Biology subjects

Ward, M. C.

Publications and source records attributed to Ward, M. C..

13 recordsLinked to original sources

bioRxiv2026

Conserved RNA-protein modules link early anthracycline responses to atrial fibrillation risk

Anthracycline chemotherapeutics increase risk for cardiovascular disease. The early molecular events that link inherited risk to cardiac dysfunction remain poorly defined. We therefore exposed iPSC-derived cardiomyocytes from six individuals to three anthracyclines and an anthracenedione, which act as topoisomerase inhibitors (TOP2i) to induce DNA damage, and generated proteomics data following three and 24 hours of TOP2i treatment. We constructed a 19 module co-expression network and identified four protein modules that associate with response to all TOP2i. Integration with a co-expression network generated from paired transcriptomic data revealed that the two most drug-responsive protein modules are preserved in the RNA network. The most preserved RNA module is enriched for p53 motifs in associated active regulatory regions and p53 target genes, which propagates to enrichment of p53 targets in the most drug-responsive preserved protein module. Integration of the protein network with genome-wide association studies across hundreds of cardiovascular traits identified a preserved protein module enriched for genetic risk of atrial fibrillation, PR interval, and longitudinal strain, suggesting that the molecular response to TOP2i is linked to genetic variation influencing cardiac electrophysiology and contractile performance. Differential protein abundance in this module associates with impaired calcium handling in cardiomyocytes thereby linking molecular effects to cellular decline. Together, these results define a TOP2i-induced gene regulatory response propagated to the proteome that underlies early cardiotoxicity, and demonstrate how a multi-level network architecture can provide insight into genetically mediated susceptibility to drug-induced stress.

genomics↗

bioRxiv2026

Progressive suppression of DNA repair genes with persistent p53 activation in Doxorubicin-treated cardiomyocytes

Doxorubicin (DOX) is an effective anti-cancer drug; however it can cause cardiotoxicity by inducing DNA double-strand breaks in cardiomyocytes. Cardiotoxicity can manifest immediately or years following treatment. Most human in vitro models of DOX-induced cardiotoxicity (DIC) focus on the acute effects of DOX treatment. To understand the long-term effects, we profiled the global gene expression response to DOX exposure over time. We treated iPSC-derived cardiomyocytes from six individuals with DOX for 24 hours and assayed responses after 0, 24 and 144 hours of recovery. DNA damage, determined by {gamma}H2AX expression, is induced following DOX treatment and is resolved by the final recovery timepoint. We identified both acute and chronic gene expression response signatures. The chronic signature, representing 501 genes, is enriched for p53 target genes and DNA damage response genes compared to acute response genes. P53 target genes are persistently activated, and DNA damage response genes are progressively downregulated over time. Our results suggest an altered cell state following repair of double-strand breaks that is distinct from pre-exposed cells. DOX response genes with persistent changes in expression can be applied to the design of toxicity biomarkers or therapeutic targets.

genomics↗

bioRxiv2025

Topoisomerase II inhibitors CX-5461 and Doxorubicin differ in their cardiotoxicity profiles

CX-5461 (CX) is a chemotherapeutic drug currently under investigation for the treatment of late-stage cancers. While CX was first described as an RNA polymerase I inhibitor, it has recently been shown to primarily inhibit the beta isoform of topoisomerase II. This isoform is also inhibited by anthracycline drugs including Doxorubicin (DOX) and mediates the toxic effects of these drugs on the heart. It is unclear whether CX will similarly cause cardiotoxicity. We therefore designed a study to test the effects of CX compared to DOX on iPSC-derived cardiomyocytes from six individuals. While both CX and DOX induce cell death in cardiomyocytes, CX is 20-fold less cytotoxic than DOX. At sub-lethal doses, DOX induces DNA damage, while CX does not. Transcriptome profiling following treatment with two sub-micromolar concentrations of both drugs over time reveals that DOX induces thousands of gene expression changes compared to hundreds induced by CX. Comparison of gene expression trajectories across drugs reveals that genes that respond to CX also respond to DOX, while most DOX response genes are drug specific. Shared response genes correspond to pathways related to chromosome segregation and DNA replication. CX does not affect the expression of any of the genes in functionally-validated loci associated with DOX-induced cardiotoxicity. Our data demonstrate that CX treatment of cardiomyocytes induces gene expression changes that mirror a subset of those induced by DOX; however these changes do not coincide with the cardiotoxicity observed with DOX treatment.

