measured by an improved
   non-invasive monitoring method [[74]30]. In brief, after one week
   acclimation, scallops were placed in the petri dish (diameter = 110.0
   cm, height = 40 cm) for heart performance measurement, and the water
   temperature (27 °C) was heated and controlled by heating rods-1000W
   (ChuangNing, Shanghai, China) during measurement. Then, CNY-70
   (Newshift, Lisbon, Portugal) optical sensors were fixed with the blue
   tack (~20 g) to the shell surface above the heart tissues of
   individuals. Infrared signal variations from CNY-70 were amplified by
   AMP-03 (Newshift, Lisbon, Portugal), followed via PowerLab 16/35
   (ADInstruments, Sydney, Australia), which filtered and transmitted the
   signal to the computer. Individuals to be tested were placed in
   seawater, and once the mantle tentacles extended (around 3 min), the
   instruments were connected for a two-minute measurement. Finally, the
   software LabChart v8.1.3 (ADInstruments, Sydney, Australia) was applied
   to record these signals and analyze the heart performance, including
   heart rate (HR) and heart amplitude (HA).

2.4. Oxidative Stress Indexes Determination

   Catalase (CAT) activity, superoxide dismutase (SOD) activity, and total
   antioxidant capacity (T-AOC) of the heart were detected for each sample
   point (n = 3, respectively). The stored heart tissues were mixed with
   saline solution to create a 10% tissue homogenate for enzyme activity
   measurement. The CAT, SOD, and T-AOC enzyme activities were determined
   following the manufacturer’s instructions of commercial kits (Nanjing
   Jiancheng Bioengineering Institute, Nanjing, China, cat. No. A007-1-1,
   cat. No. A001-3-2 and cat. No. A015-3-1). The reaction temperature of
   CAT, SOD, and T-AOC kits are 37 °C. The CAT activity was determined on
   the basis of the ammonium molybdate method [[75]31]; the SOD activity
   was measured using the WBT-1 method [[76]32]; and the T-AOC activity
   was estimated through the FRAP method [[77]33].

2.5. RNA Isolation, Library Construction and RNA-Seq Analysis

   Total RNA was isolated from the heart of three samples randomly
   selected at each time point following the protocol described by Hu et
   al. [[78]34]. The quality of the RNA, including integrity and
   concentration, was examined by 1.0% agarose gel electrophoresis and a
   NanoDrop spectrophotometer (Thermo Fisher, Waltham, MA, USA),
   respectively. A total of 27 RNA-seq libraries were constructed using an
   RNA-seq library kit (Vazyme, NR603-01, Nanjing, China) and subjected to
   sequence on Illumina HighSeq 2000 high-throughput platform provided by
   Novogene Co., Ltd. (Beijing, China).

   The raw reads were processed by the FastQC with default parameters
   [[79]35], and filtered reads were subjected to STAR v2.4.1 [[80]36] to
   align them with chromosome-level reference C. farreri genome [[81]37].
   Gene expression profiles were calculated with the form of TPM
   (Transcripts per Million) [[82]38]. Differentially expressed genes
   (DGEs) were identified using the edgeR [[83]39] with a statistically
   significant threshold of |log[2]FC (fold change)| > 1 and p value <
   0.05.

   Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathway analysis of DEGs were performed to explore gene
   function using the R package GOSeq [[84]40] and KOBAS_v2.0 [[85]41],
   respectively, and p value < 0.05 was set as the threshold for
   significantly enriched.

2.6. Quantitative Real Time-PCR Validation

   To further validate the reliability of the RNA-seq data, the expression
   level of five DEGs, including Globin1, Globin2, Transient receptor
   potential cation channel subfamily A member 1, Heat shock protein
   beta-1, and Crystallin alpha B, were detected using quantitative
   real-time PCR validation (qPCR). The Elongation factor 1 alpha, which
   is widely used as an internal reference gene in bivalves
   [[86]42,[87]43], was selected as the reference gene. The detailed
   primers are listed in [88]Table S1. The qPCR was performed using a
   LightCycler 480 system (Roche, Basel, Switzerland), and the reaction
   mixture consisted of 1× SYBR Green (Vazyme, Q131-02, Nanjing, China),
   0.2 mM primer pairs, and 2 μL of cDNA template in a total volume of 20
   μL. The program was as follows: an initial denaturation step (95 °C for
   30 s), followed by 35 cycles of denaturing at 95 °C for 5 s and
   annealing at 60 °C for 20 s. The relative expression level of DGEs was
   calculated using the 2^−ΔΔCT method [[89]44].

