Biology subjects

Wu, S. M.

Publications and source records attributed to Wu, S. M..

8 recordsLinked to original sources

bioRxiv2026

Beta-Adrenergic Stimulation and MYH7 G256E Mutant Gene Dosage Drive Hypertrophic Cardiomyopathy Phenotype Penetrance

AimsHypertrophic cardiomyopathy (HCM) is the most prevalent genetic heart disorder, characterized by significant phenotypic variability even among individuals with identical MYH7 mutations. This study aims to elucidate factors contributing to this variability and identify drivers of phenotype penetrance. We compared the baseline phenotypes of a highly penetrant MYH7 H251N mutation and the variably penetrant MYH7 G256E mutation and investigated the impact of adding beta-adrenergic stimulation and homozygosity on disease phenotype penetrance using cardiomyocytes from an isogenic line of human induced pluripotent stem cells (hiPSC-CMs). Methods and ResultsIsogenic hiPSCs with MYH7 H251N and MYH7 G256E mutations were generated using CRISPR/Cas9 technology and differentiated into cardiomyocytes (CMs). Single-cell RNA sequencing (scRNAseq) and functional analysis of contractile function revealed consistent HCM phenotype presentation in H251N CMs, whereas G256E CMs exhibited a subtle and more variable phenotype. Beta-adrenergic stimulation induced a distinct metabolic stress response in G256E CMs, characterized by impaired mitochondrial ATP upregulation. Increasing mutant gene dosage from hetero- to homozygosity led to consistent increase in hypertrophic and structural gene expression changes in G256E CMs at RNA and protein levels. These changes were distinct from the changes observed with stress response. Importantly, homozygous G256E CMs exhibited a hypercontractile functional and disorganized structural phenotype. Across multiple experimental conditions, we identified consistent increase in cardiomyocyte specific transcriptomic markers such as NPPB, APOE, PDLIM3 and ANKRD1. ConclusionsOur study highlights the use of a variably penetrant MYH7 mutation to investigate factors that influence HCM phenotype penetrance. Specifically, we found that mutant gene dosage and beta-adrenergic stimulation induce distinct HCM disease phenotypes, providing novel insights into mechanisms that may contribute to variable disease expression in HCM. Translational PerspectiveHCM is characterized by significant phenotypic variability, complicating both diagnosis and clinical management. This study explores the factors driving HCM phenotype penetrance using isogenic hiPSC-CMs with MYH7 mutations. We demonstrate that beta-adrenergic stimulation and increased mutant gene dosage significantly impact HCM disease penetrance. Beta-adrenergic stimulation triggers metabolic stress responses, while increased gene dosage leads to a hypercontractile and structurally disorganized phenotype. These findings provide insight into how specific modifiers can shape disease-associated phenotypes in HCM model systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/729411v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@16a72d7org.highwire.dtl.DTLVardef@1999ca6org.highwire.dtl.DTLVardef@1fb8752org.highwire.dtl.DTLVardef@7148b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Differential and compensatory roles for type I phosphatidylinositol-4-phosphate-5-kinase isoforms in retinal function and health.

Phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) plays important roles in development, signaling, intracellular trafficking and regulation throughout the nervous system. Using selective and combined gene ablation strategies, we have determined the roles of this lipid and the kinase isoforms of the PIP5KI family primarily responsible for its synthesis in mouse retina. In rod cells, PI(4,5)P2 localizes predominantly to the plasma membrane of inner and outer segments and is enriched in membranes near the synaptic termini. Disruption of the gene encoding the {gamma} PIP5KI isoform, Pip5k1c, throughout the developing retina, using Cre expression driven by a Six3 transcription factor-dependent promoter, yields dramatic, but not complete, loss of the protein, with no apparent effects on morphology or function through the first 3-4 months after birth. Slowly progressing photoreceptor degeneration is observed at later ages. Complete loss of the {gamma} isoform in rods, driven by the rhodopsin promoter-based iCre75 transgene, leads to no obvious developmental defects, but results in an earlier-onset rod degeneration. Germ-line ablation of neither the Pip5k1a nor the Pip5k1b gene leads to any observable morphological defects. Homozygous Pip5k1a ablation leads to functional defects in photoreceptors as revealed by reduced a-wave and b-wave amplitudes in the electroretinograms. On the background of rod-specific Pip5k1c ablation, Pip5k1a deficiency greatly accelerates retinal degeneration. These results reveal a complex interplay among PIP5KI isoforms in ensuring proper photoreceptor function and health, with apparent partial redundancy in fulfilling their critical functions. They underscore the important role of PI(4,5)P2 in neuronal signaling and homeostasis. Significance StatementPhosphatidylinositol(4,5)P2, PI(4,5)P2, plays essential roles in nervous system development and function, but its roles in retina have been unknown. This study combines biochemistry, mouse genetics, light- and electron microscopy to reveal both specific and redundant functions for PIP2 formed by different kinase isoforms in the mammalian retina. It has implications for retinal function, disease and therapy, and for the broader field of phosphoinositide regulation.

