Comparative Analysis of Isoproterenol and Lipopolysaccharide Mediated Cytoprotective Responses in the Heart

Abstract

A panoply of different stimuli activate endogenous signaling pathways that confer cardioprotection in the heart; however, it is unclear whether these stimuli engage similar or distinct biological pathways. We showed that tissue injury induced by isoproterenol (ISO), or pretreatment with the Toll-like receptor 4 agonist lipopolysaccharide (LPS), protect the heart against the myopathic effects of a subsequent exposure to ISO. Here, we employed a multiomic approach to examine ISO- and LPS-treated hearts in order to identify shared and distinct biological themes. Bulk RNA sequencing showed that ISO and LPS elicited upregulation of distinct stress response signatures that featured interferon (IFN) signaling, while converging on the widespread downregulation of pathways related to metabolism or cell growth. Multiome (RNA + ATAC) sequencing revealed that ISO-treated hearts underwent distinct chromatin remodeling, with more regions showing decreased accessibility than increased accessibility. IFN-related pathways were consistently enriched across many cell types in the heart, including endocardial cells, TNK cells, myeloid cells, and endothelial cells. Analysis of cell-cell interactions using CellChat showed that ISO stimulation enhanced communication between myeloid cells with fibroblasts, TNK cells, and pericytes. Remarkably, cell-cell interactions for cardiac myocytes were restricted to myeloid cells. Bulk ATAC sequencing of bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) demonstrated that ISO and LPS stimulation predominantly resulted in decreased chromatin accessibility in HSPCs and bone marrow stromal cells. Viewed together these studies show that although ISO and LPS engage distinct upstream signaling pathways, both converge on IFN signaling and a shared suppression of energy-intensive pathways.

New and Noteworthy

This study shows that isoproterenol and lipopolysaccharide establish cardioprotection through the activation of distinct upstream signaling and cell-cell communication networks that converge on interferon signaling in the heart. Viewed together these studies suggest an important role for immune-mediated cardioprotection as a mechanism for enhancing cardiac resilience in response to stress.

Introduction

The term cardioprotection first appeared in the literature to describe the beneficial effects of verapamil in preserving myocardial structural integrity by preventing calcium overload induced by supraphysiologic doses of β-adrenergic catecholamines (1). Murry, Jennings, and Reimer later expanded the concept of cardioprotection by demonstrating that brief, non-lethal episodes of myocardial ischemia render the heart more resistant to a subsequent episode of prolonged ischemia (2). This phenomenon, which they termed ischemic preconditioning, provided compelling evidence that the myocardium activates intrinsic cytoprotective mechanisms in response to sublethal stress, thereby limiting tissue injury and enhancing cardiac resilience to subsequent stress.

Importantly, ensuing studies from multiple laboratories demonstrated that a wide range of pharmacologic and non-pharmacologic stimuli are sufficient to activate endogenous signaling pathways that confer cardioprotection, highlighting the plasticity and biological robustness of stress-induced adaptive stress responses in the heart (reviewed in (3–5)).

We have shown previously that tissue injury triggered by isoproterenol (ISO)-mediated adrenergic stress was sufficient to protect the heart from the myopathic effects of a second ISO challenge, consistent with ISO-induced cardioprotection (6). Remarkably, pharmacologic depletion of macrophages and dendritic cells blunted the ISO-mediated cytoprotective effect, suggesting that immune-driven tissue repair processes contributed to protecting the heart from subsequent stress. Recognizing that ISO-induced cardiomyocyte necrosis results in the release of cytosolic and nuclear damage-associated molecular patterns (DAMPs) that activate signaling via endosomal and plasma membrane toll-like receptors (TLRs) (7), we hypothesized that TLR-mediated signaling would be sufficient to confer cytoprotective responses against ISO-induced myocardial injury. To this end, we recently demonstrated that lipopolysaccharide (LPS), a canonical TLR4 agonist, protects the heart from the myopathic effects of isoproterenol (ISO) through mechanisms involving both type I and type II interferon (IFN) signaling (8). The aim of the present study was to extend these findings by investigating whether ISO- and LPS-induced cytoprotection involve distinct or shared signaling pathways, with the goal of identifying unique, common, or dissimilar biological themes. Here, we show that ISO and LPS initiate distinct cytoprotective responses in the heart that converge on downstream IFN type 1 and type 2 signaling pathways.

