Detyrosinated microtubules (MTs) provide mechanical resistance that can impede the motion of contracting cardiomyocytes. However, the functional effects of MT detyrosination in heart failure or in human hearts have not previously been studied. Here we utilize mass spectrometry and single-myocyte mechanical assays to characterize changes to the cardiomyocyte cytoskeleton and their functional consequences in human heart failure. Proteomic analysis of left ventricle tissue reveals a consistent upregulation and stabilization of intermediate filaments and MTs in failing human hearts. As revealed by super-resolution imaging, failing cardiomyocytes are characterized by a dense, heavily detyrosinated MT network, which is associated with increased myocyte stiffness and impaired contractility. Pharmacological suppression of detyrosinated MTs lowers the viscoelasticity of failing myocytes and restores 40–50% of lost contractile function; reduction of MT detyrosination using a genetic approach also softens cardiomyocytes and improves contractile kinetics. Together, these data demonstrate that a modified cytoskeletal network impedes contractile function in cardiomyocytes from failing human hearts and that targeting detyrosinated MTs could represent a new inotropic strategy for improving cardiac function.

Summary Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin and, independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery.

47Pulmonary endothelial cells (ECs) are an essential component of the gas exchange 48 machinery of the lung alveolus. Despite this, the extent and function of lung EC heterogeneity 49 remains incompletely understood. Using single-cell analytics, we identify multiple EC 50 populations in the mouse lung, including macrovascular endothelium (maEC), microvascular 51 endothelium (miECs), and a new population we have termed Car4-high ECs. Car4-high ECs 52 express a unique gene signature, and ligand-receptor analysis indicates they are primed to 53 receive reparative signals from alveolar type I cells. After acute lung injury, they are 54 preferentially localized in regenerating regions of the alveolus. Influenza infection reveals the 55 emergence of a population of highly proliferative ECs that likely arise from multiple miEC 56 populations and contribute to alveolar revascularization after injury. These studies map EC 57 heterogeneity in the adult lung and characterize the response of novel EC subpopulations 58 required for tissue regeneration after acute lung injury. 59 Significance 60Using transcriptional profiling of the pulmonary vascular endothelium and confirmation at 61 the RNA and protein levels, we have revealed extensive EC heterogeneity throughout the 62 vasculature of the lungs. We show that a subpopulation of endothelium re-enters the cell cycle 63 and proliferates in response to acute injury, whereas another subpopulation is enriched in 64 vasculogenic gene expression. These data provide foundational information regarding the 65 biological complexity of lung ECs, which will contribute to the development of novel tools to 66 enhance regeneration of the lung following injury. 67 whether such EC subpopulations contribute to in vivo tissue homeostasis and response to injury 93 in the adult lung remains unknown. 94To address these questions and to define pulmonary EC heterogeneity at homeostasis 95 and during regeneration, we utilized single cell RNA sequencing (scRNA-seq) analysis of the 96 adult mouse lung, both uninjured and after acute influenza-induced viral injury. In addition to 97 identifying microvascular (miEC) and both arterial and venous macrovascular (maEC) 98 populations, we identified a new population we have termed Car4-high ECs that possess a 99 unique transcriptome. Car4-high ECs express high levels of Car4 and Cd34, are found 100 throughout the lung periphery at homeostasis, and are primed to respond to Vegfa signaling 101 based on their high expression of Vegf receptors, which corresponds to a receptor-ligand 102 interaction analysis between Car4-high ECs and AT1 cells, their epithelial co-partners in gas 103 exchange. Car4-high ECs are enriched in the regenerating zones surrounding the most 104 damaged regions of the lung following influenza-or bleomycin-induced lung injury during the 105 subsequent tissue regeneration process. Influenza injury revealed the emergence of a unique 106 population of highly proliferative ECs, which are closely related to Car4-low miECs in gene 107 expression ...

The lung alveolus is the functional unit of the respiratory system required for gas exchange. During the transition to air breathing at birth, biophysical forces are thought to shape the emerging tissue niche. However, the intercellular signaling that drives these processes remains poorly understood. Applying a multimodal approach, we identified alveolar type 1 (AT1) epithelial cells as a distinct signaling hub. Lineage tracing demonstrates that AT1 progenitors align with receptive, force-exerting myofibroblasts in a spatial and temporal manner. Through single-cell chromatin accessibility and pathway expression (SCAPE) analysis, we demonstrate that AT1-restricted ligands are required for myofibroblasts and alveolar formation. These studies show that the alignment of cell fates, mediated by biophysical and AT1-derived paracrine signals, drives the extensive tissue remodeling required for postnatal respiration.