Massive field-of-view sub-cellular traction force videography enabled by Single-Pixel Optical Tracers (SPOT).

We report a massive field-of-view and high-speed videography platform for measuring the sub-cellular traction forces of more than 10,000 biological cells over 13 mm2 at 83 frames per second. Our Single-Pixel Optical Tracers (SPOT) tool uses 2-dimensional diffraction gratings embedded into a soft substrate to convert cells' mechanical traction force into optical colors detectable by a video camera. The platform measures the sub-cellular traction forces of diverse cell types, including tightly connected tissue sheets and near isolated cells. We used this platform to explore the mechanical wave propagation in a tightly connected sheet of Neonatal Rat Ventricular Myocytes (NRVMs) and discovered that the activation time of some tissue regions are heterogeneous from the overall spiral wave behavior of the cardiac wave.

Massively Concurrent Sub-Cellular Traction Force Videography enabled by Single-Pixel Optical Tracers (SPOTs).

We report a large field-of-view and high-speed videography platform for measuring the sub-cellular traction forces of more than 10,000 biological cells over 13mm 2 at 83 frames per second. Our Single-Pixel Optical Tracers (SPOT) tool uses 2-dimensional diffraction gratings embedded into a soft substrate to convert cells' mechanical traction stress into optical colors detectable by a video camera. The platform measures the sub-cellular traction forces of diverse cell types, including tightly connected tissue sheets and near isolated cells. We used this platform to explore the mechanical wave propagation in a tightly connected sheet of Neonatal Rat Ventricular Myocytes (NRVMs) and discovered that the activation time of some tissue regions are heterogeneous from the overall spiral wave behavior of the cardiac wave. One-Sentence Summary: An optical platform for fast, concurrent measurements of cell mechanics at 83 frames per second, over a large area of 13mm 2 .

DNA methylation networks underlying mammalian traits.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

Cardiac regeneration - Past advancements, current challenges, and future directions.

Cardiovascular disease is the leading cause of mortality and morbidity worldwide. Despite improvements in the standard of care for patients with heart diseases, including innovation in pharmacotherapy and surgical interventions, none have yet been proven effective to prevent the progression to heart failure. Cardiac transplantation is the last resort for patients with severe heart failure, but donor shortages remain a roadblock. Cardiac regenerative strategies include cell-based therapeutics, gene therapy, direct reprogramming of non-cardiac cells, acellular biologics, and tissue engineering methods to restore damaged hearts. Significant advancements have been made over the past several decades within each of these fields. This review focuses on the advancements of: 1) cell-based cardiac regenerative therapies, 2) the use of noncoding RNA to induce endogenous cell proliferation, and 3) application of bioengineering methods to promote retention and integration of engrafted cells. Different cell sources have been investigated, including adult stem cells derived from bone marrow and adipose cells, cardiosphere-derived cells, skeletal myoblasts, and pluripotent stem cells. In addition to cell-based transplantation approaches, there have been accumulating interest over the past decade in inducing endogenous CM proliferation for heart regeneration, particularly with the use of noncoding RNAs such as miRNAs and lncRNAs. Bioengineering applications have focused on combining cell-transplantation approaches with fabrication of a porous, vascularized scaffold using biomaterials and advanced bio-fabrication techniques that may offer enhanced retention of transplanted cells, with the hope that these cells would better engraft with host tissue to improve cardiac function. This review summarizes the present status and future challenges of cardiac regenerative therapies.

Cardiac pericytes mediate the remodeling response to myocardial infarction.

Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-ß signaling. Deleting TGF-ß receptor 1 in chondroitin sulfate proteoglycan 4-expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.

Dual genetic tracing reveals a unique fibroblast subpopulation modulating cardiac fibrosis.