Materials and Methods

Animals

Female 9–10-week-old C57BL/6J mice (#000664) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and had ad libitum access to a standard diet. Mice were housed in the Division of Comparative Medicine, Washington University School of Medicine. Because stress-induced cardiomyopathy (Takotsubo cardiomyopathy) occurs predominately in women (~90% of cases), female mice were used for this study. We have previously reported on sexually dimorphic responses to ISO-induced myocardial in this model (9, 10). All experiments were performed following the NIH Guide for the Care and Use of Laboratory Animals, as approved by the Institutional Animal Care and Use Committee, Washington University School of Medicine (protocol #20-0392 and #22-0280).

Treatment Protocols

Mice received single intraperitoneal (i.p.) injections of 300 mg/kg isoproterenol (ISO; Sigma, St. Louis, MO), 2.5 mg/kg bacterial lipopolysaccharide (LPS; Sigma, St. Louis, MO), or diluent (endotoxin-free phosphate-buffered saline; PBS). ISO solutions were prepared fresh immediately before injection and kept on ice until use.

Bulk RNA Sequencing and Analysis

Hearts were harvested from mice treated at baseline and on days 1 and 7 following ISO or LPS administration. The inferior mid-apical region of the left ventricle was isolated from each heart and snap-frozen. This region was selected for RNA sequencing based on a prior study, which showed that these regions exhibited the most pronounced wall motion abnormalities one hour after ISO administration (11). Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Germantown, MD) according to the manufacturer’s instructions and submitted to the Genome Access Technology Center at the McDonnell Genome Institute, Washington University in St. Louis for library preparation and RNA sequencing.

Samples were prepared according to the library kit manufacturer’s protocol, indexed, pooled, and sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, CA). Basecalls and demultiplexing were performed with Illumina’s bcl2fastq2 software. RNA sequencing reads were aligned and quantitated to the Ensembl release 101 primary assembly with an Illumina DRAGEN Bio-IT on-premise server running version 3.9.3–8 software. All gene counts were then imported into the R/Bioconductor package EdgeR (12), and TMM normalization size factors were calculated to adjust for samples for differences in library size. Ribosomal genes and those not expressed at more than one count per million in at least n – 1 samples (where n is the smallest group size) were excluded from further analysis. The TMM size factors and matrix of counts were then imported into the R/Bioconductor package Limma (13). Weighted likelihoods based on the observed mean-variance relationship of every gene and sample were then calculated for all samples, and the count matrix was transformed to moderated log2 counts-per-million with Limma’s voomWithQualityWeights (14). The performance of all genes was assessed with plots of the residual standard deviation of every gene to their average log count with a robustly fitted trend line of the residuals. For principal component analysis (PCA), R prcomp() eigenvectors for the first three prinicipal components based on genes with expression having a standard deviation greater than or equal to one log2 count per million across all samples were plotted using the R plotly library. Differential expression analysis was then performed to analyze for differences between conditions. Pathway enrichment analysis was done on lists of up- and down-regulated genes with a less stringent unadjusted p-value cut-off of less than 0.05 using Enrichr, focusing on MSigDB Hallmark 2020 gene sets (15).

Single-nuclei multiome RNA and ATAC sequencing

Mice received either 300 mg/kg ISO or saline (diluent) and were euthanized 7 days later. Hearts were collected at the time of euthanasia and snap-frozen for further analysis. Nuclei were obtained from whole hearts using the Chromium Nuclei Isolation kit (10x Genomics, Pleasanton, CA). Nuclei from all three hearts in each group were pooled together before submission for multiomic, single-nuclei RNA and ATAC sequencing at the Genome Access Technology Center at the McDonnell Genome Institute, Washington University in St. Louis. The sequencing data were analyzed using the computational pipeline we previously described (8). Briefly, the pipeline was built on the Seurat and Signac frameworks. Nuclei clusters were annotated based on RNA marker genes. The analysis included differential gene expression (adjusted p-value < 0.05, log₂ fold change > 0.25), differential chromatin accessibility (p-value < 0.005, average log₂ fold change > 0.25, as defined by Signac), pathway enrichment using Enrichr, and cell-cell interaction network analysis via CellChat. To reduce the sparsity associated with single-nuclei ATAC sequencing data, we performed differential accessibility testing on aggregated pseudo-bulk profiles. For this study, we used data from a previously reported diluent-treated group (8).