neuroscience↗

bioRxiv2024

A Novel Therapeutic Approach using CXCR3 Blockade to Treat Immune Checkpoint Inhibitor-mediated Myocarditis

BackgroundImmune checkpoint inhibitors (ICIs) are successful in treating many cancers but may cause immune-related adverse events. ICI-mediated myocarditis has a high fatality rate of up to 40%, with severe cardiovascular consequences. Targeted therapies for ICI myocarditis are currently lacking. MethodsWe used a genetic mouse model of PD-1 deletion (MRL/Pdcd1-/-) along with a novel drug-treated ICI myocarditis mouse model to recapitulate the disease phenotype. We performed single-cell RNA-sequencing (scRNAseq), single-cell T-cell receptor sequencing (scTCR-seq), and cellular indexing of transcriptomes and epitopes (CITE-seq) on immune cells isolated from MRL and MRL/Pdcd1-/- mice at serial timepoints. We assessed the impact of macrophage deletion in MRL/Pdcd1-/- mice, then inhibited CXC chemokine receptor 3 (CXCR3) in ICI-treated mice to assess therapeutic effect on myocarditis phenotype. Furthermore, we delineated functional effects of CXCR3 blockade on T-cell and macrophage interactions in a transwell assay. We then correlated the results in human single-cell multi-omics data from blood and heart biopsy data from patients with ICI myocarditis. ResultsSingle-cell multi-omics demonstrated expansion of CXCL9/10+CCR2+ macrophages and CXCR3hi CD8+ effector T-lymphocytes in the hearts of MRL/Pdcd1-/- mice correlating with onset of myocarditis development. Both depletion of CXCL9/10+CCR2+ macrophages and CXCR3 blockade respectively led to decreased CXCR3hiCD8+ T-cell infiltration into the heart and significantly improved survival. A transwell assay showed that selective blockade of CXCR3 and its ligand, CXCL10 decreased CD8+ T-cell migration towards macrophages, implicating this interaction in T-cell cardiotropism towards cardiac macrophages. Cardiac biopsies from patients with confirmed ICI myocarditis demonstrated infiltrating CXCR3+ lymphocytes and CXCL9+/CXCL10+ macrophages. Both mouse cardiac immune cells and patient peripheral blood immune cells revealed expanded TCRs correlating with CXCR3hi CD8+ T-cells in ICI myocarditis samples. ConclusionsThese findings bring forth the CXCR3-CXCL9/10 axis as an attractive therapeutic target for ICI myocarditis treatment, and more broadly, as a druggable pathway in cardiac inflammation.

cell biology↗

bioRxiv2023

Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration

RationaleOver 200 mutations in the sarcomeric protein {beta}-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation. ObjectiveThis study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function. We hypothesize that the G256E mutation would alter myosin function, precipitating compensatory responses in cellular functions. MethodsWe developed a collaborative pipeline to characterize myosin function at multiple scales (protein to myofibril to cell to tissue). We also used our previously published data on other mutations to compare the degree to which myosin function was altered. ResultsAt the protein level, the G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 50.9%, suggesting more myosins available for contraction. Myofibrils isolated from hiPSC-CMs CRISPR-edited with G256E (MYH7WT/G256E) generated greater tension, had faster tension development and slower early phase relaxation, suggesting altered myosin-actin crossbridge cycling kinetics. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. Single-cell transcriptomic and metabolic profiling demonstrated upregulation of mitochondrial genes and increased mitochondrial respiration, suggesting altered bioenergetics as an early feature of HCM. ConclusionsMYH7 G256E mutation causes structural instability in the transducer region, leading to hypercontractility across scales, perhaps from increased myosin recruitment and altered crossbridge cycling. Hypercontractile function of the mutant myosin was accompanied by increased mitochondrial respiration, while cellular hypertrophy was modest in the physiological stiffness environment. We believe that this multi-scale platform will be useful to elucidate genotype-phenotype relationships underlying other genetic cardiovascular diseases.