genomics↗

bioRxiv2025

Sodium Butyrate Rescues Neurodevelopmental Deficits Following Perinatal Methadone Exposure

Prenatal opioid exposure (POE) induces long-term neurodevelopmental, behavioral and cognitive deficits for which no targeted treatments are available. The mechanisms underlying POE deficits are poorly understood, but have been linked to a range of central, peripheral, and enteric nervous system changes. Emerging evidence indicates that maternal microbiota changes may also contribute to these long-term deficits in offspring. Here we test the efficacy of the short-chain fatty acid sodium butyrate (NaB) to mitigate POE-induced deficits in a rat model. Both methadone and sodium butyrate treatments altered dam microbiota composition and function: notably methadone disrupted dam gene expression of microbial enzymes critical for butyric acid production and reduced faecal butyric acid levels. In postnatal day 9 pups, methadone increased gut barrier permeability that was reversed with NaB, and enzymatic disruptions were observed in pups at postnatal day 21 that resolved in adulthood. POE induced anxiety-like behavior in adolescence, and adult deficits in working spatial memory and attentional processing that were partially rescued in rats that had received prenatal NaB. POE was associated with decreased myelination in the hippocampus, and this was partially reversed by NaB. Together these results highlight for the first time the link between the gut-brain axis in animal models of POE. Furthermore, they provide the first indication in a rat model of NaB as a simple yet effective treatment to significantly improve the outcomes of children born with POE.

animal behavior and cognition↗

bioRxiv2025

Anthracyclines induce global changes in cardiomyocyte chromatin accessibility that overlap with cardiovascular disease loci

Breast cancer drugs including anthracyclines (ACs) and Trastuzumab increase the risk for cardiovascular diseases (CVDs) such as atrial fibrillation (AF) and heart failure (HF) that ultimately affect the heart muscle. These CVDs are associated with hundreds of genetic variants in non- coding regions of the genome. However, how these drugs affect the regulatory potential of the non-coding genome of the heart and CVD risk loci is unknown. We therefore measured global chromatin accessibility across iPSC-derived cardiomyocytes from four individuals treated with the ACs, Doxorubicin, Epirubicin, and Daunorubicin, a related non-AC, Mitoxantrone, and the monoclonal antibody Trastuzumab, or a vehicle control for three and 24 hours. We identified 155,557 high-confidence regions of open chromatin across 48 samples where the major sources of variation are associated with drug type and time. Jointly modeling the data revealed three accessibility response signatures denoted as early-acute, early-sustained, and late that correspond to 67,329 regions that open or close in response to drug treatment. Sequences associated with drug-induced chromatin opening contain motifs for DNA damage-associated transcription factors including p53 and ZBTB14, and associate with increases in active histone acetylation and gene expression. 21 AF- and HF-associated SNPs directly overlap with regions associated with drug-induced opening. A shared intronic HF and AF SNP, rs3176326, that is also an eQTL for CDKN1A in heart tissue, associates with increased chromatin accessibility, histone acetylation, and CDKN1A expression in response to all ACs. Our results demonstrate large-scale changes in chromatin accessibility in cardiomyocytes treated with ACs, which correspond to several regions harboring CVD risk loci. Author summaryAnthracyclines are a widely used class of breast cancer drugs that are linked to cardiac toxicity and the development of heart disease in some women. There are hundreds of genetic variants that associate with risk for heart disease; however their role and mechanism of action in drug- induced toxicity is unclear given that most reside in the non-coding genome. We therefore tested the effects of five breast cancer drugs on genome-wide chromatin accessibility using induced pluripotent stem cell-derived cardiomyocytes. We found tens of thousands of chromatin regions that change in accessibility after drug treatment. Regions with increased accessibility contain sequences that have been shown to bind DNA damage-associated transcription factors, and associate with increases in nearby gene expression. We find 21 heart disease-associated genetic variants in regions that increase in chromatin accessibility following treatment. This suggests that cancer drugs have large effects on the non-coding genome of heart cells including at regions associated with heart disease. This research contributes to our understanding of how genetic variants associated with disease exert their effects.