After severe heart injury, fibroblasts are activated and proliferate excessively to form scarring, leading to decreased cardiac function and eventually heart failure. It is unknown, however, whether cardiac fibroblasts are heterogeneous with respect to their degree of activation, proliferation and function during cardiac fibrosis. Here, using dual recombinase-mediated genetic lineage tracing, we find that endocardium-derived fibroblasts preferentially proliferate and expand in response to pressure overload. Fibroblast-specific proliferation tracing revealed highly regional expansion of activated fibroblasts after injury, whose pattern mirrors that of endocardium-derived fibroblast distribution in the heart. Specific ablation of endocardium-derived fibroblasts alleviates cardiac fibrosis and reduces the decline of heart function after pressure overload injury. Mechanistically, Wnt signaling promotes activation and expansion of endocardium-derived fibroblasts during cardiac remodeling. Our study identifies endocardium-derived fibroblasts as a key fibroblast subpopulation accounting for severe cardiac fibrosis after pressure overload injury and as a potential therapeutic target against cardiac fibrosis.

Colchicine for the Treatment of Cardiac Injury in Hospitalized Patients With Coronavirus Disease-19.

Introduction: The impact of colchicine on hospitalized patients with Coronavirus disease-19 (COVID-19) related cardiac injury is unknown. Materials and Methods: In this multicenter randomized controlled open-label clinical trial, we randomized hospitalized adult patients with documented COVID-19 and evidence of cardiac injury in a 1:1 ratio to either colchicine 0.6 mg po twice daily for 30 days plus standard of care or standard of care alone. Cardiac injury was defined as elevated cardiac biomarkers, new arrhythmia, new/worsened left ventricular dysfunction, or new pericardial effusion. The primary endpoint was the composite of all-cause mortality, need for mechanical ventilation, or need for mechanical circulatory support (MCS) at 90 days. Key secondary endpoints included the individual components of the primary endpoint and change in and at least 2-grade reduction in the World Health Organization (WHO) Ordinal Scale at 30 days. The trial is registered with clinicaltrials.gov (NCT04355143). Results: We enrolled 93 patients, 48 patients in the colchicine arm and 45 in the control arm. There was no significant difference in the primary outcome between the colchicine and control arms (19 vs. 15%, p = 0.78), nor in the individual components of all-cause mortality (17 vs. 15%, p = 1.0) and need for mechanical ventilation (8 vs. 5%, p = 0.68); no patients in either group required MCS. The change in (-1.8 ± 2.4 vs. -1.2 ± 2.0, p = 0.12) and at least 2-grade reduction (75 vs. 75%, p = 1.0) in the WHO ordinal scale was also similar between groups. Conclusion: Patients hospitalized with COVID-19 and evidence of cardiac injury did not benefit from colchicine therapy.

In Vitro Generation of Heart Field Specific Cardiomyocytes.

Myocardial infarction (MI) can lead to irreversible loss of cardiomyocytes (CMs), primarily localized to the left ventricle (LV) of the heart. The CMs of the LV are predominantly derived from first heart field (FHF) progenitors, whereas the majority of CMs within the right ventricle originate from the second heart field (SHF) during early cardiogenesis. Human embryonic stem cells (hESCs) serve as a valuable source of CMs for understanding early cardiac development and lineage commitment of CMs within these two heart fields that ultimately enable the development of more effective candidates for cell therapy. An ideal candidate may be FHF CMs that share the same ontogeny with the LV CMs that die after MI. We previously generated a double reporter hESC line that utilizes two important cardiac transcription factors, TBX5 and NKX2-5. TBX5 marks FHF progenitors and CMs, while NKX2-5 is expressed in nearly all myocytes of the developing heart. Here, we describe a step-by-step approach to efficiently generate FHF and SHF CMs using this double reporter hESC line. In addition, this approach can be applied to any non-genetically modified hESC lines to enrich FHF and SHF CMs. Obtaining enriched populations of these two CM subtypes provides a platform for downstream comparative analyses and in vitro studies to facilitate a deeper understanding of cardiovascular lineage commitment and the development of more effective candidates for cell therapy to treat diseases or defects that affect specific regions of the heart.

Clonal Tracing of Heart Regeneration.

