Biology subjects

O'Meara, C. C.

Publications and source records attributed to O'Meara, C. C..

5 recordsLinked to original sources

bioRxiv2025

Deletion of the polarity protein Llgl1 impairs cardiomyocyte intercalated disc integrity and permits cell cycle activity into adulthood

BackgroundCell polarity orchestrates spatial organization within tissues influencing cellular processes such as adhesion, proliferation, and differentiation. While the role of polarity proteins is well-established in epithelial systems, their function in mammalian cardiomyocytes remains poorly understood. Global deletion of the polarity protein, Llgl1, has been shown to impair cardiac trabeculation and disrupt cardiomyocyte junctions in zebrafish. Here, we investigate the role of Llgl1 in mammalian cardiomyocyte function. MethodsWe developed a mouse model whereby Llgl1 is deleted from cardiomyocytes (Llgl1CKO) and assessed cardiomyocyte intercalated disc integrity, cell cycle activity, cardiac function, and electrophysiology. We subjected mice to myocardial infarction to test if permanent Llgl1 deletion, or inducible Llgl1 deletion in adult cardiomyocytes (Llgl1TamCKO), influences the cardiac injury response. We performed RNAsequencing and in vitro knockdown and viral overexpression experiments to delineate the mechanisms by which Llgl1 influences cardiomyocyte phenotypes. ResultsLlgl1CKO mice display disrupted intercalated disc integrity indicated by reduced protein density at cardiomyocyte junctions, associated with electrical conduction defects that manifest as ventricular arrhythmia. Furthermore, Llgl1CKO mice display enhanced cardiomyocyte cell cycle activity extending into adulthood. Llgl1CKO mice and mice whereby Llgl1 is deleted by tamoxifen induction in adulthood both show improved cardiac function and reduced scar size following myocardial infarction. Mechanistically, Llgl1 alters mechanotransduction and cell cycle pathways in the heart, and regulates proper localization of N-cadherin to the cell membrane, a process that is optimized by phosphorylation of five serine residues at the C-terminus of Llgl1. ConclusionsWe elucidate a key role for Llgl1 in establishing proper intercalated disc integrity in mammalian cardiomyocytes. Disruption of intercalated discs and mechanotransduction via Llgl1 knockout is associated with cardiac arrhythmia, elevated cell cycle activity and resistance to cardiac injury. These studies shed light on mechanisms underlying intercalated disc establishment and connect disrupted cardiomyocyte junctional integrity with enhanced cell cycle activity.

cell biology↗

bioRxiv2024

Novel Insights into Post-Myocardial Infarction Cardiac Remodeling through Algorithmic Detection of Cell-Type Composition Shifts

BackgroundRecent advances in single cell sequencing have led to an increased focus on the role of cell-type composition in phenotypic presentation and disease progression. Cell-type composition research in the heart is challenging due to large, frequently multinucleated cardiomyocytes that preclude most single cell approaches from obtaining accurate measurements of cell composition. Our in silico studies reveal that ignoring cell type composition when calculating differentially expressed genes (DEGs) can have significant consequences. For example, a relatively small change in cell abundance of only 10% can result in over 25% of DEGs being false positives. MethodsWe have implemented an algorithmic approach that uses snRNAseq datasets as a reference to accurately calculate cell type compositions from bulk RNAseq datasets through robust data cleaning, gene selection, and multi-sample cross-subject and cross-cell-type deconvolution. We applied our approach to cardiomyocyte-specific 1A adrenergic receptor (CM-1A-AR) knockout mice. 8-12 week-old mice (either WT or CM-1A-KO) were subjected to permanent left coronary artery (LCA) ligation or sham surgery (n=4 per group). Transcriptomes from the infarct border zones were collected 3 days later and analyzed using our algorithm to determine cell-type abundances, corrected differential expression calculations using DESeq2, and validated these findings using RNAscope. ResultsUncorrected DEGs for the CM-1A-KO X LCA interaction term featured many cell-type specific genes such as Timp4 (fibroblasts) and Aplnr (cardiomyocytes) and overall GO enrichment for terms pertaining to cardiomyocyte differentiation (P=3.1E-4). Using our algorithm, we observe a striking loss of cardiomyocytes and gain in fibroblasts in the 1A-KO + LCA mice that was not recapitulated in WT + LCA animals, although we did observe a similar increase in macrophage abundance in both conditions. This recapitulates prior results that showed a much more severe heart failure phenotype in CM-1A-KO + LCA mice. Following correction for cell-type, our DEGs now highlight a novel set of genes enriched for GO terms such as cardiac contraction (P=3.7E-5) and actin filament organization (P=6.3E-5). ConclusionsOur algorithm identifies and corrects for cell-type abundance in bulk RNAseq datasets opening new avenues for research on novel genes and pathways as well as an improved understanding of the role of cardiac cell types in cardiovascular disease.

