Sex-specific responses to slow progressive pressure overload in a large animal model of HFpEF.

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.

Clinico-histopathologic and single-nuclei RNA-sequencing insights into cardiac injury and microthrombi in critical COVID-19.

Acute cardiac injury is prevalent in critical COVID-19 and associated with increased mortality. Its etiology remains debated, as initially presumed causes - myocarditis and cardiac necrosis - have proved uncommon. To elucidate the pathophysiology of COVID-19-associated cardiac injury, we conducted a prospective study of the first 69 consecutive COVID-19 decedents at CUIMC in New York City. Of 6 acute cardiac histopathologic features, presence of microthrombi was the most commonly detected among our cohort. We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak erythrocyte sedimentation rate and C-reactive protein were independently associated with increased odds of microthrombi, supporting an immunothrombotic etiology. Using single-nuclei RNA-sequencing analysis on 3 patients with and 4 patients without cardiac microthrombi, we discovered an enrichment of prothrombotic/antifibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling among cardiac fibroblasts in microthrombi-positive, relative to microthrombi-negative, COVID-19 hearts. Non-COVID-19, nonfailing hearts were used as reference controls. Our study identifies a specific transcriptomic signature in cardiac fibroblasts as a salient feature of microthrombi-positive COVID-19 hearts. Our findings warrant further mechanistic study as cardiac fibroblasts may represent a potential therapeutic target for COVID-19-associated cardiac microthrombi.

The genetic architecture of DNA replication timing in human pluripotent stem cells.

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome's replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) - sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.

Molecular Pathophysiology of Cardiac Injury and Cardiac Microthrombi in Fatal COVID-19: Insights from Clinico-histopathologic and Single Nuclei RNA Sequencing Analyses.

Cardiac injury is associated with critical COVID-19, yet its etiology remains debated. To elucidate the pathogenic mechanisms of COVID-19-associated cardiac injury, we conducted a single-center prospective cohort study of 69 COVID-19 decedents. Of six cardiac histopathologic features, microthrombi was the most commonly detected (n=48, 70%). We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak ESR and CRP during hospitalization were independently associated with higher odds of microthrombi. Using single nuclei RNA-sequence analysis, we discovered an enrichment of pro-thrombotic/anti-fibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling amongst cardiac fibroblasts in microthrombi-positive COVID-19 hearts relative to microthrombi-negative COVID-19. Non-COVID-19 non-failing hearts were used as reference controls. Our cumulative findings identify the specific transcriptomic changes in cardiac fibroblasts as salient features of COVID-19-associated cardiac microthrombi.

Molecular signatures of aneuploidy-driven adaptive evolution.

Alteration of normal ploidy (aneuploidy) can have a number of opposing effects, such as unbalancing protein abundances and inhibiting cell growth but also accelerating genetic diversification and rapid adaptation. The interplay of these detrimental and beneficial effects remains puzzling. Here, to understand how cells develop tolerance to aneuploidy, we subject disomic (i.e. with an extra chromosome copy) strains of yeast to long-term experimental evolution under strong selection, by forcing disomy maintenance and daily population dilution. We characterize mutations, karyotype alterations and gene expression changes, and dissect the associated molecular strategies. Cells with different extra chromosomes accumulated mutations at distinct rates and displayed diverse adaptive events. They tended to evolve towards normal ploidy through chromosomal DNA loss and gene expression changes. We identify genes with recurrent mutations and altered expression in multiple lines, revealing a variant that improves growth under genotoxic stresses. These findings support rapid evolvability of disomic strains that can be used to characterize fitness effects of mutations under different stress conditions.

Genomic methods for measuring DNA replication dynamics.

Genomic DNA replicates according to a defined temporal program in which early-replicating loci are associated with open chromatin, higher gene density, and increased gene expression levels, while late-replicating loci tend to be heterochromatic and show higher rates of genomic instability. The ability to measure DNA replication dynamics at genome scale has proven crucial for understanding the mechanisms and cellular consequences of DNA replication timing. Several methods, such as quantification of nucleotide analog incorporation and DNA copy number analyses, can accurately reconstruct the genomic replication timing profiles of various species and cell types. More recent developments have expanded the DNA replication genomic toolkit to assays that directly measure the activity of replication origins, while single-cell replication timing assays are beginning to reveal a new level of replication timing regulation. The combination of these methods, applied on a genomic scale and in multiple biological systems, promises to resolve many open questions and lead to a holistic understanding of how eukaryotic cells replicate their genomes accurately and efficiently.

Germline Structural Variations Are Preferential Sites of DNA Replication Timing Plasticity during Development.

The DNA replication timing program is modulated throughout development and is also one of the main factors influencing the distribution of mutation rates across the genome. However, the relationship between the mutagenic influence of replication timing and its developmental plasticity remains unexplored. Here, we studied the distribution of copy number variations (CNVs) and single nucleotide polymorphisms across the zebrafish genome in relation to changes in DNA replication timing during embryonic development in this model vertebrate species. We show that CNV sites exhibit strong replication timing plasticity during development, replicating significantly early during early development but significantly late during more advanced developmental stages. Reciprocally, genomic regions that changed their replication timing during development contained a higher proportion of CNVs than developmentally constant regions. Developmentally plastic CNV sites, in particular those that become delayed in their replication timing, were enriched for the clustered protocadherins, a set of genes important for neuronal development that have undergone extensive genetic and epigenetic diversification during zebrafish evolution. In contrast, single nucleotide polymorphism sites replicated consistently early throughout embryonic development, highlighting a unique aspect of the zebrafish genome. Our results uncover a hitherto unrecognized interface between development and evolution.