2.7. Weighted Gene Co-Expression Network Analysis

   A weighted gene co-expression network of 27 transcriptomes of the heart
   was constructed to identify the key co-expressed gene modules connected
   to various times under heat stress using the R package WGCNA [[90]45].
   To make the network with an approximate scale-free topology, the genes
   that were expressed in all samples were used for network construction,
   with the following parameters: minimum module size = 200, tree cutting
   height = 0.99, soft threshold power = 12. To account for multiple
   tests, false discovery rate (FDR) was calculated using the package
   WGCNA. The modules with FDR < 0.05 at least in two time points were
   selected as significant heat responding modules, and genes were
   connected if the topological overlap between them was more than 0.1.
   Cytoscape v3.8.0 was used to visualize gene co-expression networks
   [[91]46].

2.8. Data Analysis

   Statistical analysis was conducted with one-way analysis of variance
   followed by post hoc comparison of means-based Turkey’s Least
   Significant Difference (LSD) test. SPSS Statistics v23 software (IBM
   Corp, Armonk, NY, USA) was used to determine the differences among
   different heat stress treatments (p value < 0.05, n = 3).

3. Results

3.1. Heart Perfomance of C. farreri during Heat Stress

   As illustrated in [92]Figure 1, with the increase of heat stress time,
   the heart rate (HR) of C. farreri exhibited a trend of initial increase
   followed by a decrease to control levels. Specifically, the heart rate
   continuously increased, starting from 3 h, peaking at 24 h with a rate
   of 57.60 ± 4.94 bpm (beat per minute), then maintaining a relatively
   high level until a decline commenced after 6 d, eventually returning to
   control levels after 15 days of stress ([93]Figure 1A). In addition, as
   time increased, the heart amplitude (HA) reached its first peak of 0.33
   ± 0.13 V at 3 h, followed by a continuous decline to a nadir of 0.19 ±
   0.08 V at 12 h, and then a subsequent rise to a second peak of 0.44 ±
   0.05 V at 15 d, with a slight decrease observed at 30 d. However, there
   was no significant difference in HA of C. farreri during heat stress (p
   > 0.05) ([94]Figure 1B).

Figure 1.

   [95]Figure 1
   [96]Open in a new tab

   Heart rate and amplitude of C. farreri under different heat stress
   time. (A) Heart rate (HR); (B) Heart amplitude (HA). The circle shape
   represents the mean ± S.D. (n = 3). (* p value < 0.05).

3.2. Oxidative Stress Indexes

   The biochemical indexes, CAT, SOD, and T-AOC were used to evaluate the
   effects of heat stress on oxidative stress in C. farreri. The CAT
   activity initially exhibited a significant increase at 3 h, reaching
   its peak at 3.63 ± 0.63 U/mg·prot, followed by a rapid decline and
   subsequent gradual rise at 3 days, ultimately returning to baseline
   levels ([97]Figure 2A). The SOD activity increased significantly during
   heat stress, reaching peaks at 26.94 ± 0.66 U/mg·prot and 22.82 ± 5.15
   U/mg·prot at 3 h and 6 d, respectively ([98]Figure 2B). T-AOC increased
   since 3 h, reached a significant peak of 0.15 ± 0.0051 mmol/gprot at 12
   h, then rapidly declined to control level ([99]Figure 2C).

Figure 2.

   [100]Figure 2
   [101]Open in a new tab

   Antioxidant enzymes activity and total antioxidant capacity in heart of
   C. farreri under heat stress. (A) CAT activity; (B) SOD activity; (C)
   T-AOC. The triangular shape represents the mean ± S.D. (n = 3). (* p
   value < 0.05).

3.3. Heart Transcriptomes and DEGs Analysis

   A total of 1,338,825,848 raw reads were generated with an average of
   49.5 M per sample ([102]Table S2). After quality control, the
   percentage of high-quality reads ranged from 86.41% to 97.65%.
   High-quality reads were then mapped to the reference genome of C.
   farreri using the STAR, and the ratio of unique mapping of the library
   was about 71.88% on average. Then the data, combined with gene length
   information, were utilized to calculate the Transcripts per Million
   (TPM) values for gene expression.

