Protocol for the isolation and single-nuclei multiomic analyses of the human fetal epicardium.

The characterization of cell populations that reside in the outer layer of the heart has been hindered by difficulties in their isolation. Here, we present a protocol for isolation and single-nuclei multiomic analyses of the human fetal epicardium. We describe steps for microdissection, isolation, and enrichment of epicardial cells by mechanical dissociations and direct lysis. We then detail procedures for integrating transcriptome and chromatin accessibility datasets. This approach allows the analysis of diverse cell populations, marked by unique cis-regulatory elements. For complete details on the use and execution of this protocol, please refer to Travisano et al.1.

Methods for dynamic and whole volume imaging of the zebrafish heart.

Tissue development and regeneration are dynamic processes involving complex cell migration and cell-cell interactions. We have developed a protocol for complementary time-lapse and three-dimensional (3D) imaging of tissue for developmental and regeneration studies which we apply here to the zebrafish cardiac vasculature. 3D imaging of fixed specimens is used to first define the subject at high resolution then live imaging captures how it changes dynamically. Hearts from adult and juvenile zebrafish are extracted and cleaned in preparation for the different imaging modalities. For whole-mount 3D confocal imaging, single or multiple hearts with native fluorescence or immuno-labeling are prepared for stabilization or clearing, and then imaged. For live imaging, hearts are placed in a prefabricated fluidic device and set on a temperature-controlled microscope for culture and imaging over several days. This protocol allows complete visualization of morphogenic processes in a 3D context and provides the ability to follow cell behaviors to complement in vivo and fixed tissue studies. This culture and imaging protocol can be applied to different cell and tissue types. Here, we have used it to observe zebrafish coronary vasculature and the migration of coronary endothelial cells during heart regeneration.

Single-nuclei multiomic analyses identify human cardiac lymphatic endothelial cells associated with coronary arteries in the epicardium.

Cardiac lymphatic vessels play important roles in fluid homeostasis, inflammation, disease, and regeneration of the heart. The developing cardiac lymphatics in human fetal hearts are closely associated with coronary arteries, similar to those in zebrafish hearts. We identify a population of cardiac lymphatic endothelial cells (LECs) that reside in the epicardium. Single-nuclei multiomic analysis of the human fetal heart reveals the plasticity and heterogeneity of the cardiac endothelium. Furthermore, we find that VEGFC is highly expressed in arterial endothelial cells and epicardium-derived cells, providing a molecular basis for the arterial association of cardiac lymphatic development. Using a cell-type-specific integrative analysis, we identify a population of cardiac lymphatic endothelial cells marked by the PROX1 and the lymphangiocrine RELN and enriched in binding motifs of erythroblast transformation specific (ETS) variant (ETV) transcription factors. We report the in vivo molecular characterization of human cardiac lymphatics and provide a valuable resource to understand fetal heart development.

Craniofacial and cardiac defects in chd7 zebrafish mutants mimic CHARGE syndrome.

Congenital heart defects occur in almost 80% of patients with CHARGE syndrome, a sporadically occurring disease causing craniofacial and other abnormalities due to mutations in the CHD7 gene. Animal models have been generated to mimic CHARGE syndrome; however, heart defects are not extensively described in zebrafish disease models of CHARGE using morpholino injections or genetic mutants. Here, we describe the co-occurrence of craniofacial abnormalities and heart defects in zebrafish chd7 mutants. These mutant phenotypes are enhanced in the maternal zygotic mutant background. In the chd7 mutant fish, we found shortened craniofacial cartilages and extra cartilage formation. Furthermore, the length of the ventral aorta is altered in chd7 mutants. Many CHARGE patients have aortic arch anomalies. It should be noted that the aberrant branching of the first branchial arch artery is observed for the first time in chd7 fish mutants. To understand the cellular mechanism of CHARGE syndrome, neural crest cells (NCCs), that contribute to craniofacial and cardiovascular tissues, are examined using sox10:Cre lineage tracing. In contrast to its function in cranial NCCs, we found that the cardiac NCC-derived mural cells along the ventral aorta and aortic arch arteries are not affected in chd7 mutant fish. The chd7 fish mutants we generated recapitulate some of the craniofacial and cardiovascular phenotypes found in CHARGE patients and can be used to further determine the roles of CHD7.