genomics↗

bioRxiv2024

DNA damage-associated protein co-expression network in cardiomyocytes informs on tolerance to genetic variation and disease

Cardiovascular disease (CVD) is associated with both genetic variants and environmental factors. One unifying consequence of the molecular risk factors in CVD is DNA damage, which must be repaired by DNA damage response proteins. However, the impact of DNA damage on global cardiomyocyte protein abundance, and its relationship to CVD risk remains unclear. We therefore treated induced pluripotent stem cell-derived cardiomyocytes with the DNA-damaging agent Doxorubicin (DOX) and a vehicle control, and identified 4,178 proteins that contribute to a network comprising 12 co-expressed modules and 403 hub proteins with high intramodular connectivity. Five modules correlate with DOX and represent distinct biological processes including RNA processing, chromatin regulation and metabolism. DOX-correlated hub proteins are depleted for proteins that vary in expression across individuals due to genetic variation but are enriched for proteins encoded by loss-of-function intolerant genes. While proteins associated with genetic risk for CVD, such as arrhythmia are enriched in specific DOX-correlated modules, DOX-correlated hub proteins are not enriched for known CVD risk proteins. Instead, they are enriched among proteins that physically interact with CVD risk proteins. Our data demonstrate that DNA damage in cardiomyocytes induces diverse effects on biological processes through protein co-expression modules that are relevant for CVD, and that the level of protein connectivity in DNA damage-associated modules influences the tolerance to genetic variation.

genetics↗

bioRxiv2023

Anthracycline-induced cardiotoxicity associates with a shared gene expression response signature to TOP2-inhibiting breast cancer drugs in cardiomyocytes

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However, they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX), Daunorubicin (DNR), Epirubicin (EPI), and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class, or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this, we studied the effects of treatment of DOX, DNR, EPI, MTX, and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum, while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling, a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time, giving rise to four distinct gene expression response signatures, denoted as TOP2i early-acute, early-sustained, and late response genes, and non-response genes. TOP2i early response genes are enriched in chromatin regulators, which mediate AC sensitivity across breast cancer patients. However, there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability, we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed, DOX response eQTLs are enriched in genes that respond to all TOP2i. Next, we identified eight genes in loci associated with AC toxicity by GWAS or TWAS. All eight genes, including RARG and SLC28A3, respond to at least two ACs, and their expression correlates with the release of cardiotoxicity markers. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes, including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity. Author summaryAnthracycline drugs such as Doxorubicin are effective treatments for breast cancer; however, they can cause cardiotoxicity in some women. It is unclear whether women would experience the same toxicity for all drugs in this class, or whether specific drugs would be better tolerated in specific individuals. We used an in vitro system of induced pluripotent stem cell-derived cardiomyocytes from six healthy females to test the effects of five breast cancer drugs on cell heath and global gene expression. We identified a strong shared cellular and gene expression response to drugs from the same class. However, there is more variation in gene expression levels between individuals following treatment with each anthracycline compared to untreated cells. We found that genes in regions previously associated with Doxorubicin-induced cardiotoxicity in cancer patients, respond to at least two drugs in the class. This suggests that drugs in the same class induce similar effects on an individuals heart. This work contributes to our understanding of how drug response, in the context of off-target effects, varies across individuals.