   According to the differential analysis, a total of 3541 DEGs were
   identified, and the number of up-regulated genes and down-regulated
   genes in 3 h, 6 h, 12 h, 24 h, 3 d, 6 d, 15 d, and 30 d were 222, 126,
   267, 232, 136, 51, 326, 299 and 503, 385, 429, 376, 1144, 1223, 82, 50,
   respectively ([103]Figure 3). Under heat stress, the number of DEGs in
   the heart remained relatively stable within the first 24 h of heat
   stress. However, a significant increase was observed at 3 d and 6 d.
   Prior to 6 d, the number of downregulated genes substantially exceeded
   that of the upregulated ones. Beginning from 15 d of heat stress, there
   was a notable reduction in the number of DEGs, with an increase in the
   upregulated DEGs. After further categorization of the expression trends
   of 3541 DEGs, three distinct trends were identified, as illustrated in
   [104]Figure 4. In expression trend A, a significant clustering of 711
   DEGs was identified (p value < 0.05). This trend initially showed a
   decrease followed by an increase, which is contrary to the trend
   observed in heart rate changes. In expression trend B, another set of
   106 DEGs exhibited an expression trend that was similar to the trend of
   heart rate, characterized by an initial increase followed by a
   decrease. However, the peak in this trend occurred with a little delay
   compared to the heart rate peak. Additionally, there was another
   distinct expression trend C, characterized by two fluctuations of
   initially decreasing and then increasing expression. This trend was
   significantly associated with 1188 DEGs (p value< 0.05). The
   expressions of key genes from three trends in hearts of C. farreri
   under heat stress are shown in [105]Figure 5.

Figure 3.

   [106]Figure 3
   [107]Open in a new tab

   Numbers of DEGs in heart of C. farreri under heat stress. Red bar
   represents the number of up-regulated DEGs; blue bar represents the
   number of down-regulated DEGs.

Figure 4.

   [108]Figure 4
   [109]Open in a new tab

   Three expression trends of DEGs in heart of C. farreri under heat
   stress. (A) Expression trend contrary to the trend observed in HR
   changes; (B) expression trend similar to the trend of HR; (C)
   expression trend opposite to the trend of HA changes. The X-axis
   represents the time of heat stress. The Y-axis represents the log[2]FC
   of gene expression change.

Figure 5.

   [110]Figure 5
   [111]Open in a new tab

   The expressions of key genes from three trends in heart of C. farreri
   under heat stress.

3.4. Functional Enrichment Analysis of DEGs

   The GO enrichment was used to classify the possible functions of the
   DEGs in the heart of C. farreri. There were 134, 21, and 113 terms
   enriched significantly (p value < 0.05) in Biological Process (BP),
   Cellular Component (CC), and Molecular Function (MF) ([112]Table S3).
   Among them, the top 25 terms were shown in [113]Figure 6A, including
   phospholipid binding (GO:0005543, MF), ion binding (GO:0043167, MF),
   lipid binding (GO:0008289, MF), calmodulin-dependent protein kinase
   activity (GO:0004683, MF), phosphatidylserine decarboxylase activity
   (GO:0004609, MF), phosphorylase kinase activity (GO:0004689, MF),
   G-protein coupled receptor kinase activity (GO:0004703, MF),
   intracellular signal transduction (GO:0035556, BP), inositol catabolic
   process (GO:0019310, BP), etc.

Figure 6.

   [114]Figure 6
   [115]Open in a new tab

   The GO and KEGG analysis of DEGs in heart of C. farreri under heat
   stress. (A) GO annotation. The X-axis represents the annotated
   functions. The Y-axis represents the percent of enriched gene numbers.
   (B) KEGG enrichment. The X-axis represents the rich factor of KEGG
   terms. The Y-axis represents the enriched pathways. The circle shape
   represents the number of DGEs in the enriched pathways.

   DEGs of the heart were enriched in 63 pathways significantly (p value <
   0.05) according to the KEGG analysis ([116]Table S4). These pathways
   could be classified into classes such as Metabolism, Environmental
   Information Processing, Cellular Processes, and Organismal Systems.
   [117]Figure 6B lists the top 25 pathways, primarily related to the MAPK
   signaling pathway, FoxO signaling pathway, VEGF signaling pathway,
   phosphatidylinositol signaling system, the regulation of the actin
   cytoskeleton, etc.

