Epicardium and Coronary Vessels.

Congenital anomalies and acquired diseases of the coronary blood vessels are of great clinical relevance. The early diagnosis of these conditions remains, however, challenging. In order to improve our knowledge of these ailments, progress has to be achieved in the research of the molecular and cellular mechanisms that control development of the coronary vascular bed. The aim of this chapter is to provide a succint account of the key elements of coronary blood vessel development, especially in the context of the role played by the epicardium and epicardial cellular derivatives. We will discuss the importance of the epicardium in coronary blood vessel morphogenesis, from the contribution of the epicardially derived mesenchyme to these blood vessels to its role as an instructive signaling center, attempting to relate these concepts to the origin of coronary disease.

Congenital Coronary Blood Vessel Anomalies: Animal Models and the Integration of Developmental Mechanisms.

Coronary blood vessels are in charge of sustaining cardiac homeostasis. It is thus logical that coronary congenital anomalies (CCA) directly or indirectly associate with multiple cardiac conditions, including sudden death. The coronary vascular system is a sophisticated, highly patterned anatomical entity, and therefore a wide range of congenital malformations of the coronary vasculature have been described. Despite the clinical interest of CCA, very few attempts have been made to relate specific embryonic developmental mechanisms to the congenital anomalies of these blood vessels. This is so because developmental data on the morphogenesis of the coronary vascular system derive from complex studies carried out in animals (mostly transgenic mice), and are not often accessible to the clinician, who, in turn, possesses essential information on the significance of CCA. During the last decade, advances in our understanding of normal embryonic development of coronary blood vessels have provided insight into the cellular and molecular mechanisms underlying coronary arteries anomalies. These findings are the base for our attempt to offer plausible embryological explanations to a variety of CCA as based on the analysis of multiple animal models for the study of cardiac embryogenesis, and present them in an organized manner, offering to the reader developmental mechanistic explanations for the pathogenesis of these anomalies.

The Genetics of Human Congenital Coronary Vascular Anomalies.

The genetics of human congenital coronary vascular anomalies (hCCVA) remains largely underresearched. This is surprising, because although coronary vascular defects represent a relatively small proportion of human congenital heart disease (CHD), hCCVAs are clinically significant conditions. Indeed, hCCVA frequently associate to other congenital cardiac structural defects and may even result in sudden cardiac death in the adult. In this brief chapter, we will attempt to summarize our current knowledge on the topic, also proposing a rationale for the development of novel approaches to the genetics of hCCVA.

Generation of heart and vascular system in rodents by blastocyst complementation.

Generating organs from stem cells through blastocyst complementation is a promising approach to meet the clinical need for transplants. In order to generate rejection-free organs, complementation of both parenchymal and vascular cells must be achieved, as endothelial cells play a key role in graft rejection. Here, we used a lineage-specific cell ablation system to produce mouse embryos unable to form both the cardiac and vascular systems. By mouse intraspecies blastocyst complementation, we rescued heart and vascular system development separately and in combination, obtaining complemented hearts with cardiomyocytes and endothelial cells of exogenous origin. Complemented chimeras were viable and reached adult stage, showing normal cardiac function and no signs of histopathological defects in the heart. Furthermore, we implemented the cell ablation system for rat-to-mouse blastocyst complementation, obtaining xenogeneic hearts whose cardiomyocytes were completely of rat origin. These results represent an advance in the experimentation towards the in vivo generation of transplantable organs.

Development of an injectable alginate-collagen hydrogel for cardiac delivery of extracellular vesicles.

Extracellular vesicles (EVs) are nanosized particles with attractive therapeutic potential for cardiac repair. However, low retention and stability after systemic administration limit their clinical translation. As an alternative, the combination of EVs with biomaterial-based hydrogels (HGs) is being investigated to increase their exposure in the myocardium and achieve an optimal therapeutic effect. In this study, we developed and characterized a novel injectable in-situ forming HG based on alginate and collagen as a cardiac delivery vehicle for EVs. Different concentrations of alginate and collagen crosslinked with calcium gluconate were tested. Based on injectability studies, 1% alginate, 0.5 mg/mL collagen and 0.25% calcium gluconate HG was selected as the idoneous combination for cardiac administration using catheter-based systems. Rheological examination revealed that the HG possessed an internal gel structure, weak mechanical properties and low viscosity, facilitating an easy administration. In addition, EVs were successfully incorporated and homogeneously distributed in the HG. After administration in a rat model of myocardial infarction, the HG showed long-term retention in the heart and allowed for a sustained release of EVs for at least 7 days. Thus, the combination of HGs and EVs represents a promising therapeutic strategy for myocardial repair. Besides EVs delivery, the developed HG could represent a useful platform for cardiac delivery of multiple therapeutic agents.

