Molecular Mechanisms of Reduced Risk of Decompression Sickness in Deep Diving Cetaceans

doi:10.21203/rs.3.rs-3643888/v1

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Molecular Mechanisms of Reduced Risk of Decompression Sickness in Deep Diving Cetaceans

https://doi.org/10.21203/rs.3.rs-3643888/v1

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published 04 Apr, 2024

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Background

Cetaceans, having experienced prolonged adaptation to aquatic environments, have undergone evolutionary changes in their respiratory systems. This process of evolution has resulted in the emergence of distinctive phenotypic traits, notably the abundance of elastic fibers in their lungs. These fibers facilitate the collapse of cetaceans' lungs during dives and subsequent re-expansion upon surfacing, effectively reducing the risk of decompression sickness. Such adaptations play a crucial role in minimizing the risk of decompression sickness during deep dives. Nevertheless, the scientific inquiry into the mechanisms through which these unique phenotypic characteristics govern the diving behavior of marine mammals, including cetaceans, remains unresolved.

Results

This study entails an evolutionary analysis of 42 genes associated with pulmonary fibrosis across 45 mammalian species. Twenty-one genes in cetaceans exhibited accelerated evolution, featuring specific amino acid substitutions in 14 of them. Primarily linked to the development of the respiratory system and lung morphological construction, these genes play a crucial role. Moreover, among marine mammals, we identified eight genes undergoing positive selection, and the evolutionary rates of three genes significantly correlated with diving depth. Specifically, the SFTPC gene exhibited convergent amino acid substitutions. Through in vitro cellular experiments, we illustrated that convergent amino acid site mutations in SFTPC contribute positively to pulmonary fibrosis in marine mammals, thereby reducing the risk of decompression sickness during diving.

Conclusions

The study unveils pivotal genetic signals in cetaceans and other marine mammals, arising through evolution. These discoveries yield substantial biological insights into how these species mitigate the risk of decompression sickness during dives. Moreover, the research serves as a valuable reference for delving deeper into human diving physiology.

decompression sickness

lung fibrosis

amino acid substitution

molecular evolutionary analysis

cetaceans

Decompression sickness (DCS) is a multisystem disorder arising from the formation of gas bubbles in the body due to a sudden decrease in underwater pressure. Primarily occurring during activities like diving, underwater operations, and high-altitude flights necessitating a change in environmental pressure [1, 2]. DCS results from the inhalation of nitrogen at high pressure, leading to its dissolution in tissues and blood [3, 4]. Upon sudden return to a low-pressure environment, the rapid decrease in nitrogen partial pressure results in oversaturated nitrogen forming bubbles. These bubbles have the potential to block blood vessels, impact blood circulation, and inflict damage on the nervous, pulmonary, cardiovascular, and other systems [5]. Severity and symptoms of DCS vary among individuals. Mild DCS may manifest as symptoms like skin itching and fatigue, while severe cases can lead to significant central nervous system damage, potentially resulting in life-threatening outcomes such as coma and paralysis [6].

Current research indicates that numerous animals may manifest DCS symptoms after prolonged submersion. For example, humans may experience DCS symptoms during scuba diving [7–9]. Divers and free divers often engage in deep-sea diving, and sudden surfacing can result in the formation of nitrogen bubbles, leading to DCS symptoms like headaches, joint pain, and breathing difficulties [8, 9]. Certain seabirds, like penguins, can endure prolonged submersion. Upon surfacing, they may display DCS symptoms [10]. Sea turtles, another prevalent marine species, can endure extended underwater stays. Upon ascent, nitrogen gas in their bodies may form bubbles, resulting in DCS symptoms [11].

Despite DCS being well-documented in various animals and humans involved in activities with significant pressure changes, marine mammals, especially cetaceans, exhibit distinctive physiological adaptations that seem to reduce this risk [12–14]. For example, a study examining lung structures in diverse mammals revealed that cetaceans' lung parenchyma has a notably higher proportion of elastic fibers and smooth muscles compared to other mammals. This structural feature is thought to fulfill cetaceans' physiological requirements for swift and extensive gas exchange during deep dives, providing them with an advantage [13]. Another study revealed that cetaceans' alveolar walls are significantly thicker and enriched with elastic fibers, facilitating the maintenance of a relatively constant volume and shape in their lungs under high pressure. In typical mammalian lungs, comparable structural changes would lead to pulmonary fibrosis [15]. This is because mammalian lungs typically do not encounter high pressure, and these structural changes can compromise alveolar elasticity, resulting in lung tissue fibrosis [15]. Another study identified that two lung-specific genes, MAP3K19 and SEC14L3, have lost their function in cetaceans [16]. MAP3K19 belongs to the mitogen-activated protein kinase (MAPK) family and is upregulated in response to oxidative and other environmental stresses, inducing the expression of pro-inflammatory chemokines. In contrast, cetaceans exhibit the opposite phenotype, possessing a substantial amount of elastic tissue in their lungs. SEC14L3, serving as a sensor for lipid droplet packaging defects, might influence the composition of pulmonary surfactants. Alterations in pulmonary surfactants may affect cetaceans and other diving mammals [16]. The absence of MAP3K19 and SEC14L3 in cetaceans may also serve a protective function, mitigating the risk of DCS during deep dives [16]. Additionally, research on Leptonychotes weddelli revealed that their lungs collapse during deep and prolonged dives, with the degree of collapse positively linked to the maximum dive depth [17]. This is considered a mechanism for mitigating the risk of DCS during dives [17]. While we have identified macro-level mechanisms capable of reducing DCS risk in marine mammals, research at the molecular level on this matter is lacking.