3.5. Gene Coexpression Network

   All RNA-seq data from 27 samples were used for calculation of
   gene-to-gene correlations. After eliminating the unexpressed genes, a
   total of 26 modules were derived from the expression data of 24,878
   genes, with module size varying from 210 to 4620 ([118]Figure 6;
   [119]Table S5). The module yellow (M1) and module blue (M2) with FDR <
   0.05 were identified as heat stress-responsive modules, which was shown
   in [120]Figure 7 and [121]Figure 8. There were 2251 genes in M1 and
   3113 genes in M2. The number of DEGs were 163 in M1 and 478 in M2,
   respectively. GO analysis showed that genes in M1 were significantly
   enriched in membrane, transporter activity, and calcium ion binding
   ([122]Table S6). Meanwhile, the possible functions of genes in M2 were
   motor activity, protein binding, and microtubule-based process
   ([123]Table S7). KEGG analysis identified several common enrichments of
   pathways in both modules, including the calcium signaling pathway,
   vascular smooth muscle contraction, cGMP-PKG signaling pathway, and
   MAPK signaling pathway ([124]Table S1).

Figure 7.

   [125]Figure 7
   [126]Open in a new tab

   The WGCNA analysis of scallop heart transcriptome under heat stress.
   Dendrogram is produced by average linkage hierarchical clustering of
   genes based on topological overlap. Horizontal color bars represent
   different co-expression modules that are also numbered. Unassigned
   genes are labeled in grey.

Figure 8.

   [127]Figure 8
   [128]Open in a new tab

   Main response modules of WGCNA network of heart after heat treatment.
   Yellow dots belong to M1 and blue dots belong to M2.

3.6. Expression Patterns of Candidate Genes

   To verify the accuracy of transcriptome sequence data, a total of five
   genes, Heat shock protein beta-1 (HSPB1), Crystallin alpha B (CRYAB),
   Globin1, Globin2, and Transient receptor potential cation channel
   subfamily A member 1 (TRPA1) were randomly selected for RT-qPCR
   ([129]Figure 9). RT-qPCR results (green line) of these genes were
   consistent with RNA-Seq results (red line), indicating the high
   reliability of the transcriptome.

Figure 9.

   [130]Figure 9
   [131]Open in a new tab

   Quantitative real time-PCR validation. Green line represents RT-qPCR
   results; red line represents RNA-Seq results.

4. Discussion

   The heart plays a crucial role in the circulatory system by
   facilitating the transport of gases and nutrients, as well as
   maintaining homeostasis and supporting various physiological functions
   throughout different conditions and stages of life [[132]47,[133]48].
   An increase in heart rate plays a positive role in marine animals’
   response to stressful environments, such as high temperatures and low
   oxygen. In this study, when Zhikong scallops C. farreri were exposed to
   heat stress, significant increase in their heart rate were observed.
   The increase of heart rate might be a physiological regulation behavior
   of scallops to cope with acute heat stress by adjusting the supply of
   oxygen and substances. Heart rate is an important indicator for
   measuring cardiac functionality [[134]19,[135]20,[136]21]. And the
   subsequent gradual recovery of heart rate indicates that scallops can
   adapt to the temperature condition of 27 °C and eventually tend to
   homeostasis. Similar phenomena of heart rate variation have also been
   reported in other mollusks and heart rate-related indicators such as
   ABT (Arrhenius break temperature) have been used in many studies to
   assess individual stress tolerance [[137]26,[138]49]. For example, the
   ABT values of two warm water species (A. irradians and A. ventricosus)
   were found to be significantly higher than those of two cold water
   species (C. farreri and P. yessoensis), which indicated that ABT could
   serve as a reliable indicator of the thermal tolerance for scallops
   [[139]49]. In abalone, the heart rates exhibited a sharp decline below
   a certain dissolved oxygen threshold, which can be referred to as the
   breakpoint of dissolved oxygen (BPDO). Further analysis showed that
   BPDO was significant related with the survival time of individuals
   under hypoxia (~0.5 mg/L) and can be used to select hypoxic-tolerant
   individuals for further genetic breeding [[140]50]. These studies
   suggest that heart rate and the related indicators are important in
   studying stress and tolerance in mollusks. Regarding the heart
   amplitude results, although there were no significant changes under
   high-temperature stress, a fluctuating trend was observed. Notably,
   during acute heat stress before 12 h, an inverted V-shaped change was
   evident, a trend that has also been found in the heat stress of the bay
   scallop (A. irradians). The inverted “V” trend implied that the
   homeostasis of Zhikong scallops with heat stress may be maintained by
   increasing the oxygen absorption to enhance oxygen supply, during which
   the metabolic changes in poikilotherms have been reported to be linked
   to higher temperature adaptation. According to the results of HA in
   this study, the changes continued to show an inverted V-shaped trend
   during the subsequent 30-day period. Moreover, we found that HA
   increased with duration of high-temperature stress and showed a certain
   recovery trend at the end. This suggested that under long-term stress,
   the heart may adapt to high temperatures by regulating its amplitude.
   This may be that under long-term heat stress, the scallops gradually
   adapt to the high-temperature stress, thus reducing the demand for the
   large supply of oxygen, so their hearts did not need to maintain a high
   level of amplitude.