In Vivo and In Vitro Cartilage Differentiation from Embryonic Epicardial Progenitor Cells.

The presence of cartilage tissue in the embryonic and adult hearts of different vertebrate species is a well-recorded fact. However, while the embryonic neural crest has been historically considered as the main source of cardiac cartilage, recently reported results on the wide connective potential of epicardial lineage cells suggest they could also differentiate into chondrocytes. In this work, we describe the formation of cardiac cartilage clusters from proepicardial cells, both in vivo and in vitro. Our findings report, for the first time, cartilage formation from epicardial progenitor cells, and strongly support the concept of proepicardial cells as multipotent connective progenitors. These results are relevant to our understanding of cardiac cell complexity and the responses of cardiac connective tissues to pathologic stimuli.

Isolation methods of large and small extracellular vesicles derived from cardiovascular progenitors: A comparative study.

Since the discovery of the beneficial therapeutical effects of extracellular vesicles (EVs), these agents have been attracting great interest as next-generation therapies. EVs are nanosized membrane bodies secreted by all types of cells that mediate cell-cell communication. Although the classification of different subpopulations of EVs can be complex, they are broadly divided into microvesicles and exosomes based on their biogenesis and in large and small EVs based on their size. As this is an emerging field, current investigations are focused on basic aspects such as the more convenient method for EV isolation. In the present paper, we used cardiac progenitor cells (CPCs) to study and compare different cell culture conditions for EV isolation as well as two of the most commonly employed purification methods: ultracentrifugation (UC) and size-exclusion chromatography (SEC). Large and small EVs were separately analysed. We found that serum starvation of cells during the EV collecting period led to a dramatic decrease in EV secretion and major cell death. Regarding the isolation method, our findings suggest that UC and SEC gave similar EV recovery rates. Separation of large and small EV-enriched subpopulations was efficiently achieved with both purification protocols although certain difference in sample heterogeneity was observed. Noteworthy, while calnexin was abundant in large EVs, ALIX and CD63 were mainly found in small EVs. Finally, when the functionality of EVs was assessed on primary culture of adult murine cardiac fibroblasts, we found that EVs were taken up by these cells, which resulted in a pronounced reduction in the proliferative and migratory capacity of the cells. Specifically, a tendency towards a larger effect of SEC-related EVs was observed. No differences could be found between large and small EVs. Altogether, these results contribute to establish the basis for the use of EVs as therapeutic platforms, in particular in regenerative fields.

Use and Misuse of Cq in qPCR Data Analysis and Reporting.

In the analysis of quantitative PCR (qPCR) data, the quantification cycle (Cq) indicates the position of the amplification curve with respect to the cycle axis. Because Cq is directly related to the starting concentration of the target, and the difference in Cq values is related to the starting concentration ratio, the only results of qPCR analysis reported are often Cq, ΔCq or ΔΔCq values. However, reporting of Cq values ignores the fact that Cq values may differ between runs and machines, and, therefore, cannot be compared between laboratories. Moreover, Cq values are highly dependent on the PCR efficiency, which differs between assays and may differ between samples. Interpreting reported Cq values, assuming a 100% efficient PCR, may lead to assumed gene expression ratios that are 100-fold off. This review describes how differences in quantification threshold setting, PCR efficiency, starting material, PCR artefacts, pipetting errors and sampling variation are at the origin of differences and variability in Cq values and discusses the limits to the interpretation of observed Cq values. These issues can be avoided by calculating efficiency-corrected starting concentrations per reaction. The reporting of gene expression ratios and fold difference between treatments can then easily be based on these starting concentrations.

Understanding the Adult Mammalian Heart at Single-Cell RNA-Seq Resolution.