The advancement of sequencing technology and the growing wealth of mammalian genome data present an opportunity to delve into the molecular mechanisms by which cetaceans mitigate the risk of DCS during extended dives. In this study, we utilized a functional genomics approach to assess the evolution of 42 genes across 45 mammalian species, encompassing both cetaceans with high-quality genomes and their terrestrial counterparts. Through a combination of bioinformatic analysis and in vitro functional experiments, our objective was to elucidate the molecular mechanisms by which cetaceans mitigate the risk of DCS during deep dives. Our findings contribute not only to a theoretical framework for comprehending the pathophysiology and genetics of DCS in humans but also provide insights into the evolution of marine mammals.

Species coverage and sequence acquisition

In our study, we investigated 45 mammalian species with high-quality sequencing and assembly, representing a wide range of classifications (include species with a pulmonary fibrosis phenotype and their close relatives). The selected species belong to different orders including: Cetartiodactyla (Tursiops truncatus, Orcinus orca, Delphinapterus leucas, Lipotes vexillifer, Physeter catodon, Balaenoptera acutorostrata, Balaenoptera musculus, Globicephala melas, Lagenorhynchus obliquidens, Monodon monoceros, Neophocaena asiaeorientalis, Phocoena sinus, Bos taurus, Ovis aries, Sus scrofa, Bos mutus and Bos grunniens), Perissodactyla (Equus caballus and Equus asinus), Carnivora (Odobenus rosmarus, Phoca vitulina, Leptonychotes weddellii, Neomonachus schauinslandi, Callorhinus ursinus, Eumetopias jubatus, Zalophus californianus, Halichoerus grypus, Mirounga leonine, Canis familiaris, Ailuropoda melanoleuca and Ursus maritimus), Chiroptera (Rhinolophus ferrumequinum), Eulipotyphla (Erinaceus europaeus), Proboscidea (Loxodonta africana), Sirenia (Trichechus manatus), Pilosa (Choloepus didactylus), Dasyuromorphia (Sarcophilus harrisii), Ornithorhynchidae (Ornithorhynchus anatinus), Primates (Homo sapiens, Rhinopithecus roxellana and Rhinopithecus bieti), Rodentia (Mus musculus and Rattus norvegicus), and Lagomorpha (Oryctolagus cuniculus and Ochotona curzoniae) (Table S2).

The gene set associated with pulmonary fibrosis disease was collected from several previously published articles [18–21], as well as a database using the keyword ‘pulmonary fibrosis’ (https://www.informatics.jax.org/) (Table S1). The protein-coding sequences (CDS) were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/) (Table S3). In addition, custom perl scripts were used to perform BLASTn searches to further verify partially or unannotated CDS. The longest transcript was retained for each gene in this analysis. To obtain higher quality sequences, we performed multiple sequence alignments for each orthologous gene using MACSE v2 [22] combined with PRANK v.170427 [23]. The aligned sequences were then trimmed using Gblocks v0.91 [24] with default settings.

Molecular evolutionary analysis

Comparisons of nonsynonymous (dN)/synonymous (dS) substitution ratios have become a useful means for quantifying the impact of natural selection on molecular evolution [25, 26]. We employed the CODEML model implemented in the PAML 4.7 [27] software to perform codon-based maximum likelihood estimation of ω values. Values of ω < 1, = 1, and > 1 correspond to purifying selection, neutral evolution, and positive selection, respectively [28]. Considering the influences of base transition/transversion rates and codon usage in molecular evolution [29], we employed likelihood ratio tests (LRTs) to compare the hypotheses within each pair of nested models. To account for multiple testing, we utilized the Benjamini-Hochberg method [30] and set a false discovery rate (FDR) cutoff of 0.05.

To identify REGs in cetaceans, the two-ratio model in PAML was employed [27–31]. The null hypothesis (one-ratio model) assumed that genes in all-mammals evolved with the same ω, while the alternative hypothesis set two different ω values for the foreground (cetacean) and background (other mammal) species. Genes with ω (cetaceans) > ω (other mammals) were considered REGs in cetaceans [28].