Cardiomyocytes in the adult mammalian heart have a low turnover during homeostasis. After myocardial injury, there is irreversible loss of cardiomyocytes, which results in subsequent scar formation and cardiac remodeling. In order to better understand and characterize the proliferative capacity of cardiomyocytes, in vivo methods have been developed to track their fate during normal development and after injury. Lineage tracing models are of particular interest due to their ability to record cell proliferation events over a long period of time, either during development or in response to a pathological event. This paper reviews two well-studied lineage-tracing, transgenic mouse models-mosaic analysis with double markers and rainbow reporter system.

Aortic intimal resident macrophages are essential for maintenance of the non-thrombogenic intravascular state.

Leukocytes and endothelial cells frequently cooperate to resolve inflammatory events. In most cases, these interactions are transient in nature and triggered by immunological insults. Here, we report that in areas of disturbed blood flow, aortic endothelial cells permanently and intimately associate with a population of specialized macrophages that are recruited at birth from the closing ductus arteriosus and share the luminal surface with the endothelium becoming interwoven in the tunica intima. Anatomical changes that affect hemodynamics, like in patent ductus arteriosus, alter macrophage seeding to coincide with regions of disturbed flow. Aortic resident macrophages expand in situ via direct cell renewal. Induced-depletion of intimal macrophages led to thrombin-mediated endothelial cell contraction, progressive fibrin accumulation and formation of microthrombi that, once dislodged, caused blockade of vessels in several organs. Together the findings revealed that intravascular resident macrophages are essential to regulate thrombin activity and clear fibrin deposits in regions of disturbed blood flow.

Recent Advances in Designing Electroconductive Biomaterials for Cardiac Tissue Engineering.

Implantable cardiac patches and injectable hydrogels are among the most promising therapies for cardiac tissue regeneration following myocardial infarction. Incorporating electrical conductivity into these patches and hydrogels is found to be an efficient method to improve cardiac tissue function. Conductive nanomaterials such as carbon nanotube, graphene oxide, gold nanorod, as well as conductive polymers such as polyaniline, polypyrrole, and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate are appealing because they possess the electroconductive properties of semiconductors with ease of processing and have potential to restore electrical signaling propagation through the infarct area. Numerous studies have utilized these materials for regeneration of biological tissues that possess electrical activities, such as cardiac tissue. In this review, recent studies on the use of electroconductive materials for cardiac tissue engineering and their fabrication methods are summarized. Moreover, recent advances in developing electroconductive materials for delivering therapeutic agents as one of emerging approaches for treating heart diseases and regenerating damaged cardiac tissues are highlighted.

Mapping human haematopoietic stem cells from haemogenic endothelium to birth.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.

Immunoengineering strategies to enhance vascularization and tissue regeneration.

Immune cells have emerged as powerful regulators of regenerative as well as pathological processes. The vast majority of regenerative immunoengineering efforts have focused on macrophages; however, growing evidence suggests that other cells of both the innate and adaptive immune system are as important for successful revascularization and tissue repair. Moreover, spatiotemporal regulation of immune cells and their signaling have a significant impact on the regeneration speed and the extent of functional recovery. In this review, we summarize the contribution of different types of immune cells to the healing process and discuss ways to manipulate and control immune cells in favor of vascularization and tissue regeneration. In addition to cell delivery and cell-free therapies using extracellular vesicles, we discuss in situ strategies and engineering approaches to attract specific types of immune cells and modulate their phenotypes. This field is making advances to uncover the extraordinary potential of immune cells and their secretome in the regulation of vascularization and tissue remodeling. Understanding the principles of immunoregulation will help us design advanced immunoengineering platforms to harness their power for tissue regeneration.

Environmental factors influence somatic cell reprogramming to cardiomyocyte-like cells.