   Furthermore, the antioxidant capacities of the heart were measured to
   investigate the physiological responses of Zhikong scallops under heat
   stress. Two essential antioxidant enzymes, SOD and CAT, were reported
   to cooperatively ameliorate organisms’ damages resulting from heat
   stress by scavenging accumulated ROS (reactive oxygen species).
   Consistent with such similar synergistic alterations in SOD and CAT
   activities in other mollusks facing thermal threats, our findings also
   showed significant upregulation of SOD and CAT, and this protective
   reaction might prevent the possible damage of scallops from heat
   stress.

   Transcriptomics are often used to explore the molecular mechanism
   behind biological progress [[141]51]. According to the heart
   transcriptomes analysis, under heat stress, the number of DEGs over a
   30-day stress period exhibited a trend of initially increasing and then
   decreasing. The DEGs were further classified into three different
   trends, indicating different regulatory mechanisms in the heart under
   heat stress.

   Pathway enrichment analysis showed that in expression trend A, 711
   differentially expressed genes were significantly enriched in pathways
   such as limonene and pinene degradation, ascorbate and aldarate
   metabolism, and fatty acid degradation ([142]Table S8a). Limonene and
   pinene degradation were the most significant pathway (p value < 0.05).
   There were three DEGs enriched in this pathway: ALDH1A1 (aldehyde
   dehydrogenase family 1 member A1, evm.model.scaffold692443.1), ALDH1B1
   (aldehyde dehydrogenase family 1 member B1, evm.model.scaffold3743.11),
   and ALDH3B1 (aldehyde dehydrogenase family 3 member B1,
   evm.model.scaffold58593.20), all belonging to the aldehyde
   dehydrogenase (ALDH) family and were down regulated before 3 d
   ([143]Figure 5). Therefore, we speculated that this pathway was
   involved in the cardiac response of scallops to high temperature by
   regulating the antioxidant capacity of ALDH. Moreover, these three
   genes were also enriched in the ascorbate and aldarate metabolism. In
   vertebrates, aldehyde dehydrogenase is an important enzyme involved in
   the detoxification of aldehydes and the mitigation of oxidative stress
   by converting toxic lipid peroxides, such as aldehydes, into non-toxic
   acids [[144]52,[145]53]. So, aldehyde dehydrogenase helps prevent the
   accumulation of toxic aldehyde compounds, thereby protecting cells from
   oxidative stress and contributing to overall cellular health and
   longevity [[146]54,[147]55,[148]56]. In C. farreri, down-regulation of
   ALDHs was found under heat stress, which indicated a weakened function
   of this protection in the heart under heat stress. Conversely, the
   enzyme activity assays indicated a significant increase in SOD and CAT
   activities within the first 3 h, suggesting that the primary
   antioxidant mechanisms in scallops in response to heat stress are
   activated during this period. Consequently, the total antioxidant
   capacity remains stable within this timeframe. In addition, other
   oxidases in the ascorbate and aldarate metabolism pathway also showed a
   downward trend before 6 d, such as inositol oxygenase (IOX,
   evm.model.scaffold56383.6) and L-ascorbate oxidase (LAC25,
   evm.model.scaffold12189.2; LAC6, evm.model.scaffold61517.16). In
   Drosophila melanogaster, inositol oxygenase catalyzes the conversion of
   myo-inositol to glucuronic acid, which is a key step in inositol
   catabolism [[149]57]. The down-regulation of IOX in scallops under high
   temperatures may suggest that heat stress inhibits inositol catabolism.
   Finally, L-ascorbate oxidase is involved in the oxidation of L-ascorbic
   acid to dehydroascorbic acid, thus it is crucial in maintaining the
   redox balance within cells [[150]58,[151]59]. As L-ascorbic acid can
   effectively remove free radicals in the body and reduce the damage of
   free radicals to cells, thereby protecting cells from oxidative stress,
   so the down-regulation of its oxidase was conducive to maintaining the
   function of the heart of C. farreri in high-temperature environments.
   Overall, in the heart of C. farreri under heat stress, the expression
   levels of these oxidases decreased, whereas the activities of
   antioxidant enzymes such as SOD and CAT increased. Concurrently, heart
   rate increased before 6 d. These observations suggest that an enhanced
   antioxidant capacity may play a crucial role in maintaining cardiac
   physiology, thereby promoting survival and adaptability to heat stress
   conditions.