To test whether positively selected sites were limited to a specific lineage, we used branch-site model and free-ratio model in the CODEML program to analyze the all-mammals dataset. The branch-site model can detect positive selection at specific sites along a specific branch, in both the null hypothesis (Ma0) and alternative hypothesis (Ma), distinctions in ω values are permitted among foreground and background branches and within sites in each gene. Under the Ma0 model, all species and sites are assumed to have ω values that do not exceed 1, indicating the absence of positive selection signals. In contrast, the Ma model permits certain sites in the foreground branch (cetaceans) to have ω values greater than 1, indicating the presence of positively selected sites [32, 33]. Whereas the free-ratio model allows an independent ω value for each branch [27], we compared the free-ratio model that assumes an independent ω ratio for each branch with the null one-ratio model with the same ω for all branches. When the value of dN or dS in each ω value is less than 0.0002, we considered it as an outlier “n/a” [34]. The relative goodness-of-fit of the nested models was determined using LRTs. All nested models were statistically different from the null model (P < 0.05) [35].

Identification of specific and convergent amino acids.

We used the Fasparser 2.0 [36] to detect specific amino acids in cetaceans and convergent amino acids in marine mammals, respectively. Human canonical sequences in UniProt (https://www.uniprot.org/) [37] were taken as references for the locations of amino acids.

Mapping of positively selected sites and cetacean-specific sites onto protein structures

To investigate the impact of positively selected sites and cetacean-specific sites on protein function. We used Swiss-Model (https://swissmodel.expasy.org/) [38] to predict the 3D structure, then used the Pfam (http://pfam.xfam.org/) [39] website to search for domains of each protein. Finally, the EzMol (http://www.sbg.bio.ic.ac.uk/~ezmol/) [40] was used to annotate the positively selective sites and functional domains in the obtained 3D structure.

Association analysis between gene evolution and diving depth

In order to investigate the potential relationship between the gene evolutionary rate (ω) and diving depth, we utilized root-to-tip ω, which refers to the average ω value accumulated from the last common ancestor to the extant species and serves as a selection indicator encompassing the evolutionary history of the gene within a given species [41, 42]. The evolutionary rate of each gene was calculated using codeml in PAML [27] with a free-ratio model. The diving depth data of marine mammals were collected from previously published sources (Table S7). PGLS regression [43] was used to analyse the relationship between log (root-to-tip ω) and log (diving depth). The lambda (λ) value estimated by the maximum likelihood method was used as a quantitative measure of phylogenetic signals [44]. All statistical analyses were performed using R in the package Caper [45].

Gene functions and signaling pathway annotation and enrichment

The genes were subjected to GO functional [46] enrichment analyses using the Metascape (http://metascape.org) [47]. In Metascape, we set Homo sapiens as the "Input as species" and "Analysis as species" and used a cutoff of p-value < 0.05.

Cell culture, lentiviral production and infection

Human NSCLC cell line A549 cells was purchased from ATCC and cultured according to the protocols of the ATCC. All culture media were purchased from WISENT, cell culture dishes were purchased from Jet Biofil. The sequence of Whale-SFTPC was generated in Beijing Tsingke Biotech Co. Ltd. Lentiviral vectors (Lentivirus-mCherry-Puro-CMV-N3xFlag-BI-SFTPC/mutSFTPC, Lentivirus-mCherry-Puro-CMV-N3xFlag-Mouse-SFTPC/mutSFTPC) were produced by Corues Biotechnology Company (Nanjing, China), and lentiviral infection protocol followed the company’s instructions. The sequence is shown in the TableS11.

Dna extraction and pcr amplification

Fresh mouse lung tissue samples were used, mouse RNA was extracted according to the traditional phenol-chloroform protocol [82], and the cDNA of mouse SFTPC was obtained by PCR amplification. PCR profiles were 95°C/5 min, 95°C-30 s, 70°C-30 s, 72°C-40 s, and 72°C/5 min cycles, repeated 35 times. PCR products were directly sequenced using an ABI 3730 DNA sequencer (Sangon Biotech, Shanghai Co, Ltd). The construction of mouse and cetacean SFTPC gene mutants with individual amino acid mutations (I123V, V123I) followed the instructions of the Beyotime (Shanghai, China) Quick Mutation kit. All primers used for amplification are listed in Table S10.

Antibodies

Antibodies used were as follows: antibodies against Cleaved-Caspase 3 (cat# 9664), was purchased from Cell Signaling Technology, Inc., Danvers, MA, USA; antibodies against MMP7 (cat# A20701), ACTA2 (cat# A17910) were purchased from ABclonal (Wuhan,China) ;antibodies against SFTPC (cat#10774-1-AP) and beta-Tubulin (cat# 10094-1-AP) were purchased from Proteintech Group, Rosemont, IL, USA.