Bulk ATAC sequencing

Mice received either 300 mg/kg ISO, 2.5 mg/kg LPS, or saline (diluent) and were euthanized 7 days later. The marrow from both femurs were flushed to obtain bone marrow-derived hematopoietic stem and progenitor cells (HSPCs), sent for sequencing to the Genome Engineering & Stem Cell Center and Genome Technology Access Center at the McDonnell Genome Institute, Washington University in St. Louis, and analyzed as previously described (8). Briefly, after peak identification, read count quantification and normalization, differential accessibility analysis was done by DESeq2 (v1.46.0), with differentially accessible peaks (adjusted p-value < 0.05, log₂ fold change >2/<−2) annotated using HOMER by assigning each peak to its nearest transcription start site regardless of genomic distance using the mm10 genome reference and pathway enrichment analysis done by EnrichR with the Gene Ontology Biological Process 2025 database. To determine whether genes recurrently mutated in clonal hematopoiesis (CH) exhibited features of active transcriptional regulation, we also analyzed chromatin accessibility at loci corresponding to a curated list of murine orthologs of CH-associated genes (Tet2, Dnmt3a, Asxl1, Jak2, Trp53, Ppm1d, Sf3b1, Srsf2, U2af1, Idh1, Idh2, Cbl, Runx1, Gnb1, Zrsr2, Bcor, Setbp, Foxo3). Peaks were associated with these genes based on proximity to their annotated transcription start sites, as described above. Increased accessibility at these regulatory regions was interpreted as evidence of a transcriptionally permissive chromatin state, potentially supporting gene activation.

Statistical analysis

All statistical analyses were performed as part of differential expression and differential accessibility analysis pipelines, as detailed in the previous sections. The p-values considered statistically significant have been indicated in these sections as well.

Results

Bulk RNA sequencing of hearts from LPS and ISO-treated mice

Mice were treated with LPS or ISO on day 0 (baseline) and then followed for 7 days (Figure 1A). Bulk RNA sequencing was performed on LV myocardial samples obtained at baseline (prior to injection) and on days 1 and 7 after treatment. Principal component analysis of the gene transcripts (PCA) at baseline, day 1, and day 7 revealed different time-dependent transcriptional profiles for the ISO- and LPS-treated hearts. On day 1, gene expression clusters for both ISO- and LPS-treated hearts occupied distinct regions along the PC3 axis relative to baseline. However, by day 7, the PC3 coordinates of ISO-treated hearts shifted back toward baseline values. In contrast, the PC3 coordinates for LPS-treated hearts remained distinct from baseline and also diverged from their day 1 profile along the PC1 axis (Figure 1B). Together, these findings demonstrate that ISO and LPS induce distinct, time-dependent transcriptional responses to tissue injury, with ISO triggering a transient gene expression program that resolves toward baseline, whereas LPS elicits a transcriptional program that persists and evolves over time.

Figure 1. Bulk RNA sequencing analysis of ISO- and LPS-treated hearts.

(A) Mice were injected i.p. with 300 mg/kg isoproterenol (ISO), 2.5 mg/kg lipopolysaccharide (LPS), or diluent control (PBS). Hearts were collected at baseline (day 0) and on days 1 and 7, with the inferior mid-apical region of the left ventricle used for bulk RNA sequencing. (n=3–4/group) (B) Principal component analysis plots of the ISO- and LPS-treated samples across time points. (C) Number of differentially expressed genes (DEGs) across different groups, relative to control (P<0.05, log2 fold change >/<0). (D) Venn diagrams showing the number of common and unique upregulated DEGs between the ISO and LPS groups on days 1 and 7. (E) Pathway enrichment analysis of upregulated gene lists in the ISO and LPS groups on day 1 and (F) on day 7.