Direct cardiac reprogramming, which refers to somatic cell (i.e. fibroblast) fate conversion to cardiomyocyte-like cell without transitioning through an intermediate pluripotent state, provides a novel therapeutic strategy for heart regeneration by converting resident cardiac fibroblasts to cardiomyocytes in situ. However, several limitations need to be addressed prior to clinical translation of this technology. They include low efficiency of reprogramming, heterogeneity of starting fibroblasts, functional immaturity of induced cardiomyocytes (iCMs), virus immunogenicity and toxicity, incomplete understanding of changes in the epigenetic landscape as fibroblasts undergo reprogramming, and the environmental factors that influence fate conversion. Several studies have demonstrated that a combination of enforced expression of cardiac transcription factors along with certain cytokines and growth factors in the presence of favorable environmental cues (including extracellular matrix, topography, and mechanical properties) enhance the efficiency and quality of direct reprogramming. This paper reviews the literature on the influence of the microenvironment on direct cardiac reprogramming in vitro and in vivo.

Isolation and characterization of human embryonic stem cell-derived heart field-specific cardiomyocytes unravels new insights into their transcriptional and electrophysiological profiles.

AIMS: We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart field-specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations. METHODS AND RESULTS: Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enable purification of cardiomyocytes and first heart field cardiomyocytes, respectively. CONCLUSIONS: We describe a high-yield approach for isolation and characterization of human embryonic stem cell-derived heart field-specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modelling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart- or chamber-specific congenital heart defects.

Cell proliferation fate mapping reveals regional cardiomyocyte cell-cycle activity in subendocardial muscle of left ventricle.

Cardiac regeneration involves the generation of new cardiomyocytes from cycling cardiomyocytes. Understanding cell-cycle activity of pre-existing cardiomyocytes provides valuable information to heart repair and regeneration. However, the anatomical locations and in situ dynamics of cycling cardiomyocytes remain unclear. Here we develop a genetic approach for a temporally seamless recording of cardiomyocyte-specific cell-cycle activity in vivo. We find that the majority of cycling cardiomyocytes are positioned in the subendocardial muscle of the left ventricle, especially in the papillary muscles. Clonal analysis revealed that a subset of cycling cardiomyocytes have undergone cell division. Myocardial infarction and cardiac pressure overload induce regional patterns of cycling cardiomyocytes. Mechanistically, cardiomyocyte cell cycle activity requires the Hippo pathway effector YAP. These genetic fate-mapping studies advance our basic understanding of cardiomyocyte cell cycle activity and generation in cardiac homeostasis, repair, and regeneration.

Validity of echocardiography for detection of left ventricular thrombus with surgical validation in patients awaiting durable left ventricular assist device.

OBJECTIVE: Unrecognized left ventricular thrombi (LVT) can have devastating clinical implications and precludes patients with end-stage heart failure from undergoing left ventricular assist device (LVAD) implantation without cardiopulmonary bypass assistance. We assessed the reliability of an echocardiogram to diagnose LVT in patients with end-stage heart disease who underwent LVAD implantation. METHODS: A single-center retrospective study evaluated 232 consecutive adult patients requiring implantation of durable LVADs between 2005 and 2019. The validity of preoperative transthoracic echocardiogram (TTE) and intraoperative transesophageal echocardiogram (TEE) for diagnosing LVT was compared to direct inspection at the time of LVAD implantation. RESULTS: There were 232 patients that underwent LVAD implantation, with 226 patients (97%) receiving a preoperative TTE. Of those 226 patients, 32 patients (14%) received ultrasound enhancing agents (UEA). Intraoperative TEE images were available in 195 patients (84%). The sensitivity of TTE without UEA was 22% and specificity was 90% for detecting LVT, compared to 50% and 86%, respectively, for TTE with UEA. For intraoperative TEE, the sensitivity and specificity were 46% and 96%, respectively. The false omission rate ranged from 4% to 8% for all modalities of echocardiography. CONCLUSION: Among patients undergoing LVAD implantation, preoperative TTE and intraoperative TEE had poor sensitivity for LVT detection. Up to 8% of echocardiograms were incorrectly concluded to be negative for LVT on surgical validation. The low sensitivity and positive predictive value for diagnosing LVT suggest that echocardiography has limited reliability in this cohort of patients who are at high risk of LVT formation and its subsequent complications.