bioinformatics↗

bioRxiv2024

Physiological role and mechanisms of action for a long noncoding haplotype region

Most common sequence variants associated with human traits are in noncoding regions of the genome, form haplotypes with other noncoding variants, and exhibit small effect sizes in the general population. Determining the physiological roles and mechanisms of action for these noncoding variants, particularly large haplotypes containing multiple variants, is both critical and challenging. To address this challenge, we developed an approach that integrates physiological studies in genetically engineered and phenotypically permissive animal models, precise editing of large haplotypes in human induced pluripotent stem cells (hiPSCs), and targeted chromatin conformation analysis. We applied this approach to examine the blood pressure associated rs1173771 locus, which includes a haplotype containing 11 single nucleotide polymorphisms (SNPs) spanning 17.4 kbp. Deleting the orthologous noncoding region in the genome of the Dahl salt-sensitive rat attenuated the salt-induced increase in systolic blood pressure by nearly 10 mmHg. This attenuation of hypertension appeared to be mediated by upregulation of the adjacent gene Npr3 (natriuretic peptide receptor 3) in arteries, enhancing vasodilation. The blood pressure-elevating and -lowering haplotypes were precisely reconstituted in hiPSCs using an efficient, two-step genome editing technique. The blood pressure-elevating haplotype decreased NPR3 expression in endothelial cells and vascular smooth muscle cells derived from the edited, isogenic hiPSCs. The influence of the haplotype was partially recapitulated by the sentinel SNP rs1173771. Additionally, the blood pressure-elevating haplotype showed significantly greater chromatin interactions with the NPR3 promoter region. This study illustrates the feasibility of ascertaining the physiological roles and mechanisms of action for large noncoding haplotypes. Our efficient, integrated, and targeted approach can be applied to investigate other noncoding variants.

genetics↗

bioRxiv2022

Myofibroblast Ccn3 is regulated by Yap and Wwtr1 and contributes to adverse cardiac outcomes

While Yap and Wwtr1 regulate resident cardiac fibroblast to myofibroblast differentiation following cardiac injury, their role specifically in activated myofibroblasts remains unexplored. Here we assess the pathophysiological and cellular consequence of genetic depletion of Yap alone (Yapfl/fl;PostnMCM) or Yap and Wwtr1 (Yapfl/fl;Wwtr1fl/+;PostnMCM) in adult mouse myofibroblasts following myocardial infarction and identify and validate novel downstream factors specifically in cardiac myofibroblasts that mediate pathological remodeling. Following myocardial infarction, depletion of Yap in myofibroblasts had minimal effect on heart function while depletion of Yap/Wwtr1 resulted in smaller scars, reduced interstitial fibrosis, and improved ejection fraction and fractional shortening. Single cell RNA sequencing of interstitial cardiac cells 7 days post infarction showed suppression of pro-fibrotic genes in fibroblasts derived from Yapfl/fl,Wwtr1fl/+;PostnMCM hearts. In vivo myofibroblast depletion of Yap/Wwtr1 as well in vitro knockdown of Yap/Wwtr1 dramatically decreased RNA and protein expression of the matricellular factor Ccn3. Administration of recombinant CCN3 to adult mice following myocardial infarction remarkably aggravated cardiac function and scarring. CCN3 administration drove myocardial gene expression of pro-fibrotic genes in infarcted left ventricles implicating CCN3 as a novel driver of cardiac fibrotic processes following myocardial infarction.

physiology↗

bioRxiv2022

Cardiomyocyte ploidy is dynamic during postnatal development and varies across genetic backgrounds

Somatic polyploidization, an adaptation by which cells increase their DNA content to support cell and organ growth, is observed in many mammalian cell types, including cardiomyocytes. Although polyploidization is beneficial in many contexts, progression to a polyploid state is often accompanied by a loss of proliferative capacity. Recent work suggests that heterogeneity in cardiomyocyte ploidy is highly influenced by genetic diversity. However, the developmental course by which cardiomyocytes reach their final ploidy state has only been investigated in select genetic backgrounds. Here, we assessed cardiomyocyte number, cell cycle activity, and ploidy dynamics across two divergent inbred mouse strains; C57Bl/6J and A/J. Both strains are born and reach adulthood with a comparable number of cardiomyocytes, however the end composition of ploidy classes and developmental progression to reach the final state and number differ substantially. In addition to corroborating previous findings that identified Tnni3k as a mediator of cardiomyocyte ploidy, we also uncover a novel role for Runx1 and Tnni3k in ploidy dynamics and cardiomyocyte cytokinesis. These data provide novel insight into the developmental path to cardiomyocyte ploidy states and challenge the paradigm that polyploidization and hypertrophy are the only mechanisms for growth in the mouse heart after the first week of life.

developmental biology↗