Western blotting

Collect the cells and lyse them in cell lysis buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% NonidetP-40, 0.5% sodium deoxycholate, and 1% protease inhibitor cocktails,MCE), centrifuge the cell lysate at 4°C, 14,000 rpm for 15 min, In Western blot studies, protein concentrations of samples were determined using the Bradford protein method, and 20 µg of proteins for each sample were separated by SDS-PAGE followed by transfer to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h at room temperature with 5% nonfat milk in the TBST buffer and incubated with a specific primary antibody diluted in TBST buffer overnight at 4 ℃. After extensive washes by TBST buffer, the membranes were incubated with a corresponding secondary antibody for 1 h at room temperature, and the immunoreactive bands were visualized by using an ECL kit (Vazyme Biotech Co. Ltd., Nanjing, China).

Cell viability assay

Cell proliferation was assessed through CCK8 assays. Cells were briefly seeded at 3 × 103 cells/well in triplicate in a 96-well plate and cultured overnight. At 24 h and 48 h, 10 µL of CCK8 solution (Vazyme Biotech Co. Ltd., Nanjing, China) was added to each well, followed by incubation at 450 nm for an additional 2 h using a microplate reader to measure absorbance. Cell viability for each treatment was determined as a percentage of its OD450 nm compared to the vehicle control, calculated using GraphPad Prism 8.0 software.

Statistical analysis

Experiments were carried out at least 3 times unless otherwise stated. The software GraphPad Prism 8.0 was used for statistical analysis and the data are shown as mean ± S.D. Student’s t-test and one way ANOVA were employed to assess the statistical significance of differences between data sets. A p-value of lower than 0.05 was considered to be significant. p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Rapidly evolving genes and positively selected genes in cetaceans

Positive selection and rapid evolution of specific genes result from the accumulation of genetic variation in response to environmental changes, a component of Biological Adaptation to Natural Selection [48]. In this study, we employed the Consensus Tree from TimeTree (Fig. S1) and the CODEML model in PAML to detect rapidly evolving genes (REGs) and positively selected genes (PSGs) across 45 mammalian species, designating cetaceans as the foreground species and other mammals as the background. Employing branch model analyses with CODEML, we identified 21 REGs in cetaceans, adjusting for multiple testing using the FDR method (adjusted p-value < 0.05) (Table S4). The evolutionary rates of these genes were notably higher in cetaceans than in other mammalian species (Fig. 1A), implying unique selective pressures on cetaceans. Enrichment analysis further disclosed that REGs in cetaceans were linked to respiratory gaseous exchange by the respiratory system, diseases associated with surfactant metabolism, blood vessel development, and blood vessel morphogenesis (Fig. 1B).

The Branch model (One-ratio vs Free-ratio) and Branch-site models were utilized to identify positive selection in seven cetacean genes [49] (Table S5, Table S6). Four of these genes (SFTPB, SFTPC, MMP7, HLA-DRB1) exhibited positive selection in the background branch species as well (Fig. 1C). To establish the relationship between the proposed positively selected amino-acid sites and protein function, we conducted a search on the UniProt database to map these sites onto their respective 3D structures. The results revealed that certain positively selected amino-acid sites were located within crucial functional domains of proteins (Fig. S2). Specifically, site 40 and 69 of MMP7 exhibit positive selection in the Putative peptidoglycan binding domain, and sites 104, 115, 121, 131 are situated in the Matrixin domain. Previous reports indicate that MMP7 overexpression induces apoptosis of alveolar epithelial cells and migration of interstitial cells, leading to the destruction of the alveolar wall and the development of pulmonary fibrosis [21, 50] (Fig. S2).

Specific amino acid substitutions in cetaceans

Fixed amino acid replacements within a specific group of mammals may contribute to explaining a particular phenotype [34]. We detected 28 cetacean-specific amino acid substitutions in 14 proteins (Fig. 2). Enrichment analysis unveiled associations of the genes encoding these proteins with inflammatory response, lung development, and respiratory system development (Fig. S3). Mapping the cetacean-specific sites to their respective 3D structures was done to assess their functional relevance. The results revealed that certain sites were located within crucial functional domains of proteins (Fig. S4). As an example, the cetacean SERPINE1 protein exhibited a specific amino acid site (53) within the Serpin (serine protease inhibitor) domain. Previous reports indicate that mutations or functional defects in the Serpin domain may result in the loss of its ability to inhibit protease activity, contributing to its functional abnormality in pulmonary fibrosis [51, 52](Fig. S4).

Association between gene evolution and diving depth

Evaluating the statistical association between genes and normal phenotypic variation is a well-regarded strategy for investigating the phenotypic implications of an ongoing selection signature [53]. Phylogenetic generalized least squares (PGLS) regressions were conducted to explicitly explore the connection between the evolutionary rate of genes and diving depth. Regression analyses demonstrated a significant positive association between log10 (root-to-tip ω) and log10 (diving-depth m) in three genes, namely BDKRB1 (R2 = 0.1760, p = 0.0460), PINK1 (R2 = 0.0990, p = 0.0282), and RTEL1 (R2 = 0.2090, p = 0.0401) (Fig. 4, Table S7, Table S8).