Intramyocardial Injection for the Study of Cardiac Lymphatic Function in Zebrafish.

Zebrafish have proved to be an important model for studying cardiovascular formation and function during postembryonic development and regeneration. The present protocol describes a method for injecting fluorescent tracers into the zebrafish myocardium to study interstitial fluid and debris uptake into cardiac lymphatic vessels. To do so, microspheres (200 nm diameter) and quantum dots (<10 nm diameter) are introduced into the myocardium of live zebrafish, which can be tracked using ex vivo confocal microscopy. These tracers are then tracked intermittently over several hours to follow clearance from the myocardium into cardiac lymphatic vessels. Quantum dots are transported through cardiac lymphatic vessels away from the heart, while larger microspheres remain at the injection site for over three weeks. This method of intramyocardial injection can be extended to other uses, including the injection of encapsulated MS or hydrogels to locally release cells, proteins, or compounds of interest to a targeted region of the heart.

Heterogeneous pdgfrb+ cells regulate coronary vessel development and revascularization during heart regeneration.

Endothelial cells emerge from the atrioventricular canal to form coronary blood vessels in juvenile zebrafish hearts. We find that pdgfrb is first expressed in the epicardium around the atrioventricular canal and later becomes localized mainly in the mural cells. pdgfrb mutant fish show severe defects in mural cell recruitment and coronary vessel development. Single-cell RNA sequencing analyses identified pdgfrb+ cells as epicardium-derived cells (EPDCs) and mural cells. Mural cells associated with coronary arteries also express cxcl12b and smooth muscle cell markers. Interestingly, these mural cells remain associated with coronary arteries even in the absence of Pdgfrß, although smooth muscle gene expression is downregulated. We find that pdgfrb expression dynamically changes in EPDCs of regenerating hearts. Differential gene expression analyses of pdgfrb+ EPDCs and mural cells suggest that they express genes that are important for regeneration after heart injuries. mdka was identified as a highly upregulated gene in pdgfrb+ cells during heart regeneration. However, pdgfrb but not mdka mutants show defects in heart regeneration after amputation. Our results demonstrate that heterogeneous pdgfrb+ cells are essential for coronary development and heart regeneration.

GLUT1 overexpression enhances glucose metabolism and promotes neonatal heart regeneration.

The mammalian heart switches its main metabolic substrate from glucose to fatty acids shortly after birth. This metabolic switch coincides with the loss of regenerative capacity in the heart. However, it is unknown whether glucose metabolism regulates heart regeneration. Here, we report that glucose metabolism is a determinant of regenerative capacity in the neonatal mammalian heart. Cardiac-specific overexpression of Glut1, the embryonic form of constitutively active glucose transporter, resulted in an increase in glucose uptake and concomitant accumulation of glycogen storage in postnatal heart. Upon cryoinjury, Glut1 transgenic hearts showed higher regenerative capacity with less fibrosis than non-transgenic control hearts. Interestingly, flow cytometry analysis revealed two distinct populations of ventricular cardiomyocytes: Tnnt2-high and Tnnt2-low cardiomyocytes, the latter of which showed significantly higher mitotic activity in response to high intracellular glucose in Glut1 transgenic hearts. Metabolic profiling shows that Glut1-transgenic hearts have a significant increase in the glucose metabolites including nucleotides upon injury. Inhibition of the nucleotide biosynthesis abrogated the regenerative advantage of high intra-cardiomyocyte glucose level, suggesting that the glucose enhances the cardiomyocyte regeneration through the supply of nucleotides. Our data suggest that the increase in glucose metabolism promotes cardiac regeneration in neonatal mouse heart.

The Lymphatic System in Zebrafish Heart Development, Regeneration and Disease Modeling.

