Single-cell transcriptomic dynamics of scallop heart reveals the heterogeneous response to heat stress

Abstract

Background: Animals with open circulatory systems are highly vulnerable to environmental temperature fluctuations, making them particularly threatened by global warming. However, research on the cellular heterogeneity of heart responses to elevated temperatures in animals with open circulatory systems remains limited.

Results: Here, we conducted a comprehensive investigation of the morphology, metabolism and scRNA-seq of the heart in a molluscan model, Argopecten irradians, under heat stress. Our results unraveled that the severity of cardiac structure damage increased progressively with rising temperature, accompanied by widespread mitochondrial dysfunction and neurohumoral response. We identified two subpopulations within cardiomyocytes (CMs), including ventricular myocytes (VMs) and atrial myocytes (AMs), which exhibited specialized functional roles in response to thermal stress. Specifically, AMs enhanced cell-cell communications with the immune-like cells and fibroblasts to contribute to maintaining cardiac homeostasis under heat stress. Whereas, VMs displayed enhanced energy supply and differentiation potential to withstand thermal challenges. Furthermore, RNA interference targeting the most heat-responsive gene, PLRP2-like, resulted in a significant reduction in heat tolerance and triglyceride accumulation in scallops.

Conclusions: Our study investigated the heterogeneous response of the scallop heart to high temperatures, revealing distinct response patterns between VMs and AMs. We further identified a key gene, AiPLRP2-like, which exhibits unique cellular localization patterns compared to its mammalian counterpart and may play a pivotal role in regulating cardiac thermotolerance in organisms with open circulatory systems. These findings provide novel insights into the theoretical framework and evolutionary adaptations of marine invertebrate hearts in response to environmental temperature fluctuations.

Keywords: Heart; Heat stress; Mollusks; Open circulatory systems; PLRP2; ScRNA-seq.

Fig. 1

Impacts of heat stress on cardiac performance, morphology and metabolism of scallop hearts. A Heart rates (left) and representative cardiograms (right) of bay scallops under heat stress. B Scanning and Transmission electron microscopy (SEM and TEM) analysis. The characters F, H, S in SEM (I-III) stand for fissure, hole, and shrinking, respectively. The characters Z, MyF, and SpR in TEM (I-III) represent the cardiac structure Z-lines, Myofiber, and Sarcoplasmic reticulum, respectively. C In vivo contents of Norepinephrine (NE), Gamma-aminobutyric acid (GABA), acetylcholine (ACh), and activities of Acetylcholinesterase (AChE) in scallop hearts after exposure to heat stress (n = 4, mean ± SD). (D) The enzyme activities of PHD, LDH and PK, and ROS content of scallop hearts (n = 4, mean ± SD). The error bars represent SDs. The statistical significance was determined by a two-sided t-test and the P-values were shown above the plot

Fig. 2

scRNA-seq analysis of heat untreated/treated scallop hearts. A Scallop heart collection from control and heat exposure groups, and sequenced with the microfluidics via 10 × Genomic platform. B UMAP plot analysis of 16 cell clusters in control (NG) and heat exposure (MG and DG) groups. C The heatmap (left) representing the top10 enriched genes among the 16 clusters. The cluster numbers correspond to the cluster shown in UMAP visualization. The umap (right) shows the expression patterns of the selected marker genes for each cell type and each dot in the graph represents a cell. The red rectangle emphasizes the umap of cardiomyocytes. D Differentially expressed genes (DEGs) and (E) relative abundance in 16 cell cluster of heart cells after heat exposure

