Development of the human heart.

In 2014, an extensive review discussing the major steps of cardiac development focusing on growth, formation of primary and chamber myocardium and the development of the cardiac electrical system, was published. Molecular genetic lineage analyses have since furthered our insight in the developmental origin of the various component parts of the heart, which currently can be unambiguously identified by their unique molecular phenotype. Moreover, genetic, molecular and cell biological analyses have driven insights into the mechanisms underlying the development of the different cardiac components. Here, we build on our previous review and provide an insight into the molecular mechanistic revelations that have forwarded the field of cardiac development. Despite the enormous advances in our knowledge over the last decade, the development of congenital cardiac malformations remains poorly understood. The challenge for the next decade will be to evaluate the different developmental processes using newly developed molecular genetic techniques to further unveil the gene regulatory networks operational during normal and abnormal cardiac development.

The Role of the Epicardium During Heart Development and Repair.

The heart is lined by a single layer of mesothelial cells called the epicardium that provides important cellular contributions for embryonic heart formation. The epicardium harbors a population of progenitor cells that undergo epithelial-to-mesenchymal transition displaying characteristic conversion of planar epithelial cells into multipolar and invasive mesenchymal cells before differentiating into nonmyocyte cardiac lineages, such as vascular smooth muscle cells, pericytes, and fibroblasts. The epicardium is also a source of paracrine cues that are essential for fetal cardiac growth, coronary vessel patterning, and regenerative heart repair. Although the epicardium becomes dormant after birth, cardiac injury reactivates developmental gene programs that stimulate epithelial-to-mesenchymal transition; however, it is not clear how the epicardium contributes to disease progression or repair in the adult. In this review, we will summarize the molecular mechanisms that control epicardium-derived progenitor cell migration, and the functional contributions of the epicardium to heart formation and cardiomyopathy. Future perspectives will be presented to highlight emerging therapeutic strategies aimed at harnessing the regenerative potential of the fetal epicardium for cardiac repair.

Identification of Human Very Small Embryonic like Stem Cells (VSELS) in Human Heart Tissue Among Young and Old Individuals.

Very Small Embryonic-Like (VSEL) stem cells are a proposed pluripotent population, residing in adult tissues. VSELs have been described in multiple tissues including bone marrow, cord blood, and gonads. They exhibit multiple characteristics of embryonic stem cells including the ability to differentiate into cellular lineages of all three germ layers, including cardiomyocytes and vascular endothelial cells. However, their presence in adult solid organs such as heart in humans has not been established. VSELs are valuable source of stem cells for tissue regeneration and replacement of cells for turnover and usual wear-and-tear. The purpose of our study was to explore the existence of human VSELs (huVSELs) in human heart tissue and examine the changes in their prevalence with aging and cardiac disease. Human heart tissue, collected from healthy and ischemic heart disease subjects was examined for the prevalence of VSELS, defined as CD45-/CD133+/SSEA4+. Both epicardial and endocardial tissues were examined comparing VSEL numbers across different age groups. Our data confirm the existence of huVSELs in adult hearts with decreasing prevalence during aging. This is the first evidence of huVSELs in adult cardiac tissue. Cardiac huVSELs could be further explored in future studies to characterize their primitive potential and therapeutic potential in regenerative studies.

Transcriptomic evidence that cortisol alters perinatal epicardial adipose tissue maturation.

Cortisol administration during late gestation in ewes, modeling maternal stress, resulted in transcriptomic changes suggesting altered maturation and metabolic changes to the offspring heart. This study investigates the effects of cortisol on epicardial adipose tissue (EAT), a visceral fat pad associated with adverse cardiovascular conditions in adults. Pregnant ewes were treated with either 1 mg·kg-1·day-1 cortisol from 115 days gestation to term and EAT collected from term fetuses (control: n = 8, maternal cortisol 1 mg·kg-1·day-1: n = 6). To compare the effects of cortisol to the normal maturation in EAT, we also modeled the normal changes in gene expression in EAT at the transition from in utero to postnatal life using the EAT from control fetuses and from two-week-old lambs (control: n = 7). Transcriptomic modeling was used to identify pathways altered by maternal cortisol overexposure. Transcriptomic modeling confirmed the brown fat phenotype of EAT at term and a transition toward white fat at 2 wk of age in EAT of control fetuses/lambs and highlighted a role of immune responses, including complement coagulation, and serotonin in this transition. Maternal cortisol (1 mg·kg-1·day-1) increased the lipid peroxidation product 4-hydroxynonenal in EAT of term fetuses but did not affect the number of activated macrophages or size of the lipid droplets in the depot; transcriptomics suggested an earlier metabolic maturation of EAT via, in part, increased immune responses.