Heart disease remains the single largest cause of death in developed countries, and novel therapeutic interventions are desperately needed to alleviate this growing burden. The cardiac lymphatic system is the long-overlooked counterpart of the coronary blood vasculature, but its important roles in homeostasis and disease are becoming increasingly apparent. Recently, the cardiac lymphatic vasculature in zebrafish has been described and its role in supporting the potent regenerative response of zebrafish heart tissue investigated. In this review, we discuss these findings in the wider context of lymphatic development, evolution and the promise of this system to open new therapeutic avenues to treat myocardial infarction and other cardiopathologies.

Apical Resection and Cryoinjury of Neonatal Mouse Heart.

Neonatal mouse hearts have a regenerative capacity similar to adult zebrafish. Different cardiac injury models have been established to investigate the regenerative capacity of neonatal mouse hearts, including ventricular amputation, cryoinjury, and ligation of a major coronary artery. While the ventricular resection model can be utilized to study how tissue forms and regenerates de novo, cryoinjury and coronary artery ligation are methods that might better mimic myocardial infarction by creating tissue damage and necrosis as opposed to the removal of healthy tissue in the ventricular amputation model. Here we describe methods of creating ventricular resection and cardiac cryoinjury in newborn mice.

Mononuclear diploid cardiomyocytes support neonatal mouse heart regeneration in response to paracrine IGF2 signaling.

Injury to the newborn mouse heart is efficiently regenerated, but this capacity is lost by one week after birth. We found that IGF2, an important mitogen in heart development, is required for neonatal heart regeneration. IGF2 originates from the endocardium/endothelium and is transduced in cardiomyocytes by the insulin receptor. Following injury on postnatal day 1, absence of IGF2 abolished injury-induced cell cycle entry during the early part of the first postnatal week. Consequently, regeneration failed despite the later presence of additional cell cycle-inducing activities 7 days following injury. Most cardiomyocytes transition from mononuclear diploid to polyploid during the first postnatal week. Regeneration was rescued in Igf2-deficient neonates in three different contexts that elevate the percentage of mononuclear diploid cardiomyocytes beyond postnatal day 7. Thus, IGF2 is a paracrine-acting mitogen for heart regeneration during the early postnatal period, and IGF2-deficiency unmasks the dependence of this process on proliferation-competent mononuclear diploid cardiomyocytes.

Extended culture and imaging of normal and regenerating adult zebrafish hearts in a fluidic device.

Myocardial infarction and heart failure are leading causes of death worldwide, in large part because adult human myocardium has extremely limited regeneration capacity. Zebrafish are a powerful model for identifying new strategies for human cardiac repair because their hearts regenerate after relatively severe injuries. Zebrafish are also relatively scalable and compatible with many genetic tools. However, characterizing the regeneration process in live adult zebrafish hearts has proved challenging because adult fish are opaque, preventing live imaging in vivo. An alternative strategy is to explant and culture intact adult zebrafish hearts and investigate them ex vivo. However, explanted hearts maintained in conventional culture conditions experience rapid declines in morphology and physiology. To overcome these limitations, we designed and fabricated a fluidic device for culturing explanted adult zebrafish hearts with constant media perfusion that is also compatible with live imaging. We then compared the morphology and calcium activity of hearts cultured in the device, hearts cultured statically in dishes, and freshly explanted hearts. After one week of culture, hearts in the device experienced significantly less morphological degradation compared to hearts cultured in dishes. Hearts cultured in devices for one week also maintained capture rates similar to fresh hearts, unlike hearts cultured in dishes. We then cultured explanted injured hearts in the device and used live imaging techniques to continuously record the myocardial revascularization process over several days, demonstrating how our device is compatible with long-term live imaging and thereby enables unprecedented visual access to the multi-day process of adult zebrafish heart regeneration.

Late developing cardiac lymphatic vasculature supports adult zebrafish heart function and regeneration.