Functional convergence of the SFTPC among marine mammals

Initial observations revealed convergent/parallel amino acid substitutions in the SFTPC protein among 23 marine mammals (excluding Trichechus manatus). At position 123 of the SFTPC protein, marine mammals exhibit Valine, while terrestrial mammals display Isoleucine (Fig. 3A). To ascertain the relationship between this convergent site and protein function, we mapped the site onto the three-dimensional structure of the SFTPC protein. The results indicated that the site is situated in the BRICHOS domain of the SFTPC protein (Fig. 3B).

Correlation between SFTPC gene mutations and protein expression of pulmonary fibrosis markers

We investigated whether convergent evolutionary site variations in the SFTPC gene in marine mammals enhance pulmonary fibrosis, leading to lung collapse during diving and potentially reducing the risk of decompression sickness. Various SFTPC variants were overexpressed, and western blotting techniques were utilized to assess whether variations in SFTPC gene loci induce alterations in the expression levels of fibrotic marker proteins, including MMP7 (Matrix Metalloproteinase 7) and α-SMA (Alpha-Smooth Muscle Actin). The results show that in whales, the expression levels of MMP7 and α-SMA proteins in wild-type individuals are higher than those in the control and mouse wild-type. Additionally, in whale mutants, there is a decrease in the expression of both MMP7 and α-SMA proteins compared to their wild-type counterparts. In mouse mutants, there is an increase in MMP7 protein expression relative to their wild-type, but no significant change in α-SMA protein expression (Fig. 5A).

Correlation between SFTPC gene mutations and cell proliferation and apoptosis abilities

The regulatory mechanisms connecting pulmonary fibrosis and endoplasmic reticulum (ER) stress are intricately linked [54]. Subsequently, we conducted a thorough investigation to determine whether lung fibrosis resulting from SFTPC gene mutations influences cellular proliferation and apoptosis during the ER stress phase. Initially, we evaluated cell proliferation levels after overexpressing various SFTPC variants. Our results indicate that in cell proliferation assays, the five experimental groups exhibited higher cell proliferation capabilities at 24 and 48 hours in both wild-type and mutant mice compared to wild-type and mutant whales. No significant changes were observed in proliferation capabilities between wild-type and mutant whales compared to the control and between wild-type and mutant mice (Fig. 5B, Table S9). We then analyzed alterations in the expression levels of the apoptotic effector, Cleaved-caspase 3, using protein assays. We observed that, compared to the control, the expression level of Cleaved-caspase 3 increased in wild-type whales, and it was higher in wild-type mice compared to wild-type whales (Fig. 5C).

Cetaceans, mammalian species that have fully transitioned from land to an aquatic lifestyle, have developed adaptive changes in their respiratory system in response to the low-oxygen environment of the ocean. The lungs, among these adaptations, serve as a crucial interface between the body and the environment during respiration under low-oxygen conditions. Research has demonstrated that the lungs of cetaceans, specifically whale species, have undergone adaptive evolution related to resistance against oxidative stress [25]. Understanding the molecular evolutionary mechanisms that drive diving adaptations in whales is essential for comprehending the treatment of diving-related disorders. Proposals suggest that the lungs of cetaceans have developed specific features, including a notable presence of elastic fibers, to adapt to diving and breath-holding. Our research in this study indicates that genes linked to pulmonary fibrosis have undergone adaptive evolution in marine mammals, such as whales. This adaptation is crucial for their adjustment to hypoxic environments and the mitigation of the risk of DCS.

The adaptation of cetaceans to diving and breath-holding is the result of coevolution of multiple genes

Cetaceans, a group of mammals, have transitioned from land to a fully aquatic lifestyle. Owing to the hypoxic ocean environment, their respiratory system has undergone a series of adaptive changes. Among these adaptations, the lungs serve as a crucial interface between the body and the environment during respiration in a hypoxic environment. Studies have demonstrated that the lungs of cetaceans have undergone a series of adaptations related to resistance against oxidative stress [55]. Suggestions propose that the lungs of cetaceans have developed specific characteristics, including a substantial amount of elastic fibers, to adapt to diving and breath-holding.

Our study results indicate accelerated evolution and specific amino acid substitutions in multiple genes related to lung fibrosis in cetaceans. Functional enrichment analysis revealed their main enrichment in processes such as respiratory gas exchange, cellular response to DNA damage stimuli, pulmonary development, and inflammatory response. This suggests that these genes not only contribute to lung fibrosis in cetaceans but are also closely linked to their adaptation to hypoxic environments, diving, and breath-holding. For example, the RXFP1 gene encodes a receptor protein belonging to the relaxin/insulin-like peptide family receptor. In a mouse experiment, RXFP1 expression alleviated symptoms of lung fibrosis and pulmonary arterial hypertension [56]. The PLAU gene encodes an enzymatic protein known as plasminogen activator. Research has demonstrated the pivotal role of PLAU in hypoxia adaptation for cetaceans [57]. This study primarily employed a genome-wide large-scale screen, determining the association of the PLAU gene with hypoxia adaptation in porpoises and its likely prominent role in hypoxic responses [57, 58]. Similarly, numerous genes play roles in lung development, such as ABCA3. The AP3B1 gene encodes the β3A subunit of the adaptor protein complex 3 (AP-3), and mutations in the AP3B1 gene result in Hermansky-Pudlak syndrome (HPS) subtypes HPS-2 and HPS-10. Pulmonary fibrosis develops in HPS-1, HPS-2, and HPS-4 [59, 60]. Growing evidence suggests that pulmonary fibrosis, as a particular phenotype, may be a crucial protective mechanism in whales against diving-related illnesses. Overall, the adaptive evolution of genes associated with pulmonary fibrosis may be a crucial mechanism for cetaceans to adapt to deep-diving lifestyles.