Relative to baseline, the total number of differentially expressed genes (DEGs) in the ISO-treated hearts on day 1 was numerically greater (n=10,193) than for LPS-treated hearts (n=9,434) (Figure 1C; Supplemental Tables S1-S2). In agreement with the PCA findings, ISO-treated hearts exhibited substantially fewer DEGs on day 7 (n=1,240) when compared to the LPS-treated hearts (n=4,490). Analysis of the top 10 enriched pathways for the ISO- and LPS-stimulated hearts on day 1 revealed 30% overlap of shared biological themes (TNF signaling via NF-κB, IL-6/JAK/STA3 signaling, cMyc target genes) (Figure 1E) and 20% overlap of the shared biological themes on day 7 (IFN alpha response, IFN gamma) (Figure 1F). Pathway enrichment analysis of downregulated DEGs (log₂ fold change < 0, P < 0.05) showed a proportionally greater overlap between ISO and LPS treatments at both timepoints (Figure 2A). Remarkably, there was an 80% overlap of the enriched pathways for downregulated DEGs in the ISO- and LPS-stimulated hearts on day 1 (Figure 2B) and a 50% overlap of the enriched pathways for downregulated DEGs in the ISO and LPS-stimulated hearts on day 7 (Figure 2C). Notably, many of the pathways that were downregulated on day 1 and day 7 in ISO- and LPS-stimulated hearts were related to metabolism (glycolytic, fatty acid, oxidative phosphorylation) or cell growth (hedgehog signaling, mTORC1). Viewed together, the diversity of the upregulated transcriptional pathways suggests that ISO and LPS stimulate distinct stress response programs, whereas the overlap of downregulated transcriptional pathways implies that both forms of tissue injury converge on a common set of biological processes that are broadly suppressed to facilitate adaptation, such as reducing energy-demanding functions, limiting proliferation.

Figure 2. Bulk RNA sequencing analysis of downregulated genes in ISO- and LPS-treated hearts.

Analysis continued from Figure 1. (A) Venn diagrams showing the number of common and unique downregulated DEGs between the ISO and LPS groups on days 1 and 7. (B) Pathway enrichment analysis of upregulated genes in the ISO and LPS groups on day 1 and (C) on day 7.

Multiomic analysis of ISO-stimulation

We recently demonstrated that pretreatment with ISO and LPS was sufficient to confer protection against the myopathic effects of a subsequent ISO challenge, consistent with a pre-conditioning effect (8, 16). Notably, before administering the ISO challenge, ISO and LPS-treated mice did not show signs of cardiac dysfunction by echocardiography. Single-nucleus ATAC-seq (snATAC-seq) and snRNA-seq revealed that LPS pretreatment protects against ISO-induced myocardial injury, at least in part, through upregulation of IFN response pathways across multiple cell types in the heart, most notably cardiac myocytes and stromal cells (8). To investigate the putative cell types involved in ISO-induced cardiac cytoprotection, we performed snATAC-seq and snRNA-seq on mouse hearts harvested 7 days after ISO stimulation (Figure 3A).

Figure 3. Multiome analysis of ISO-treated hearts.

(A) Mice were injected i.p. with 300 mg/kg isoproterenol (ISO) or diluent (PBS/CTRL). Whole hearts were collected at baseline (day 0) and on day 7 for multiome single-nuclei RNA+ATAC sequencing. (pooled n=3/group) (B) UMAP showing the identified cell clusters from sequencing. (C) Frequencies of the identified cell types in ISO and CTRL hearts. (D) Number of differentially expressed genes (adjusted P<0.05, log2 fold change >0.25/<−0.25) and (E) differentially accessible peaks across different cell types in the ISO group, relative to control (P<0.005, log2 fold change >0.25/<−0.25). Pathway enrichment analysis on lists of genes linked to accessible chromatin regions in (F) fibroblast, (G) endocardium, (H) TNK cell, (I) cardiac myocyte, (J) myeloid, (K) pericyte, and (L) endothelium cell types; the top 5 pathways are shown.