During the last decade, extensive efforts have been made to comprehend cardiac cell genetic and functional diversity. Such knowledge allows for the definition of the cardiac cellular interactome as a reasonable strategy to increase our understanding of the normal and pathologic heart. Previous experimental approaches including cell lineage tracing, flow cytometry, and bulk RNA-Seq have often tackled the analysis of cardiac cell diversity as based on the assumption that cell types can be identified by the expression of a single gene. More recently, however, the emergence of single-cell RNA-Seq technology has led us to explore the diversity of individual cells, enabling the cardiovascular research community to redefine cardiac cell subpopulations and identify relevant ones, and even novel cell types, through their cell-specific transcriptomic signatures in an unbiased manner. These findings are changing our understanding of cell composition and in consequence the identification of potential therapeutic targets for different cardiac diseases. In this review, we provide an overview of the continuously changing cardiac cellular landscape, traveling from the pre-single-cell RNA-Seq times to the single cell-RNA-Seq revolution, and discuss the utilities and limitations of this technology.

Extracellular Vesicle-Based Therapeutics for Heart Repair.

Extracellular vesicles (EVs) are constituted by a group of heterogeneous membrane vesicles secreted by most cell types that play a crucial role in cell-cell communication. In recent years, EVs have been postulated as a relevant novel therapeutic option for cardiovascular diseases, including myocardial infarction (MI), partially outperforming cell therapy. EVs may present several desirable features, such as no tumorigenicity, low immunogenic potential, high stability, and fine cardiac reparative efficacy. Furthermore, the natural origin of EVs makes them exceptional vehicles for drug delivery. EVs may overcome many of the limitations associated with current drug delivery systems (DDS), as they can travel long distances in body fluids, cross biological barriers, and deliver their cargo to recipient cells, among others. Here, we provide an overview of the most recent discoveries regarding the therapeutic potential of EVs for addressing cardiac damage after MI. In addition, we review the use of bioengineered EVs for targeted cardiac delivery and present some recent advances for exploiting EVs as DDS. Finally, we also discuss some of the most crucial aspects that should be addressed before a widespread translation to the clinical arena.

Bone marrow contribution to the heart from development to adulthood.

Cardiac chamber walls contain large numbers of non-contractile interstitial cells, including fibroblasts, endothelial cells, pericytes and significant populations of blood lineage-derived cells. Blood cells first colonize heart tissues a few days before birth, although their recruitment from the bloodstream to the cardiac interstitium is continuous and extends throughout adult life. The bone marrow, as the major hematopoietic site of adult individuals, is in charge of renewing all circulating cell types, and it therefore plays a pivotal role in the incorporation of blood cells to the heart. Bone marrow-derived cells are instrumental to tissue homeostasis in the steady-state heart, and are major effectors in cardiac disease progression. This review will provide a comprehensive approach to bone marrow-derived blood cell functions in the heart, and discuss aspects related to hot topics in the cardiovascular field like cell-based heart regeneration strategies.

Single-Cell RNA Sequencing Analysis Reveals a Crucial Role for CTHRC1 (Collagen Triple Helix Repeat Containing 1) Cardiac Fibroblasts After Myocardial Infarction.

BACKGROUND: Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function. METHODS: Collagen1α1-GFP (green fluorescent protein)-positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing. RESULTS: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor-ß signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. CONCLUSIONS: We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies CTHRC1 as a novel regulator of the healing scar process and a target for future translational studies.

Functional modulation of atrio-ventricular conduction by enhanced late sodium current and calcium-dependent mechanisms in Scn5a1798insD/+ mice.