The high abundance of elastic fibers in the lungs is a common strategy among marine mammals to reduce the risk of DCS in hypoxic environments

The mechanism employed by marine mammals, cetaceans included, to mitigate the risk of DCS during prolonged dives has long been a subject of investigation. While there is ample macroscopic evidence indicating an enrichment of elastic fibers in the lungs of these animals, potentially aiding in adapting to hypoxic environments and reducing the risk of DCS during diving respiration, the majority of supporting evidence is derived from anatomical and physiological ecological studies. There is a notable dearth of molecular evidence from an evolutionary perspective.

By comparing two models (branch model and branch-site model) using PAML, a prevalence of positive selection was found in lung fibrosis-related genes among marine mammals, with the majority of positively selected sites situated in crucial functional protein domains. In humans and other mammals, elastic fibers in the lungs constitute a minimal proportion [61], ensuring optimal elasticity and morphological stability for efficient gas exchange and respiratory function. While an abundance of elastic fibers in the lungs may induce lung fibrosis in typical mammals, resulting in respiratory challenges, marine mammals do not experience such issues. This phenomenon may be attributed to cetaceans' possession of a dual capillary network in the lungs [13, 15], enhancing gas exchange between alveoli and capillary blood, averting respiratory distress despite fibrosis. The elevated fibrosis in cetacean lungs suggests that lung fibrosis-related genes have undergone adaptive evolution in marine mammals to facilitate their diving and respiratory processes. Eight lung fibrosis-related genes were identified: BDKRB1, CHRM3, HLA-DRB1, MMP7, PLAU, SETD2, SFTPB, and SFTPC. BDKRB1 encodes the bradykinin B1 receptor, binding to the peptide hormone bradykinin and engaging in various inflammatory response processes. Studies have demonstrated that the deletion of BDKRB1 in mice inhibits fibrosis [62], suggesting the receptor's role in promoting fibrosis and its potential significance in mammalian lung fibrosis. SFTPB and SFTPC genes encode vital components of pulmonary surfactant, crucial for maintaining alveolar surface tension and lung tissue architecture. Research indicates that mutations in these genes among marine mammals may be linked to adaptations to hypoxic conditions during diving [63]. Mutations in the CHRM3, HLA-DRB1, PLAU, and SETD2 genes are closely linked to the pathogenesis of pulmonary fibrosis [18, 58, 64, 65]. The MMP7 gene, serving as a biomarker for pulmonary fibrosis, has been demonstrated to contribute to the development of lung fibrosis through its elevated expression [21, 50].

Simultaneously, correlation analysis reveals a positive relationship between the evolutionary rates of the BDKRB1, PINK1, and RTEL1 genes and maximum diving depth. The functions of these genes are linked to the formation of pulmonary fibrosis, implying their significant role in the diving respiration process of cetaceans. For example, research has demonstrated that mutations in the RTEL1 gene contribute to an animal's adaptation to hypoxic conditions [66]. In mice, the reduction of PINK1 expression in lung epithelial cells resulted in mitochondrial depolarization and the expression of profibrotic factors. Collectively, these findings illustrate that the adaptive evolution of pulmonary fibrosis-related genes in marine mammals enhances their ability to thrive in diving conditions, reducing the risk of decompression sickness [67].

The evolution of SFTPC may have contributed to pulmonary fibrosis in marine mammals