   In expression trends B, 106 differentially expressed genes were
   predominantly involved in significant pathways like base excision
   repair, gap junction, chloroalkane, and chloroalkene degradation, among
   which base excision repair was the most significant (p value < 0.05)
   ([152]Table S8b). A/G-specific adenine DNA glycosylase (ADG,
   evm.model.scaffold4427.19) and DNA polymerase I (DPI,
   evm.model.scaffold38817.12) were enriched in the base excision repair
   pathway and were up-regulated during the heat stress ([153]Figure 5).
   A/G-specific adenine DNA glycosylase is an enzyme that plays a crucial
   role in the base excision repair (BER) pathway, which is responsible
   for correcting damaged or inappropriate bases in DNA [[154]60,[155]61].
   In an experimental heart failure (HF) model in rats, similar to our
   results, significant up-regulation of DNA glycosylase activities was
   observed, and this up-regulation initiated the BER pathway, which could
   play an important role during HF by counteracting the effect of
   genotoxic stress, structural damage of tissue, and myocardial
   remodeling [[156]62]. Our results showed that two of the key enzymes,
   A/G-specific adenine DNA glycosylase and DNA polymerase I, were
   up-regulated in the BER process, which enhanced the correction of
   damaged or inappropriate bases in DNA to resist heat stress in the
   heart of C. farreri under heat stress. In the gap junction pathway,
   tubulin alpha-3 (Tuba3a, evm.model.scaffold55215.10) and tubulin beta-1
   (Tubb1, evm.model.scaffold42195.19) were up-regulated. They are both
   critical components of microtubules, which work together to form
   microtubules through dynamic polymerization and depolymerization
   processes driven by the binding and hydrolysis of GTP (guanosine
   triphosphate) [[157]61,[158]62]. Under heat stress, the up-regulation
   of these genes in the scallop heart may contribute to the maintenance
   of cellular shape and integrity, intracellular transport, and cell
   division.

   The expression trend C showed two fluctuations of decreasing and
   increasing, in which axon guidance, chemokine signaling pathway, and
   ErbB signaling pathway was most significantly enriched (p value < 0.05)
   ([159]Table S8c). Five genes were enriched in all three pathways:
   Mitogen-activated protein kinase 3 (MAPK3, evm.model.scaffold61513.34),
   p21-activated kinase 1 (PAK1, evm.model.scaffold56431.21),
   Phosphatidylinositol-4,5-bisphosphate 3 kinase (PI3K,
   evm.model.scaffold33891.7; evm.model.scaffold58593.22), and
   1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase (PLC,
   evm.model.scaffold47691.35). Mitogen-activated protein kinases (MAPK)
   comprise a family of ubiquitous proline-directed,
   serine/threonine-protein kinases, which lie in protein kinase cascades
   and participate in signal transduction pathways that could control
   intracellular events including acute responses to environmental stress
   [[160]63,[161]64].