Figure 3B shows a UMAP plot of the snRNA-seq data, and Figure 3C summarizes the frequencies of the various cell types in the diluent- and ISO-treated mouse hearts. We observed the expected cell clusters in the ISO-stimulated hearts with an expansion of the myeloid cell compartment in the ISO-treated group (Figures 3B, 3C), consistent with our prior observations in ISO-stimulated hearts (11). ISO stimulation resulted in an increased number of DEGs in all cell groups in the heart, with the greatest number for cardiac myocytes > fibroblasts > endocardial cells > myeloid cells > endothelial cells > pericytes > T and NK cells (Figure 3D). Although cardiac myocytes exhibited the greatest number of DEGs, the majority were downregulated, in contrast to other cell types, which demonstrated a more heterogeneous distribution of upregulated and downregulated DEGs. Next, we interrogated the snATAC-seq data to determine whether there were differences in the number of regions with increased chromatin accessibility across the different cell types. As shown in Figure 3E, ISO stimulation led to an increase in the number of differentially accessible chromatin peaks across all cell types. Fibroblasts exhibited the greatest number of changes in chromatin accessibility, followed by endocardial cells, TNK cells, cardiac myocytes, myeloid cells, pericytes, and endothelial cells. The majority of the overall changes in ATAC-seq peaks corresponded to decreased chromatin accessibility in ISO-treated hearts relative to diluent-treated controls. We then integrated the snRNA-seq and snATAC-seq data sets to explore the genes that were linked to regulatory regions with increased accessibility, followed by pathway analysis. As illustrated in Figures 3F–3L, each cell type in the heart displayed a distinct set of upregulated pathways. Notably, IFN-related pathways were consistently enriched across many of the cell groups, including endocardial cells, TNK cells, myeloid cells, and endothelial cells, consistent with our bulk RNA-sequencing data on day 7 (Figure 1F).

ISO stimulation is associated with changes in myocardial intercellular communication networks

The multiomic snRNA-seq and snATAC-seq data in ISO-simulated hearts (Figure 3) revealed differences in gene expression and chromatin peak accessibility across all cell types within the heart, with the most significant changes observed in cardiac myocytes and fibroblasts. These observations suggested a potential model wherein ISO simulation provokes coordinated autocrine and paracrine responses by myocytes and non-myocytes in the heart. To begin to explore this possibility, we used unsupervised CellChat (17) to analyze intercellular communication networks in the ISO-treated hearts. Analysis of the cell-cell communication networks in the ISO-stimulated hearts revealed an increase in the number and strength of interactions between myeloid cells and fibroblasts and, to a lesser extent, TNK cells and pericytes. There was also an increase in autocrine signaling in the myeloid cell population (Figures 4A–4C). Intriguingly, the only cellular interaction for cardiac myocytes was with myeloid cells, which is consistent with our prior observation that ISO-induced expansion of immune cell clusters was necessary, at least in part, for ISO-induced preconditioning (6).

Figure 4. Analysis of cell-cell communication networks in ISO-treated hearts.

CellChat was used to analyze interactions across cell types from the multiome sequencing dataset. (A) Network maps showing the degree of communication between cell types in the ISO-treated heart relative to the diluent control, lines representing the number of interactions, or (B) interaction strength. (C) Heat maps of the network maps are in (A) and (B), with sender cells on the y-axis and receiver cells on the x-axis.

ISO and LPS stimulation provokes changes in chromatin accessibility in bone marrow HSPCs

Cardiac injury leads to epigenetic changes in hematopoietic and non-hematopoietic cell types in the bone marrow through the release of DAMPs, cytokines and chemokines that activate type I IFN responses in bone marrow hematopoietic stem and progenitor cells (HSPCs) (18). Further, ISO affects the bone marrow niche by modulating the expression of cytokines that modulate HSPC mobilization (19). Given that type I and type II IFN response genes were upregulated in the heart following ISO and LPS stimulation (Figure 1), we asked whether ISO and LPS stimulation resulted in changes in chromatin accessibility in HSPCs and bone marrow stromal cells by performing bulk ATAC-seq on HSPCs harvested from the bone marrow of mice 7 days after stimulation with ISO or LPS. Mice treated with diluent served as the appropriate control group (Figure 5A).