Differential roles of insulin like growth factor 1 receptor and insulin receptor during embryonic heart development.

BACKGROUND: The embryonic day E10-13 period of mouse heart development is characterized by robust cardiomyocyte proliferation that creates the compact zone of thickened ventricular wall myocardium. This process is initiated by the formation of the epicardium on the outer heart surface, which releases insulin-like growth factor 2 (IGF2) as the primary cardiomyocyte mitogen. Two receptors mediate IGF2 signaling, the IGF1R and the insulin receptor (INSR). RESULTS: In this study, we addressed the relative roles of the two IGF2 receptors in mouse heart development. We find that both receptors are expressed in the mouse heart during the E10-13 period, although IGF1R is much more prominently activated by IGF2 than INSR. Genetic manipulation indicates that only Igf1r is required for embryonic ventricular wall morphogenesis. INSR is not hyperactivated in the absence of IGF1R, and INSR does not compensate functionally for IGF1R in the absence of the latter. CONCLUSIONS: These results define the molecular components that are responsible for a major burst of cardiomyocyte proliferation during heart development. These results may also be relevant to understanding the efficiency of regeneration of the mammalian heart after neonatal and adult injury.

Does cardiac development provide heart research with novel therapeutic approaches?

Embryonic heart progenitors arise at specific spatiotemporal periods that contribute to the formation of distinct cardiac structures. In mammals, the embryonic and fetal heart is hypoxic by comparison to the adult heart. In parallel, the cellular metabolism of the cardiac tissue, including progenitors, undergoes a glycolytic to oxidative switch that contributes to cardiac maturation. While oxidative metabolism is energy efficient, the glycolytic-hypoxic state may serve to maintain cardiac progenitor potential. Consistent with this proposal, the adult epicardium has been shown to contain a reservoir of quiescent cardiac progenitors that are activated in response to heart injury and are hypoxic by comparison to adjacent cardiac tissues. In this review, we discuss the development and potential of the adult epicardium and how this knowledge may provide future therapeutic approaches for cardiac repair.

Head-to-head comparison of two engineered cardiac grafts for myocardial repair: From scaffold characterization to pre-clinical testing.

Cardiac tissue engineering, which combines cells and supportive scaffolds, is an emerging treatment for restoring cardiac function after myocardial infarction (MI), although, the optimal construct remains a challenge. We developed two engineered cardiac grafts, based on decellularized scaffolds from myocardial and pericardial tissues and repopulated them with adipose tissue mesenchymal stem cells (ATMSCs). The structure, macromechanical and micromechanical scaffold properties were preserved upon the decellularization and recellularization processes, except for recellularized myocardium micromechanics that was ∼2-fold stiffer than native tissue and decellularized scaffolds. Proteome characterization of the two acellular matrices showed enrichment of matrisome proteins and major cardiac extracellular matrix components, considerably higher for the recellularized pericardium. Moreover, the pericardial scaffold demonstrated better cell penetrance and retention, as well as a bigger pore size. Both engineered cardiac grafts were further evaluated in pre-clinical MI swine models. Forty days after graft implantation, swine treated with the engineered cardiac grafts showed significant ventricular function recovery. Irrespective of the scaffold origin or cell recolonization, all scaffolds integrated with the underlying myocardium and showed signs of neovascularization and nerve sprouting. Collectively, engineered cardiac grafts -with pericardial or myocardial scaffolds- were effective in restoring cardiac function post-MI, and pericardial scaffolds showed better structural integrity and recolonization capability.

Analysis of cardiomyocyte clonal expansion during mouse heart development and injury.

The cellular mechanisms driving cardiac tissue formation remain poorly understood, largely due to the structural and functional complexity of the heart. It is unclear whether newly generated myocytes originate from cardiac stem/progenitor cells or from pre-existing cardiomyocytes that re-enter the cell cycle. Here, we identify the source of new cardiomyocytes during mouse development and after injury. Our findings suggest that cardiac progenitors maintain proliferative potential and are the main source of cardiomyocytes during development; however, the onset of αMHC expression leads to reduced cycling capacity. Single-cell RNA sequencing reveals a proliferative, "progenitor-like" population abundant in early embryonic stages that decreases to minimal levels postnatally. Furthermore, cardiac injury by ligation of the left anterior descending artery was found to activate cardiomyocyte proliferation in neonatal but not adult mice. Our data suggest that clonal dominance of differentiating progenitors mediates cardiac development, while a distinct subpopulation of cardiomyocytes may have the potential for limited proliferation during late embryonic development and shortly after birth.

Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells.

Human pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (pro)epicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (pro)epicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (pro)epicardial-like cells displaying functional properties (adhesion and spreading over the myocardium) of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (pro)epicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration.

3D Printed Pericardium Hydrogels To Promote Wound Healing in Vascular Applications.

Vascular grafts that can support total replacement and maintenance by the body of the injured vessel would improve outcomes of major surgical reconstructions. Building scaffolds using components of the native vessel can encourage biological recognition by native cells as well as mimic mechanical characteristics of the native vessel. Evidence is emerging that incorporating predetermined building-blocks into a tissue engineering scaffold may oversimplify the environment and ignore critical structures and binding sites essential to development at the implant. We propose the development of a 3D-printable and degradable hybrid scaffold by combining polyethylene glycol (PEG)acrylate and homogenized pericardium matrix (HPM) to achieve appropriate biological environment as well as structural support. It was hypothesized that incorporation of HPM into PEG hydrogels would affect modulus of the scaffold and that the modulus and biological component would reduce the inflammatory signals produced from arriving macrophages and nearby endothelial cells. HPM was found to provide a number of tissue specific structural proteins including collagen, fibronectin, and glycosaminoglycans. HPM and PEGacrylate formed a hybrid hydrogel with significantly distinct modulus depending on concentration of either component, which resulted in scaffolds with stiffness between 0.5 and 20 kPa. The formed hybrid hydrogel was confirmed through a reduction in primary amines post-cross-linking. Using these hybrid scaffolds, rat bone marrow derived macrophages developed an M2 phenotype in response to low amounts (0.03%, w/v) of HPM in culture but responded with inflammatory phenotypes to high concentrations (0.3%, w/v). When cultured together with endothelial cells, both M1 and M2 macrophages were detected, along with a combination of both inflammatory and healing cytokines. However, the expression of inflammatory cytokines TNFα and IL1ß was significantly (p < 0.05) lower with hybrid hydrogels compared to single component PEG or HPM hydrogels. This reduction in inflammatory cytokines could impact the healing environment that persists at the implantation site. Finally, using this developed hybrid hydrogel, models of neonatal vasculature were manufactured using digital light projection (DLP) 3D printing. The structural control achieved with this novel biomaterial suggests a promising new tool in vascular graft development and research, with potential for complex structures for use in congenital heart defect reconstruction.

Non-muscle myosin IIB (Myh10) is required for epicardial function and coronary vessel formation during mammalian development.

The coronary vasculature is an essential vessel network providing the blood supply to the heart. Disruptions in coronary blood flow contribute to cardiac disease, a major cause of premature death worldwide. The generation of treatments for cardiovascular disease will be aided by a deeper understanding of the developmental processes that underpin coronary vessel formation. From an ENU mutagenesis screen, we have isolated a mouse mutant displaying embryonic hydrocephalus and cardiac defects (EHC). Positional cloning and candidate gene analysis revealed that the EHC phenotype results from a point mutation in a splice donor site of the Myh10 gene, which encodes NMHC IIB. Complementation testing confirmed that the Myh10 mutation causes the EHC phenotype. Characterisation of the EHC cardiac defects revealed abnormalities in myocardial development, consistent with observations from previously generated NMHC IIB null mouse lines. Analysis of the EHC mutant hearts also identified defects in the formation of the coronary vasculature. We attribute the coronary vessel abnormalities to defective epicardial cell function, as the EHC epicardium displays an abnormal cell morphology, reduced capacity to undergo epithelial-mesenchymal transition (EMT), and impaired migration of epicardial-derived cells (EPDCs) into the myocardium. Our studies on the EHC mutant demonstrate a requirement for NMHC IIB in epicardial function and coronary vessel formation, highlighting the importance of this protein in cardiac development and ultimately, embryonic survival.

Epithelial tension in the second heart field promotes mouse heart tube elongation.

Extension of the vertebrate heart tube is driven by progressive addition of second heart field (SHF) progenitor cells to the poles of the heart. Defects in this process cause a spectrum of congenital anomalies. SHF cells form an epithelial layer in splanchnic mesoderm in the dorsal wall of the pericardial cavity. Here we report oriented cell elongation, polarized actomyosin distribution and nuclear YAP/TAZ in a proliferative centre in the posterior dorsal pericardial wall during heart tube extension. These parameters are indicative of mechanical stress, further supported by analysis of cell shape changes in wound assays. Time course and mutant analysis identifies SHF deployment as a source of epithelial tension. Moreover, cell division and oriented growth in the dorsal pericardial wall align with the axis of cell elongation, suggesting that epithelial tension in turn contributes to heart tube extension. Our results implicate tissue-level forces in the regulation of heart tube extension.