   P21-activated kinases (PAK) are key effectors of the small GTPases Rac1
   and Cdc42, as well as Src family kinases. PAK1 has several
   well-documented roles such as cell proliferation, adhesion, and
   migration [[162]65]. In particular, PAK1 has been founded in cardiac
   physiology and cardio protection. For example, PAK1 roles in the
   regulation of excitability and contractility of the heart
   [[163]66,[164]67,[165]68]. PAK1 was identified in cardiomyocyte Ca^2+
   handling, and it was demonstrated that it functions through unique
   mechanisms involving regulation of the post-transcriptional activity of
   key Ca^2+-handling proteins, including the expression of Ca^2+-ATPase
   [[166]69]. In this study, under heat stress, expression of MAPK3 and
   PAK1 in scallop hearts showed two fluctuations of decreasing and
   increasing, which may regulate cardiac excitability and contractility,
   thus influencing the heart amplitudes that showed a contrary expression
   trend. PI3K is a critical player in the signal transduction pathways
   initiated by growth factors, hormones, and other extracellular stimuli.
   It converts phosphatidylinositol-4,5-bisphosphate (PIP2) into
   phosphatidylinositol-3,4,5-trisphosphate (PIP3) [[167]70,[168]71].
   While PLC hydrolyzes PIP2 to generate two important secondary
   messengers: inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)
   [[169]72]. IP3 binds to IP3 receptors (IP3R) on the endoplasmic
   reticulum, reducing the release of calcium ions from the ER into the
   cytoplasm [[170]73,[171]74]. The elevation of intracellular free
   calcium ion concentration may modulate cardiac contraction,
   corresponding to heart rate variation in C. farreri under heat stress.

   WGCNA analysis was applied to classify the possible functions of the
   expressed genes, which may drive C. farreri response to heat stress.
   Although the genes were divided into two modules, [172]Figure 8 showed
   a close relationship between them. KEGG pathway analysis revealed that
   these modules shared overlapping pathways, including calcium signaling
   pathway, vascular smooth muscle contraction, cGMP-PKG signaling
   pathway, and MAPK signaling pathway, as shown in [173]Tables S6 and S7.

   The calcium signaling pathway was widely related to signal transduction
   as well as environmental stress responses of organisms
   [[174]75,[175]76,[176]77]. G protein-coupled receptors (GPCRs) are a
   class of cell surface receptors that regulate various signaling
   pathways inside cells by responding to external signals
   [[177]78,[178]79,[179]80]. GPCRs are involved in the calcium signaling
   pathway and can regulate the concentration of calcium ions in the cell.
   A typical example is that certain GPCRs can activate G proteins to
   initiate the hydrolysis of phosphatidylinositol-4,5-diphosphate (PIP2),
   so that inositol-1,4,5-triphosphate (IP3) is produced [[180]80]. In
   this study, after exposure to heat stress, GPCR
   (M1-evm.model.scaffold42667.6, M1-evm.model.scaffold48689.2,
   M2-evm.model.scaffold35009.3, M2-evm.model.scaffold40853.21.1) in the
   calcium signaling pathway was significantly down-regulated in the heart
   of C. farreri after exposure to heat stress, which may lead to the
   decrease of intracellular IP3. And at the same time, the IP3R genes
   (M1-evm.model.scaffold62249.5, M2-evm.model.scaffold63833.21,
   M2-evm.model.scaffold726937.1, M2-evm.model.scaffold61517.13) of the
   heart showed a sustained down-regulated expression trend, which may
   reduce the accumulation of calcium ions in the ER and at the same time
   increase the release of calcium ions from the ER into the cytoplasm,
   ultimately leading to an increase in intracellular levels of free
   calcium ions. Therefore, the calcium signaling pathway regulated the
   concentration of intracellular free calcium ions, and scallops may
   respond to heat stress by regulating intracellular calcium ion
   concentration.