Figure 5. Bulk ATAC sequencing analysis of ISO- and LPS-treated bone marrow-derived hematopoietic stem and progenitor cells (HSPCs).

(A) Mice were injected i.p. with 300 mg/kg isoproterenol (ISO), 2.5 mg/kg lipopolysaccharide (LPS), or diluent control (PBS). Bone marrow-derived HSPCs were collected at day 7 for bulk ATAC sequencing. (n=3/group) (B) Volcano plots showing peaks with significantly increased and decreased accessibility in the ISO and LPS groups relative to control. (C) Venn diagrams showing the number of common and unique peaks with increased or decreased accessibility between the ISO and LPS groups on day 7 (adjusted P<0.05, log2 fold change >2/<−2). (D) Pathway enrichment analysis of genes associated with peaks of increased or decreased accessibility in the ISO and LPS groups; the top 5 pathways are shown.

The volcano plots in Figure 5B show that there was an asymmetric distribution of differentially accessible peaks, with a greater number of downregulated versus upregulated peaks. Figure 5C illustrates a Venn diagram of the differentially accessible peaks (DAPs) in HSPCs and stromal cells harvested from the ISO- and LPS-stimulated mice. As shown, there was a ~4.1-fold greater number of DAPs (adj. P < 0.05, log2FC >2/<−2) in the HSPCs harvested from LPS-stimulated mice when compared with ISO-stimulated mice. The number of DAPs with decreased accessibility exceeded those with increased accessibility by ~3.9-fold following LPS stimulation and ~18.1-fold following ISO stimulation.

Pathway enrichment analysis of genes exhibiting increased chromatin accessibility in the hematopoietic and stromal compartments of the bone marrow from ISO- and LPS-stimulated mice revealed no overlap among the top five GO biological process pathways (Figure 5D). On the other hand, genes with decreased chromatin accessibility showed minimal overlap, including pathways such as B cell receptor signaling. These findings suggest that innate immune training of HSPCs by ISO and LPS engages distinct activation programs but may converge on transcriptional repression mechanisms, which is consistent with the pattern of DAPs with decreased accessibility for the majority of cell types in the ISO-stimulated hearts (Figure 3E).

To assess whether genes recurrently mutated in clonal hematopoiesis (CH) exhibited evidence of transcriptional activation in response to LPS or ISO stimulation, we analyzed chromatin accessibility at loci corresponding to a curated list of murine orthologs of CH-associated genes (Tet2, Dnmt3a, Asxl1, Jak2, Trp53, Ppm1d, Sf3b1, Srsf2, U2af1, Idh1, Idh2, Cbl, Runx1, Gnb1, Zrsr2, Bcor, Setbp1) in the bulk ATAC-seq dataset. Whereas there was no increased chromatin accessibility for CH-related genes in the ISO-stimulated mice, we notice increased chromatin accessibility at the U2af1, Jak2, Tet2, and Cbl loci in bone marrow aspirates from LPS-stimulated mice (Supplemental Table S3). These genes are canonical drivers of clonal hematopoiesis and encode factors involved in RNA splicing (U2af1), epigenetic remodeling (Tet2), cytokine signal transduction (Jak2), and negative regulation of tyrosine kinase signaling (Cbl). We also noticed increased chromatin accessibility in regions linked with Foxo3, a gene known to regulate HSPC quiescence. The observed increase in chromatin accessibility at these loci suggests a transcriptionally permissive chromatin state. While these genes are not canonical regulators of cytoprotection, their dysregulation may perturb signaling and transcriptional networks in ways that may necessitate activation of compensatory stress responses that help preserve HSPC function under inflammatory and oxidative stress, albeit at the expense of creating a milieu that supports clonal expansion.

Discussion

The nervous and immune systems, which coevolved under selective pressures imposed by tissue injury and infection, play a critical role in coordinating host responses to environmental threats. Although traditionally viewed as distinct biological systems, accumulating evidence suggests that both systems act in a coordinated manner, especially in the context of cardiac injury and tissue repair. Relevant to this discussion, we have shown that ISO-mediated sterile tissue injury protected the heart from the myopathic effects of a second ISO challenge (6), whereas pretreatment with LPS, a pathogen-associated stress response, also protected the heart from the myopathic effects of ISO (8). Here, we sought to determine whether ISO and LPS-induced cytoprotection is conveyed through distinct or overlapping transcriptional programs.