Pulmonary surfactant, composed of lipids and proteins, is crucial for alveolar stability, surface tension reduction, and gas exchange in the lungs. SFTPC, encoding a hydrophobic protein in type II alveolar epithelial cells, is integral to these functions. The BRICHOS domain (residues 94–197) in SP-C prevents protein aggregation, ensuring proper folding and stabilization [68, 69]. The BRICHOS domain assists in the proper folding and stabilization of SP-C, preventing the formation of toxic aggregates in the alveoli and enabling its normal biological function. Previous research has found that mutations in the SFTPC gene are associated with several familial and sporadic interstitial lung diseases, including pulmonary fibrosis. These mutations can lead to abnormalities in the structure and function of the SFTPC protein, affecting the composition and function of pulmonary surfactant, ultimately resulting in lung tissue damage and fibrosis. Several studies have linked mutations in SFTPC within the BRICHOS domain of the proprotein (proSP-C) to interstitial lung diseases. For example, DNA sequence analyses of the surfactant protein C gene in children with nonspecific interstitial pneumonia and adults with usual interstitial pneumonia have identified a common heterozygous mutation in exon 5. This mutation causes a Leu188 to Gln188 change in the carboxy-terminal region of prosurfactant protein C, potentially impacting peptide processing. These observations suggest that individuals with this specific mutation in the surfactant protein C gene may have an increased risk of various types of interstitial lung diseases [70]. Using SP-CΔexon4 as a model molecule, a study provides evidence supporting the concept that misfolded BRICHOS mutant isoforms could induce cell injury by disrupting two separate but mechanistically linked subcellular systems, the unfolded protein response (UPR) and the ubiquitin-proteasome system (UPS) [69]. In summary, mutations occurring in the BRICHOS domain of surfactant protein C have been associated with interstitial lung diseases, including pulmonary fibrosis. These mutations can lead to abnormal protein structure and function, affecting the composition and function of pulmonary surfactant, and contributing to lung tissue damage and fibrosis [54, 71–74].

Moreover, our investigation demonstrates that a mutation at position 123 of the SFTPC gene, situated within the BRICHOS domain in whales, induces an upregulation of the lung fibrosis marker proteins MMP7 and α-SMA. Comparative analysis reveals heightened MMP7 protein expression in whales compared to wild-type mice. Rescue experiments further indicate increased MMP7 expression in mutant mice compared to their wild-type counterparts, whereas in whale mutants, MMP7 expression is reduced compared to wild-type mice. Similarly, α-SMA protein expression is elevated in whales relative to wild-type mice, with no increase observed in mutant mice compared to wild-type. Consistent with previous research, it is recognized that heightened MMP7 and α-SMA expression contributes to the advancement of pulmonary fibrosis [21, 50, 75]. This fibrotic development may contribute to a reduced risk of DCS in cetaceans during their diving activities [16]. In both wild-type and mutant mice, the expression levels of α-SMA protein vary. We believe that amino acid substitutions may have varying effects on protein stability and function in different organisms, and can also affect interactions with other proteins, thereby leading to differences in protein expression levels [76]. The abnormal excessive apoptosis of alveolar epithelial cells is one of the important characteristics of the development of COPD [77, 78]. Therefore, we further investigate the levels of cell proliferation and apoptosis. The cell proliferation assay indicated that at 24 and 48-hour intervals, the proliferation of cetacean wild-type and mutant A549 cells showed no significant change compared to the blank control and murine wild-type and mutant variants. However, apoptosis assays revealed an increase in Cleaved-caspase 3 expression in both wild-type and mutant groups of cetaceans and mice compared to the control, indicating apoptosis occurred in all experimental groups. Notably, more apoptosis was observed in the murine wild-type cells compared to the cetacean mutant variants. Our findings further corroborate previous discoveries that pulmonary fibrosis-induced damage to alveolar epithelial cells leads to an increase in apoptosis levels, while not affecting proliferation levels [79, 80]. Compared to the wild-type cells of mice, there is a reduced level of apoptosis in the wild-type cells of whales. We hypothesize that this could be attributed to adaptive mechanisms evolved by cetaceans due to their prolonged underwater lifestyle. These mechanisms, occurring alongside pulmonary fibrosis in whales, reduce the damage to alveolar epithelial cells, thereby facilitating their respiratory function during diving activities. These results suggest that marine mammals, including whales, have adapted to their long-term aquatic environment by evolving a distinctive phenotype in their lungs. This phenotype involves a significant thickening of the alveolar walls (the walls of the air sacs) and an abundance of elastic fibers [81]. This adaptation facilitates lung collapse during the diving process, thereby reducing the risk of decompression sickness [18].

To elucidate the molecular mechanisms employed by cetaceans to mitigate the risk of decompression sickness during deep dives, we conducted a comprehensive investigation into 42 lung fibrosis-related genes across representative mammalian lineages. Our findings unveil accelerated evolution, specific amino acid substitutions, and positive selection in multiple lung fibrosis-related genes within cetaceans. This suggests an evolution in their respiratory systems, facilitating diving and minimizing the risk of decompression sickness. Convergent positive selection and amino acid substitutions were also observed in marine mammals. Combined with in vitro experimental validation of the SFTPC gene, these results indicate the contribution of lung fibrosis genes to the capacity of marine mammals to reduce the risk of decompression sickness during diving. Additionally, a significant correlation between gene evolution rate and diving depth was detected, supporting the selective gas exchange hypothesis of diving in marine mammals at the molecular level. In conclusion, the evolutionary patterns of lung fibrosis-related genes provide novel insights into the molecular mechanisms through which cetaceans mitigate the risk of decompression sickness during deep dives. This research not only enhances our understanding of the molecular evolutionary adaptations of cetaceans to aquatic life but also holds promise for developing strategies to prevent and treat decompression sickness in humans.