Myc overexpression enhances of epicardial contribution to the developing heart and promotes extensive expansion of the cardiomyocyte population.

Myc is an essential regulator of cell growth and proliferation. Myc overexpression promotes the homeostatic expansion of cardiomyocyte populations by cell competition, however whether this applies to other cardiac lineages remains unknown. The epicardium contributes signals and cells to the developing and adult injured heart and exploring strategies for modulating its activity is of great interest. Using inducible genetic mosaics, we overexpressed Myc in the epicardium and determined the differential expansion of Myc-overexpressing cells with respect to their wild type counterparts. Myc-overexpressing cells overcolonized all epicardial-derived lineages and showed increased ability to invade the myocardium and populate the vasculature. We also found massive colonization of the myocardium by Wt1Cre-derived Myc-overexpressing cells, with preservation of cardiac development. Detailed analyses showed that this contribution is unlikely to derive from Cre activity in early cardiomyocytes but does not either derive from established epicardial cells, suggesting that early precursors expressing Wt1Cre originate the recombined cardiomyocytes. Myc overexpression does not modify the initial distribution of Wt1Cre-recombined cardiomyocytes, indicating that it does not stimulate the incorporation of early expressing Wt1Cre lineages to the myocardium, but differentially expands this initial population. We propose that strategies using epicardial lineages for heart repair may benefit from promoting cell competitive ability.

The roadmap of WT1 protein expression in the human fetal heart.

The transcription factor Wilms' Tumor-1 (WT1) is essential for cardiac development. Deletion of Wt1 in mice results in disturbed epicardial and myocardial formation and lack of cardiac vasculature, causing embryonic lethality. Little is known about the role of WT1 in the human fetal heart. Therefore, as a first step, we analyzed the expression pattern of WT1 protein during human cardiac development from week 4 till week 20. WT1 expression was apparent in epicardial, endothelial and endocardial cells in a spatiotemporal manner. The expression of WT1 follows a pattern starting at the epicardium and extending towards the lumen of the heart, with differences in timing and expression levels between the atria and ventricles. The expression of WT1 in cardiac arterial endothelial cells reduces in time, whereas WT1 expression in the endothelial cells of cardiac veins and capillaries remains present at all stages studied. This study provides for the first time a detailed description of the expression of WT1 protein during human cardiac development, which indicates an important role for WT1 also in human cardiogenesis.

Bioengineered FSTL1 Patches Restore Cardiac Function Following Myocardial Infarction.

Improving the limited ability of the heart to regenerate after infarction is crucial. Researchers now demonstrate that delivery of follistatin-like 1 (FSTL1) into injured hearts via collagen patches stimulates cardiomyocyte proliferation and cardiac functional recovery. These findings highlight the epicardium as a source of novel regenerative factors and biomimetic nanomaterials in cardiac translational medicine.

Epicardial FSTL1 reconstitution regenerates the adult mammalian heart.

The elucidation of factors that activate the regeneration of the adult mammalian heart is of major scientific and therapeutic importance. Here we found that epicardial cells contain a potent cardiogenic activity identified as follistatin-like 1 (Fstl1). Epicardial Fstl1 declines following myocardial infarction and is replaced by myocardial expression. Myocardial Fstl1 does not promote regeneration, either basally or upon transgenic overexpression. Application of the human Fstl1 protein (FSTL1) via an epicardial patch stimulates cell cycle entry and division of pre-existing cardiomyocytes, improving cardiac function and survival in mouse and swine models of myocardial infarction. The data suggest that the loss of epicardial FSTL1 is a maladaptive response to injury, and that its restoration would be an effective way to reverse myocardial death and remodelling following myocardial infarction in humans.

Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation.

RATIONALE: Fibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. OBJECTIVE: We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. METHODS AND RESULTS: We showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. CONCLUSIONS: The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

The epicardium signals the way towards heart regeneration.

From historical studies of developing chick hearts to recent advances in regenerative injury models, the epicardium has arisen as a key player in heart genesis and repair. The epicardium provides paracrine signals to nurture growth of the developing heart from mid-gestation, and epicardium-derived cells act as progenitors of numerous cardiac cell types. Interference with either process is terminal for heart development and embryogenesis. In adulthood, the dormant epicardium reinstates an embryonic gene programme in response to injury. Furthermore, injury-induced epicardial signalling is essential for heart regeneration in zebrafish. Given these critical roles in development, injury response and heart regeneration, the application of epicardial signals following adult heart injury could offer therapeutic strategies for the treatment of ischaemic heart disease and heart failure.