molecular biology↗

bioRxiv2021

Dual TBX5-Lineage and MYL2 Reporter System For Identification of Left Ventricular Cardiomyocytes During Human Induced Pluripotent Stem Cell Differentiation

During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation stage human embryos. Human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF-progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.

developmental biology↗

bioRxiv2021

Abnormal Differentiation and Proliferation of Coronary Arterial Endothelium in Hypoplastic Left Heart Syndrome

Hypoplastic left heart syndrome (HLHS) is a severe form of single ventricle congenital heart disease characterized by the underdevelopment of the left ventricle. Early serial postmortem examinations revealed high rate of coronary artery abnormalities in HLHS fetal hearts, such as thickened wall, kinking arteries and ventriculo-coronary arterial connection. However, it is unclear if there is an intrinsic defect in the HLHS coronary vessels and what the underlying molecular mechanism is. Here, we profiled both human fetal heart with an underdeveloped left ventricle (ULV) and ECs differentiated from induced pluripotent stem cells (iPSCs) derived from HLHS patients at single cell resolution. CD144+/NPR3-vascular ECs were selected and further classified as venous, arterial and late arterial subclusters. To study the arterial EC phenotype, we specifically generated iPSC-arterial ECs (AECs, CD34+CDH5+CXCR4+NT5E-/low) derived from three HLHS patients and three age-matched healthy controls. Gene ontology analysis revealed that ULV late arterial EC subcluster showed specific defects in endothelial development, proliferation, and Notch signaling compared to control. Consistently, HLHS iPSCs exhibited impaired AEC differentiation shown as the reduced CXCR4+NT5E-/low AEC progenitor population. Mature HLHS iPSC-AECs also exhibited increased G0/G1 cell cycle arrest with decreased expression of cell cycle related genes (e.g., Ki67, CCND1/2). Additionally, NOTCH targeted genes (e.g., DLL4, HEY1, GJA5) were found suppressed in both ULV AECs and HLHS iPSC-AECs compared to control. We also found the HLHS de novo mutation gene KMT2D directly regulated the transcription of NOTCH targeted genes participating in arterial differentiation and cell proliferation, contributing to the HLHS AEC dysfunctionalities. Intriguingly, the treatment of NOTCH ligand JAG1 improved cell proliferation of HLHS AECs and upregulated G1/S transition genes downstream of NOTCH pathway. In summary, our results revealed that KMT2D directly regulated transcription activity of NOTCH signaling, contributing to the poor differentiation and low proliferation of HLHS coronary AECs.

developmental biology↗

bioRxiv2019

Single-Cell RNA-Seq Reveals Endocardial Defect in Hypoplastic Left Heart Syndrome

Hypoplastic left heart syndrome (HLHS) is one of the most challenging forms of congenital heart diseases. Previous studies were mainly focused on intrinsic defects in myocardium. However, this does not sufficiently explain the abnormal development of the cardiac valve, septum, and vasculature, known to originate from the endocardium. Here, using single-cell RNA profiling, induced pluripotent stem cells, and human fetal heart tissue with an underdeveloped left ventricle, we identified a developmentally impaired endocardial population in HLHS. The intrinsic endocardial deficits contributed to abnormal endothelial to mesenchymal transition, NOTCH signaling, and extracellular matrix organization, all of which are key factors in valve formation. Consequently, endocardial abnormalities conferred reduced proliferation and maturation of cardiomyocytes through a disrupted fibronectin-integrin interaction. Several recently described HLHS de novo mutations were associated with abnormal endocardial gene and FN1 regulation and expression. Our studies provide a rationale for considering endocardial function in future regenerative strategies for HLHS.

developmental biology↗

bioRxiv2019

Toxicogenomic identification of repositioned therapy for a monogenic disease

The cost of drug development from initial concept to FDA approval has been estimated to be about 2.6 billion USD.1 This cost precludes development of targeted therapies for rare diseases such as monogenetic cardiomyopathies. As part of the Library of Integrated Network-based Cellular Signatures (LINCS) program funded by the NIH, the Broad Institute of MIT has publicly released transcriptional profiles quantifying the effects of more than 25,000 perturbagens on the expression of 978 genes in up to 77 cell lines.2 Transcriptomics has been shown to be a powerful tool in repurposing drugs3,4 and this dataset affords us the unique opportunity to systematically identify small molecule mimics or inhibitors of specific genes, thereby identifying novel treatments for genetic disorders. In this report, we take this approach to identify a novel drug therapy for a monogenic form of familial dilated cardiomyopathy with the transcriptional profile of FDA approved drugs. This approach could potentially be replicated for a wide range of monogenic diseases.

genomics↗