REGs

rapidly evolving genes

PSGs

positively selected genes

COPD

chronic obstructive pulmonary disease

DCS

Decompression sickness

SFTPC

Surfactant Protein C

PAML

Phylogenetic analysis by maximum likelihood

BLAST

Basic local alignment search tool

PGLS

Phylogenetic generalized least squares

LRT

Likelihood ratio test

FDR

False discovery rate

Gene Ontology

Ethics approval and consent to participate

All animal samples were collected legally and in accordance with the policy of the Animal Research and Ethics Committee of Nanjing Normal University. All animal experiments were approved by the Animal Research and Ethics Committee of Nanjing Normal University (NO: IACUC-20230402). There was no use of human participants, data, or tissues. We confirmed that all methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.

Consent for publication

Not applicable

Availability of data and materials

The data generated and analyzed during this study are included in this article and its additional files, including 11 tables and 11 figures. Gene ids in MGI database are available in Table S1, genome versions of NCBI database in this study are available in Table S2. Other data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by the National Natural Science Foundation of China (grant nos. 32270442, 31872219, 31370401, 32030011, 31630071, and 31772448), the National Key Research and Development Program of China (grant no. 2022YFF1301602) and the Postgraduate Research& Practice Innovation Program (grant nos. KYCX23_1747, KYCX23_1740 and KYCX23_1741). The funding bodies for this study had no role in the design of the study, collection of data, data analysis and interpretation, or in writing the manuscript.

Authors' contributions

WR, GY and SX conceived the study and assisted with manuscript revision. BG and YZ collected the data, BG performed the molecular evolution analysis and wrote the paper. YS, YZ and YW conducted cell-based experiments. All authors read and approved the final manuscript.

Acknowledgements

We thank Dr. Tianzhen Wu, Dr. Xu Zhou and all other members of Jiangsu Key Laboratory for Biodiversity and Biotechnology for suggestions and support during the project.

Author information

Authors and affiliations

Jiangsu Key Laboratory for Biodiverity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, 210023, China

Boxiong Guo, Yixuan Sun, Yuehua Wang, Ya Zhang, Yu Zheng, Shixia Xu, Guang Yang & Wenhua Ren*

Corresponding author

Correspondence to Wenhua Ren.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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No competing interests reported.

Additionalfile1.docx
Additional file 1: Fig. S1. The phylogeny of 45 mammals used in PAML (and in other subsequent evolutionary analyses such as PGLS) using TimeTree ( href="http://www.timetree.org/">http://www.timetree.org/). Fig. S2. The positively selected sites obtained using the branch-site model are mapped onto the three-dimensional structure of the gene. The sites with red numbers are located in the important functional domains of the gene. The figures were created using EzMOL. Fig. S3. GO enrichment terms for fourteen specific amino acid substitutions protein in cetaceans. The 20 related terms in biological process are shown. Fig. S4. cetacean-special amino acid substitutions mapped on the three-dimensional structure of genes. The sites with red numbers are located in the important functional domains of the gene. The figures were created using EzMOL. Fig. S5. Full-length blots/gels of SFTPC, MMP7, α-SMA and Tubulin (A, B, C and D). Fig. S6. Full-length blots/gels of Cleaved-Caspase 3 and Tubulin (A and B).
Additionalfile2.xls
Additional file 2: Table S1. 42 pulmonary-fibrosis-related genes and their function. Table S2. Taxonomic information and genome assembly quality assessment of the species used in this study. Table S3. Sequence accession information of 42 genes from 45 mammalian species used in this study. Table S4. A Branch-model analysis utilized the whale clade as the foreground branch and identified 21 genes associated with pulmonary fibrosis that underwent accelerated evolution. Table S5. The Branch-site model detected five PSGs. Table S6. The Branch model detected seven PSGs. Table S7. Diving data of marine mammals used in this study. Table S8. Association analysis between gene evolution and diving depth. Table S9. Assessing the cell viability of A549 cells at 0h, 24h, and 48h using CCK-8 assay. Table S10. Primers used in this study. Table S11. Sequences of different SFTPC variants.

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Molecular Mechanisms of Reduced Risk of Decompression Sickness in Deep Diving Cetaceans

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Abstract

Figures

Background

Methods and materials

Species coverage and sequence acquisition

Molecular evolutionary analysis

Mapping of positively selected sites and cetacean-specific sites onto protein structures

Association analysis between gene evolution and diving depth

Gene functions and signaling pathway annotation and enrichment

Cell culture, lentiviral production and infection

Dna extraction and pcr amplification

Antibodies

Western blotting

Cell viability assay

Statistical analysis

Results

Rapidly evolving genes and positively selected genes in cetaceans

Specific amino acid substitutions in cetaceans

Association between gene evolution and diving depth

Discussion

Conclusions

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