Fig. 3

Energy changes in scallop heart after heat exposure. A ATP content variation in nine tissues (including heart, hemolymph, mantle, kidney, gill, hepatopancreas, gonad, muscle, and foot) of scallop in control (NG) and heat exposed groups (MG and DG). The pink and purple bars represent the percentage of energy consumption from NG to MG, and MG to DG out of the total energy consumption from NG. The blue bars represent the residual energy of ATP content. B The relative percentage of Mito-Tracker Green fluorescence levels (left), and the ratio of the red fluorescence to the green fluorescence that marked by JC-1 (right). C Representative mitochondrial status revealed by TEM analysis. The white rectangle framed and zoomed the representative mitochondria in NG, MG and DG. D The bubble shape represents the relative expression of mitophagy marker gene in 16 cell clusters from NG to MG and MG to DG. The significance and regulatory trend are illustrated by the size and color legend, respectively

Fig. 4

Ventricular myocytes (VMs) and atrial myocytes (AMs) exhibited distinct regulatory patterns in energy metabolism pathways under heat stress. A KEGG enrichment analysis of DEGs in VMs and AMs. B Predicted diagram of the energy metabolism pathway in VMs and AMs, including glycolysis, TCA cycle, and (C) oxidative phosphorylation process. The black bolded fonts indicate the key enzyme in the process and the yellow bolded fonts represent the genes displayed significant expression changes under heat stress. The size and color of bubble illustrate the significance and regulatory trend of pathway genes, respectively

Fig. 5

Cell–cell communications between cardiomyocytes (CMs) and other cell clusters. A Heatmap represents the quantity of ligand − receptor pairs, depicted by varying colors, among the 16 cell clusters in NG, MG and DG. B, C Significant alterations in ligand-receptor pairs between cardiomyocytes (6 for AMs and 12 for VMs) and other cell clusters were identified. Upregulated genes are denoted by red triangles, while downregulated genes are indicated by blue triangles. D Average log₂FC of DEGs in (B, C) were shown. The significance and regulatory trend are illustrated by the size and color legend, respectively. Specifically, during the first stage, the heightened communication between AMs and ILCs primarily implicated signaling pathways related to retinoic acid (ALDH1A1-RORB), glutamate (GLS + SLC1A2-GRM1), and prostaglandins (PTGES3-PTGER4). During the second stage, the reduced intercellular communication between AMs and other cell populations predominantly involved BMP signaling (BMP5-BMPR1A + BMPR2/ACVR2B) with ILCs, neuroligin signaling (NLGN3-NRXN2) and calsyntenin signaling (CLSTN1-NRXN2) with FCs, and WNT signaling (WNT5B-FZD7 + LRP6) with CPCs, respectively. The intercellular communication between both AMs and VMs and FCs, mediated by melatonin signaling (ASMT-MTNR1A) exhibited a progressive increase during the two continuous stages

Fig. 6

Pseudotime differentiation trajectories of CMs and CPCs. A Pseudotime ordering of AMs (cluster 12, orange), VMs (cluster 6, blue) and CPCs (cluster 9, purple) in NG, MG and DG. B The density distribution of AMs, VMs and CPCs over pseudotime in three conditions. C Average log₂FC of NKX2-5, MLC and GATA4 were shown. The significance and regulatory trend are illustrated by the size and color legend, respectively

Fig. 7

Identification and characterization of AiPLRP2-like in bay scallop. A Visualization of differentially expressed genes in VMs (cluster 6) and AMs (cluster 12) between MG and DG. B. The phylogenetic tree was constructed based on PLRP2s (-likes) sequences from bay scallop and other representative species (invertebrates and vertebrates shown with light red, light yellow and blue backgrounds, respectively. C Predicted protein domain architecture of AiPLRP2-like and other representative species PLPR2s (-likes) determined by SMART analysis. D In situ hybridization analysis of AiPLRP2-like in NG (I), DG-without probe (II), and DG (III). E Alterations in HR, ABT and AiPLRP2-like relative expression at the resting and thermal stage at 24 h post injection of siRNA- AiPLRP2-like and PBS. F The changes of TG content in NIG and SIG during resting and thermal, respectively. The vertical bars mentioned-above represent the mean ± SD (n = 4) and the statistical significance was determined by a two-sided t-test and the P-values were shown above the plot