AIMS: SCN5A mutations are associated with arrhythmia syndromes, including Brugada syndrome, long QT syndrome type 3 (LQT3), and cardiac conduction disease. Long QT syndrome type 3 patients display atrio-ventricular (AV) conduction slowing which may contribute to arrhythmogenesis. We here investigated the as yet unknown underlying mechanisms. METHODS AND RESULTS: We assessed electrophysiological and molecular alterations underlying AV-conduction abnormalities in mice carrying the Scn5a1798insD/+ mutation. Langendorff-perfused Scn5a1798insD/+ hearts showed prolonged AV-conduction compared to wild type (WT) without changes in atrial and His-ventricular (HV) conduction. The late sodium current (INa,L) inhibitor ranolazine (RAN) normalized AV-conduction in Scn5a1798insD/+ mice, likely by preventing the mutation-induced increase in intracellular sodium ([Na+]i) and calcium ([Ca2+]i) concentrations. Indeed, further enhancement of [Na+]i and [Ca2+]i by the Na+/K+-ATPase inhibitor ouabain caused excessive increase in AV-conduction time in Scn5a1798insD/+ hearts. Scn5a1798insD/+ mice from the 129P2 strain displayed more severe AV-conduction abnormalities than FVB/N-Scn5a1798insD/+ mice, in line with their larger mutation-induced INa,L. Transverse aortic constriction (TAC) caused excessive prolongation of AV-conduction in FVB/N-Scn5a1798insD/+ mice (while HV-intervals remained unchanged), which was prevented by chronic RAN treatment. Scn5a1798insD/+-TAC hearts showed decreased mRNA levels of conduction genes in the AV-nodal region, but no structural changes in the AV-node or His bundle. In Scn5a1798insD/+-TAC mice deficient for the transcription factor Nfatc2 (effector of the calcium-calcineurin pathway), AV-conduction and conduction gene expression were restored to WT levels. CONCLUSIONS: Our findings indicate a detrimental role for enhanced INa,L and consequent calcium dysregulation on AV-conduction in Scn5a1798insD/+ mice, providing evidence for a functional mechanism underlying AV-conduction disturbances secondary to gain-of-function SCN5A mutations.

Effect of heart ischemia and administration route on biodistribution and transduction efficiency of AAV9 vectors.

Adeno-associated viruses (AAV) have become one of the most promising tools for gene transfer in clinics. Among all the serotypes, AAV9 has been described as the most efficient for cardiac transduction. In order to achieve optimal therapeutic delivery in heart disease, we have explored AAV9 transduction efficiency in an infarcted heart using different routes of administration and promoters, including a cardiac-specific one. AAV9 vectors carrying luciferase or green fluorescence protein under the control of the ubiquitous elongation-factor-1-alpha or the cardiac-specific troponin-T (TnT) promoters were administered by intramyocardial or intravenous injection, either in healthy or myocardial-infarcted mice. The transduction efficacy and specificity, the time-course expression, and the safety of each vector were tested. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV-TnT, which was significantly higher after intramyocardial injection regardless of ischemia-induction. On the contrary, high hepatic transgene expression levels were detected with the elongation-factor-1-alpha-promoter, independently of the administration route and heart damage. Moreover, tissue-specific green fluorescence protein expression was found in cardiomyocytes with the TnT vector, whereas minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that myocardial infarction greatly increased the transcriptional activity of AAV genomes. Our findings show that the use of cardiac promoters allows for specific and stable cardiac gene expression, which is optimal and robust when intramyocardially injected. Furthermore, our data indicate that the pathological status of the tissue can alter the transcriptional activity of AAV genomes, an aspect that should be carefully evaluated for clinical applications.

Removal of artifact bias from qPCR results using DNA melting curve analysis.

Quantitative PCR (qPCR) allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, analyses using validated Sybr Green I-based assays regularly amplify both the correct product and an artifact. Amplification of more than 1 product can be recognized when melting curve analysis is performed after the qPCR. Currently, such reactions need to be excluded from further analysis because the quantification result is considered meaningless. However, when the fraction of the fluorescence associated with the correct product can be determined, the quantitative result of the qPCR analysis can be corrected. The main assumptions of this correction model are: 1) the melting peak of the correct product can be identified, 2) the PCR efficiencies of all amplified products are similar, 3) the relative size of the melting peaks reflects the relative concentrations of the products, and 4) the relative concentrations do not change as the reaction reaches plateau. These assumptions were validated in a series of model experiments. The results show that the quantitative results can be corrected. Implementation of a correction for the presence of artifact amplification in the analysis of qPCR data leads to more reliable quantitative results in qPCR experiments.-Ruijter, J. M., Ruiz-Villalba, A., van den Hoff, A. J. J., Gunst, Q. D., Wittwer, C. T., van den Hoff, M. J. B. Removal of artifact bias from qPCR results using DNA melting curve analysis.

Development of the Myocardial Interstitium.

The space between cardiac myocytes is commonly referred-to as the cardiac interstitium (CI). The CI is a unique, complex and dynamic microenvironment in which multiple cell types, extracellular matrix molecules, and instructive signals interact to crucially support heart homeostasis and promote cardiac responses to normal and pathologic stimuli. Despite the biomedical and clinical relevance of the CI, its detailed cellular structure remains to be elucidated. In this review, we will dissect the organization of the cardiac interstitium by following its changing cellular and molecular composition from embryonic developmental stages to adulthood, providing a systematic analysis of the biological components of the CI. The main goal of this review is to contribute to our understanding of the CI roles in health and disease. Anat Rec, 302:58-68, 2019. © 2018 Wiley Periodicals, Inc.