The results of this study show that ISO and LPS induce distinct, time-dependent transcriptional responses that ultimately converge on the activation of type I and II IFN-related transcriptional programs (Figures 1B, 1E), which we have shown are sufficient to prevent ISO-mediated myopathic effects (8). Remarkably, there was a substantial (50–80%) overlap of ISO and LPS-induced pathways enriched for downregulated DEGs related to metabolism (glycolysis, fatty acid oxidation, oxidative phosphorylation) and cell growth (hedgehog signaling, mTORC1), consistent with adaptive, energy-sparing responses (Figures 2B, 2C). Analysis of the mutliomic snRNAseq and snATAC-seq data sets in ISO-stimulated hearts revealed two important findings with respect to chromatin accessibility and gene regulation for different cell types in the heart. First, ISO stimulation resulted in decreased chromatin accessibility for most cell types in the heart, including fibroblasts, endocardial cells, and cardiac myocytes (Figure 3E). Second, pathway analysis for the cell types with regions of increased chromatin accessibility revealed enrichment of genes related to IFN-related pathways (Figures 3F–3L), consistent with our bulk RNA-seq data. Multiomic analysis of cell-cell communication networks in ISO-stimulated hearts suggested increased interactions between myeloid cells and fibroblasts and, to a lesser extent, with TNK cells and pericytes (Figure 4). Intriguingly, the predominant cell-cell interaction for cardiac myocytes was with myeloid cells, which is consistent with our prior observation that the depletion of macrophages and dendritic cells blunted ISO-induced preconditioning in the heart (6). The findings with respect to cell-cell communication networks in ISO-treated hearts differ from our prior report of cell-cell interactions in LPS-stimulated hearts (8), which demonstrated that LPS provoked an increase in the number and strength of interactions between cardiac myocytes and fibroblasts, TNK cells, myeloid cells, pericytes, and fibroblasts, as well as an increase in autocrine signaling in cardiac myocytes. Viewed together, these observations suggest that ISO and LPS confer cytoprotection in the heart through shared mechanisms that involve the coordinated upregulation of IFN-related signaling pathways and the downregulation of ATP-dependent cell proliferative and metabolic pathways. However, the manner in which cell-cell communication networks mediate cytoprotective responses in ISO- and LPS-stimulated hearts reveals distinct biological mechanisms. That is, whereas ISO induces cytoprotection primarily by decreasing cardiac myocyte chromatin accessibility and by limiting cell-cell interactions of cardiac myocytes to myeloid cells, LPS confers cytoprotection by increasing chromatin accessibility in cardiac myocytes, as well as by increasing the number and strength of cell-cell interactions between cardiac myocytes and multiple different cell types in the heart. Intriguingly, our results also suggest that ISO and LPS stimulation modulate chromatin structure in hematopoietic and stromal compartment bone marrow cells in a manner that reduces chromatin accessibility, which would promote quiescence of HSPCs (Figure 5). In this regard, LPS stimulation leads to a ~ 3.9-fold greater decrease in the number of accessible chromatin peaks than with ISO, possibly reflecting a more persistent functional reprogramming of HSPCs (Figure 5C).