genomics↗

bioRxiv2021

Functional dynamic genetic effects on gene regulation are specific to particular cell types and environmental conditions

Genetic effects on gene expression and splicing can be modulated by cellular and environmental factors; yet interactions between genotypes, cell type and treatment have not been comprehensively studied together. We used an induced pluripotent stem cell system to study multiple cell types derived from the same individuals and exposed them to a large panel of treatments. Cellular responses involved different genes and pathways for gene expression and splicing processes, and were also highly variable across cell types and treatments. For thousands of genes, we identified variable allelic expression across contexts, and characterized different types of gene-environment interactions. Many of these GxE genes are associated with complex traits. We characterized promoter functional and evolutionary features that distinguish genes with elevated allelic imbalance mean and variance. More than 47% of the genes with dynamic regulatory interactions were missed by GTEx, but we identified them using a suitable allelic imbalance study design. This indicates the importance of exploring multiple treatments to reveal previously unrecognized regulatory loci that may be important for disease.

genomics↗

bioRxiv2020

Dynamic effects of genetic variation on gene expression revealed following hypoxic stress in cardiomyocytes

One life-threatening outcome of cardiovascular disease is myocardial infarction, where cardiomyocytes are deprived of oxygen. To study inter-individual differences in response to hypoxia, we established an in vitro model of induced pluripotent stem cell-derived cardiomyocytes from 15 individuals. We measured gene expression levels, chromatin accessibility, and methylation levels in four culturing conditions that correspond to normoxia, hypoxia and short or long-term re-oxygenation. We characterized thousands of gene regulatory changes as the cells transition between conditions. Using available genotypes, we identified 1,573 genes with a cis expression quantitative locus (eQTL) in at least one condition, as well as 367 dynamic eQTLs, which are classified as eQTLs in at least one, but not in all conditions. A subset of genes with dynamic eQTLs is associated with complex traits and disease. Our data demonstrate how dynamic genetic effects on gene expression, which are likely relevant for disease, can be uncovered under stress.

genomics↗

bioRxiv2019

Human iPSC gene signatures and X chromosome dosage impact response to WNT inhibition and cardiac differentiation fate

Non-genetic variability in human induced pluripotent stem cell (iPSC) lines impacts their differentiation outcome, limiting their utility for genetic studies and clinical applications. Despite the importance of understanding how non-genetic molecular variability influences iPSC differentiation outcome, large-scale studies capable of addressing this question have not yet been conducted. Here, we performed 258 directed differentiations of 191 iPSC lines using established protocols to generate iPSC-derived cardiovascular progenitor cells (iPSC-CVPCs). We observed cellular heterogeneity across the iPSC-CVPC samples due to varying fractions of two cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Analyzing the transcriptomes of CM-fated and EPDC-fated iPSCs discovered that 91 signature genes and X chromosome dosage differences influence WNT inhibition response during differentiation and are associated with cardiac fate. Analysis of an independent set of 39 iPSCs differentiated to the cardiac lineage confirmed shared sex and transcriptional differences that impact cardiac fate outcome. The scale and systematic approach of our study enabled novel insights into how iPSC transcriptional and X chromosome gene dosage differences influence WNT signaling during differentiation and hence cardiac cell fate.

genomics↗

bioRxiv2018

Inter-species differences in response to hypoxia in iPSC-derived cardiomyocytes from humans and chimpanzees