   Furthermore, regulation of actin cytoskeleton was specifically enriched
   (p value < 0.05) in M2 ([181]Table S7). In this pathway, 34 DEGs were
   enriched, such as actin (evm.model.scaffold14907.7), actin-related
   protein 2 (ARP2, evm.model.scaffold13457.11), myosin light chain kinase
   (MLCK, evm.model.scaffold53609.10, evm.model.scaffold63779.11,
   evm.model.scaffold1989.106.1, evm.model.scaffold63699.58), and myosin
   light chain phosphatase (MLCP, evm.model.scaffold55531.13). In the
   heart, actin is a key cytoskeletal protein involved in the contraction
   and relaxation of cardiomyocytes. It regulates the movement of
   cardiomyocytes by forming myosin complexes with myosin and troponin
   [[182]81]. ARP2 participates in regulating cytoskeletal recombination
   and cell motility in cardiomyocytes. Together with actin, it plays a
   role in the contraction and relaxation of cardiomyocytes
   [[183]82,[184]83]. MLCK and MLCP regulates myosin light chain (MLC)
   phosphorylation, which is determined by the balance of its
   phosphorylation and dephosphorylation activities and plays important
   roles in regulating the contraction process of cardiomyocytes
   [[185]84,[186]85,[187]86]. These regulatory factors influence the
   contractility and elasticity of cardiomyocytes, consequently impacting
   the amplitude of cardiac activity, which exhibited a biphasic trend
   characterized by an initial increase followed by a decrease. This
   observation suggests that regulation of protein phosphorylation plays a
   critical role in the heart of scallops responding to heat stress.

5. Conclusions

   In the present study, we investigated the potential mechanisms
   underlying the cardiac response to heat stress in C. farreri. Our
   finding indicated that high temperature significantly affects the
   cardiac function of Zhikong scallops, which was proved by both heart
   rate and antioxidant capacity of C. farreri initially increased and
   subsequently returned to baseline levels at the end of 30-day heat
   stress period. The down-regulation of oxidases, coupled with the
   up-regulation of antioxidant enzymes such as SOD and CAT, in the heart
   of C. farreri proved that antioxidant mechanisms are essential for
   maintaining cardiac function and promoting survival under heat stress.
   Additionally, up-regulation of key enzymes involved in the base
   excision repair and gap junction pathways suggested that the heart of
   C. farreri under heat stress enhanced DNA repair and microtubule
   integrity to preserve cellular structure and function. Furthermore, the
   variable expression of key signaling molecules and cytoskeletal
   regulators suggested that the calcium signaling pathway, vascular
   smooth muscle contraction, cGMP-PKG signaling pathway, and MAPK
   signaling pathway played roles in modulating cardiomyocyte contraction
   in C. farreri under heat stress. Collectively, our findings provide
   novel insights into the cardiac response of the scallops to heat stress
   and enhance our understanding of potential adaptative mechanisms in
   scallops in the context of global warming.

Supplementary Materials

   The following supporting information can be downloaded at:
   [188]https://www.mdpi.com/article/10.3390/antiox13101217/s1, Table S1:
   Primers for quantitative real time-PCR validation; Table S2: Summary of
   RNA-Seq sequencing information in heart of C. farreri after heat
   stress; Table S3: GO analysis of DEGs; Table S4: KEGG analysis of DEGs;
   Table S5: FDR of modules and module size by WGCNA analysis in C.
   farreri heart after heat stress; Table S6: GO and KEGG analysis of M1;
   Table S7: GO and KEGG analysis of M2; Table S8: KEGG analysis of three
   expression trend.
   [189]antioxidants-13-01217-s001.zip^ (239.8KB, zip)

Author Contributions

   Conceptualization, Z.Y., J.H., Z.B. and X.H.; Methodology, X.W., Z.Y.,
   C.P. and H.Y.; Software, X.W.; Validation, X.W.; Formal analysis, X.W.;
   Investigation, X.W., C.P., H.Y., C.C. and Q.X.; Data curation, X.W.,
   Z.Y., C.P., H.Y., C.C. and Q.X.; Writing—original draft, X.W., Z.Y. and
   X.H.; Writing—review and editing, Z.Y. and X.H.; Visualization, X.W.
   and Z.Y.; Supervision, J.H., Z.B. and X.H.; Project administration,
   Z.B. and X.H.; Funding acquisition, Z.B. and X.H. All authors have read
   and agreed to the published version of the manuscript.

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The data presented in this study are available in the article.

Conflicts of Interest

   The authors declare no conflicts of interest.

Funding Statement

   This research was funded by the grants of the National Key Research and
   Development Program of China (2022YFD2400302), Earmarked Fund for
   Agriculture Seed Improvement Project of Shandong Province
   (2021LZGC027), the Earmarked Fund for Modern Agro-industry Technology
   Research System (CARS-49), and National Infrastructure of Fishery
   Germplasm Resources.

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References