Cytoprotection and Biological Robustness

Although ISO and LPS-induced cytoprotection are mediated, respectively, by β-adrenergic and TLR4-mediated signaling networks, both signaling pathways ultimately converge on common downstream effector mechanisms that include the upregulation of IFN-mediated transcriptional programs and downregulation of transcriptional pathways related to energy-intensive processes, such as cell proliferation and metabolism. The convergence of distinct signaling cascades on essential cytoprotective and reparative tissue responses is a cardinal feature of biological robustness (20, 21). As proposed by Kitano, robustness is a fundamental property of complex biological systems that are required to maintain functional stability in the face of different environmental and internal perturbations (20, 22). Kitano proposes that robustness is shaped by natural selection that favors biological traits that enhance system stability and adaptability (20, 22). Robustness in complex biological systems often arises from redundancy, feedback control, modularity, and pathway convergence (20). In the context of tissue injury, robustness ensures that critical homeostatic functions, such as tissue repair and resolution of inflammation are preserved even when the initiating stimuli are different in nature, intensity, or duration (20, 22). As shown in the present study both bacterial endotoxin and β-adrenergic stimulation engage distinct upstream signaling cascades, yet both confer tissue protection through overlapping downstream mechanisms (22). Such convergence not only buffers the system against variability in the external environment but also minimizes the risk of excessive inflammation and collateral tissue damage, thereby preserving tissue integrity and function (21). Moreover, ISO and LPS had effects on the bone marrow in addition to the heart. This indicates the potential for a modular response towards cytoprotection, wherein support across tissues strengthens the system against further destabilization due to injury. Nonetheless, it should be recognized that biological robustness does not necessarily imply that perturbations are resolved in a manner that restores the system to its original state. As we have shown in our work, both ISO and LPS-induced cytoprotection were marked by distinct changes in gene expression, chromatin accessibility, and cell-cell communication, which aligns with the idea that robustness can be achieved by bringing the system to a new state of functional stability.

Limitations of the study

The present work should be considered in the context of several limitations. First, because stress-induced (Takotsubo) cardiomyopathy is predominately observed in women (~90% of cases), we focused our studies on female mice. Because we have shown that male mice display a similar, albeit slightly exaggerated cardiac response to ISO-mediated injury (11), we cannot exclude the formal possibility that the ISO-induced transcriptional and epigenetic changes observed might have been even more pronounced in males. Second, we performed our multiome and bulk ATAC sequencing experiments on a single timepoint at day 7 following ISO or LPS treatment, which prevents comparisons on the temporal dynamics of the observed responses or their long-term durability. Third, we did not perform functional validation studies of our sequencing studies especially regarding the observed differences in cell-cell communication induced by ISO or LPS. Although CellChat provides reliable insights into overall patterns of cell–cell communication within a dataset, it relies solely on transcriptomic information and not protein-protein interactions. Therefore, these findings should be regarded as provisional and will require further validation. Finally, although we discovered an effect on bone marrow HSPCs as a result of ISO and LPS treatment, we did not perform experiments to see if these had corresponding effects on the transcriptomic level, or on HSPC phenotypes such as proliferation and differentiation.

Conclusion

This study provides a comparative analysis of ISO and LPS-induced cytoprotective responses in the heart. Our results demonstrate that disparate upstream stimuli converge on common IFN-stimulated genes, suggesting that the IFN signaling network may serve as a shared regulatory pathway for adaptive stress responses in the heart. These findings are consistent with prior studies, which have demonstrated that IFNs activate a broad array of signaling cascades, including the canonical JAK-STAT pathway and several noncanonical pathways such as PI3K/Akt and NF-κB, all of which can promote cell survival under certain conditions (23–25). Given that IFN signaling can also induce pro-apoptotic responses, depending on cell type, stimulus, and microenvironment, it is unlikely that IFN signaling represents a final common cytoprotective pathway in the heart but rather represents one of multiple signaling cascades that can modulate cell survival or death depending on the biological context. This statement notwithstanding, these results underscore the possibility that IFN-driven epigenetic changes function as a priming signal, conditioning the heart to be more resilient to subsequent stress by maintaining chromatin in a poised configuration that facilitates rapid transcriptional activation of cytoprotective genes upon re-exposure to stress. Viewed together, the observations in the present study also highlight the importance of immune preconditioning in the heart, consistent with our prior observation that pharmacologic depletion of monocyte/macrophages and dendritic cells abrogates ISO-induced preconditioning (6). Further work will be required to delineate whether immune-mediated epigenetic and transcriptional adaptations can be harnessed therapeutically to enhance cardiac resilience in different disease settings.

Supplementary Material

Supplemental Tables S1–S3: DOI: 10.17632/ph7f7swnhr.1

Data Availability

All data used for this article are provided in the article and the online supplementary material. Bulk RNA, multiome, and bulk ATAC sequencing data can be accessed at GSE307900, GSE305273, and GSE305274, respectively.