Hagfish and lamprey Hox genes reveal conservation of temporal colinearity in vertebrates.

Hox genes exert fundamental roles for proper regional specification along the main rostro-caudal axis of animal embryos. They are generally expressed in restricted spatial domains according to their position in the cluster (spatial colinearity)-a feature that is conserved across bilaterians. In jawed vertebrates (gnathostomes), the position in the cluster also determines the onset of expression of Hox genes (a feature known as whole-cluster temporal colinearity (WTC)), while in invertebrates this phenomenon is displayed as a subcluster-level temporal colinearity. However, little is known about the expression profile of Hox genes in jawless vertebrates (cyclostomes); therefore, the evolutionary origin of WTC, as seen in gnathostomes, remains a mystery. Here, we show that Hox genes in cyclostomes are expressed according to WTC during development. We investigated the Hox repertoire and Hox gene expression profiles in three different species-a hagfish, a lamprey and a shark-encompassing the two major groups of vertebrates, and found that these are expressed following a whole-cluster, temporally staggered pattern, indicating that WTC has been conserved during the past 500 million years despite drastically different genome evolution and morphological outputs between jawless and jawed vertebrates.

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR).

Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to Cq or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input. To explore the range of input variations that occur in the normal use of the Cre assay these conditions were mimicked in a complete two-way design of template -plasmid DNA- and non-template -mouse cDNA- concentrations. These experiments showed that the frequency of the amplification of the correct product and the artifact, as well as the valid quantification of the correct product, depended on the concentration of the non-template cDNA. This finding questions the interpretation of dilution series in which template as well as non-template concentrations are simultaneously decreasing. Repetition of this cDNA concentration experiment with other templates revealed that exact reproduction qPCR experiments was affected by the time it takes to complete the pipetting of a qPCR plate. Long bench times were observed to lead to significantly more artifacts. However, the measurement of artifact-associated fluorescence can be avoided by inclusion of a small heating step after the elongation phase in the amplification protocol. Taken together, this trouble-shooting journey showed that reliability and reproducibility of qPCR experiments not only depends on standardization and reporting of the biochemistry and technical aspects but also on hitherto neglected factors as sample dilution and waiting times in the laboratory work flow.

Reference genes for gene expression studies in the mouse heart.

To be accurate, quantitative Polymerase Chain Reaction (qPCR) studies require a set of stable reference genes for normalization. This is especially critical in cardiac research because of the diversity of the clinical and experimental conditions in the field. We analyzed the stability of previously described as potential reference genes in different subsets of cardiac tissues, each representing a different field in cardiac research. The qPCR dataset was based on 119 different tissue samples derived from cardiac development to pathology in mouse adult hearts. These samples were grouped into 47 tissue types. The stability of 9 candidate genes was analyzed in each of 12 experimental conditions comprising different groupings of these tissue types. Expression stability was determined with the geNorm module of qbase+. This analysis showed that different sets of two or three reference genes are required for analysis of qPCR data in different experimental conditions in murine cardiac research.

Extracardiac septum transversum/proepicardial endothelial cells pattern embryonic coronary arterio-venous connections.

Recent reports suggest that mammalian embryonic coronary endothelium (CoE) originates from the sinus venosus and ventricular endocardium. However, the contribution of extracardiac cells to CoE is thought to be minor and nonsignificant for coronary formation. Using classic (Wt1(Cre)) and previously undescribed (G2-Gata4(Cre)) transgenic mouse models for the study of coronary vascular development, we show that extracardiac septum transversum/proepicardium (ST/PE)-derived endothelial cells are required for the formation of ventricular coronary arterio-venous vascular connections. Our results indicate that at least 20% of embryonic coronary arterial and capillary endothelial cells derive from the ST/PE compartment. Moreover, we show that conditional deletion of the ST/PE lineage-specific Wilms' tumor suppressor gene (Wt1) in the ST/PE of G2-Gata4(Cre) mice and in the endothelium of Tie2(Cre) mice disrupts embryonic coronary transmural patterning, leading to embryonic death. Taken together, our results demonstrate that ST/PE-derived endothelial cells contribute significantly to and are required for proper coronary vascular morphogenesis.