Despite anatomical similarities, there appear to be differences in susceptibility to cardiovascular disease between primates. For example, humans are prone to ischemia-induced myocardial infarction unlike chimpanzees, which tend to suffer from fibrotic disease. However, it is challenging to determine the relative contributions of genetic and environmental effects to complex disease phenotypes within and between primates. The ability to differentiate cardiomyocytes from induced pluripotent stem cells (iPSCs), now allows for direct inter-species comparisons of the gene regulatory response to disease-relevant perturbations. A consequence of ischemia is oxygen deprivation. Therefore, in order to understand human-specific regulatory adaptations in the heart, and to potentially gain insight into the evolution of disease susceptibility and resistance, we developed a model of hypoxia in human and chimpanzee cardiomyocytes. We differentiated eight human and seven chimpanzee iPSC lines into cardiomyocytes under normoxic conditions, and subjected these cells to 6 hours of hypoxia, followed by 6 or 24 hours of re-oxygenation. We collected genome-wide gene expression data as well as measurements of cellular stress at each time-point. The overall cellular and transcriptional response to hypoxic stress is generally conserved across species. Supporting the functional importance of precise regulatory response to hypoxia, we found that genes that respond to hypoxic stress in both species are depleted for association with expression quantitative trait loci (eQTLs) in the heart, and cardiovascular-related genes. We also identified hundereds of inter-species regulatory differences in our study. In particular, RASD1, which is associated with coronary artery disease, is up-regulated specifically in humans following hypoxia.

genomics↗

bioRxiv2017

Silencing Of Transposable Elements May Not Be A Major Driver Of Regulatory Evolution In Primate Induced Pluripotent Stem Cells

Transposable elements (TEs) comprise a substantial proportion of primate genomes. The regulatory potential of TEs can result in deleterious effects, especially during development. It has been suggested that, in pluripotent stem cells, TEs are targeted for silencing by KRAB-ZNF proteins, which recruit the TRIM28-SETDB1 complex, to deposit the repressive histone modification H3K9me3. TEs, in turn, can acquire mutations that allow them to evade detection by the host, and hence KRAB-ZNF proteins need to rapidly evolve to counteract them. To investigate the short-term evolution of TE silencing, we profiled the genome-wide distribution of H3K9me3 in induced pluripotent stem cells from ten human and seven chimpanzee individuals. We performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) for H3K9me3, as well as total RNA sequencing. We focused specifically on cross-species H3K9me3 ChIP-seq data that mapped to four million orthologous TEs. We found that, depending on the TE class, 10-60% of elements are marked by H3K9me3, with SVA, LTR and LINE elements marked most frequently. We found little evidence of inter-species differences in TE silencing, with as many as 80% of orthologous, putatively silenced, TEs marked at similar levels in humans and chimpanzees. Our data suggest limited species-specificity of TE silencing across six million years of primate evolution. Interestingly, the minority of TEs enriched for H3K9me3 in one species are not more likely to be associated with gene expression divergence of nearby orthologous genes. We conclude that orthologous TEs may not play a major role in driving gene regulatory divergence between humans and chimpanzees.

genomics↗

bioRxiv2016

Impact of regulatory variation across human iPSCs and differentiated cells

Induced pluripotent stem cells (iPSCs) are an essential tool for studying cellular differentiation and cell types that are otherwise difficult to access. We investigated the use of iPSCs and iPSC-derived cells to study the impact of genetic variation across different cell types and as models for studies of complex disease. We established a panel of iPSCs from 58 well-studied Yoruba lymphoblastoid cell lines (LCLs); 14 of these lines were further differentiated into cardiomyocytes. We characterized regulatory variation across individuals and cell types by measuring gene expression, chromatin accessibility and DNA methylation. Regulatory variation between individuals is lower in iPSCs than in the differentiated cell types, consistent with the intuition that developmental processes are generally canalized. While most cell type-specific regulatory quantitative trait loci (QTLs) lie in chromatin that is open only in the affected cell types, we found that 20% of cell type-specific QTLs are in shared open chromatin. Finally, we developed a deep neural network to predict open chromatin regions from DNA sequence alone and were able to use the sequences of segregating haplotypes to predict the effects of common SNPs on cell type-specific chromatin accessibility.

genomics↗