The cardiac lymphatic vascular system and its potentially critical functions in heart patients have been largely underappreciated, in part due to a lack of experimentally accessible systems. We here demonstrate that cardiac lymphatic vessels develop in young adult zebrafish, using coronary arteries to guide their expansion down the ventricle. Mechanistically, we show that in cxcr4a mutants with defective coronary artery development, cardiac lymphatic vessels fail to expand onto the ventricle. In regenerating adult zebrafish hearts the lymphatic vasculature undergoes extensive lymphangiogenesis in response to a cryoinjury. A significant defect in reducing the scar size after cryoinjury is observed in zebrafish with impaired Vegfc/Vegfr3 signaling that fail to develop intact cardiac lymphatic vessels. These results suggest that the cardiac lymphatic system can influence the regenerative potential of the myocardium.

Monitoring of Adult Zebrafish Heart Regeneration Using High-Frequency Ultrasound Spectral Doppler and Nakagami Imaging.

This paper reports the feasibility of Nakagami imaging in monitoring the regeneration process of zebrafish hearts in a noninvasive manner. In addition, spectral Doppler waveforms that are typically used to access the diastolic function were measured to validate the performance of Nakagami imaging. A 30-MHz high-frequency ultrasound array transducer was used to acquire backscattered echo signal for spectral Doppler and Nakagami imaging. The performances of both methods were validated with flow and tissue-mimicking phantom experiments. For in vivo experiments, both spectral Doppler and Nakagami imaging were simultaneously obtained from adult zebrafish with amputated hearts. Longitudinal measurements were performed for five zebrafish. From the experiments, the E/A ratio measured using spectral Doppler imaging increased at 3 days post-amputation (3 dpa) and then decreased to the value before amputation, which were consistent with previous studies. Similar results were obtained from the Nakagami imaging where the Nakagami parameter value increased at 3 dpa and decreased to its original value. These results suggested that the Nakagami and spectral Doppler imaging would be useful techniques in monitoring the regeneration of heart or tissues.

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.

Coronary Vasculature in Cardiac Development and Regeneration.

Functional coronary circulation is essential for a healthy heart in warm-blooded vertebrates, and coronary diseases can have a fatal consequence. Despite the growing interest, the knowledge about the coronary vessel development and the roles of new coronary vessel formation during heart regeneration is still limited. It is demonstrated that early revascularization is required for efficient heart regeneration. In this comprehensive review, we first describe the coronary vessel formation from an evolutionary perspective. We further discuss the cell origins of coronary endothelial cells and perivascular cells and summarize the critical signaling pathways regulating coronary vessel development. Lastly, we focus on the current knowledge about the molecular mechanisms regulating heart regeneration in zebrafish, a genetically tractable vertebrate model with a regenerative adult heart and well-developed coronary system.

Tunable assembly of protein-microdomains in living vertebrate embryos.

Subcellular events such as trafficking and signaling are regulated by self-assembled protein complexes inside the cell. The ability to rapidly and reversibly manipulate these protein complexes would likely enhance studies of their mechanisms and their roles in biological function and disease manifestation.[1, 2] This manuscript reports that thermally-responsive elastin-like polypeptides (ELPs) linked to fluorescent proteins can regulate the self-assembly and disassembly of protein microdomains within the individual cells of zebrafish embryos. By exploring a library of fluorescent ELP proteins, this reports demonstrates that ELPs can co-assemble different fluorescent proteins inside of embryos. By tuning ELP length and sequence, fluorescent protein microdomains can be assembled at different temperatures, in varying sizes, or for desired periods of time. For the first time in a multicellular living embryo, these studies demonstrate that temperature-mediated ELP assembly can reversibly manipulate assembly of subcellular protein complexes, which may have applications in the study and manipulation of in vivo biological functions.

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration.

Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.

Short bowel syndrome results in increased gene expression associated with proliferation, inflammation, bile acid synthesis and immune system activation: RNA sequencing a zebrafish SBS model.

BACKGROUND: Much of the morbidity associated with short bowel syndrome (SBS) is attributed to effects of decreased enteral nutrition and administration of total parenteral nutrition (TPN). We hypothesized that acute SBS alone has significant effects on gene expression beyond epithelial proliferation, and tested this in a zebrafish SBS model. METHODS: In a model of SBS in zebrafish (laparotomy, proximal stoma, distal ligation, n = 29) or sham (laparotomy alone, n = 28) surgery, RNA-Seq was performed after 2 weeks. The proximal intestine was harvested and RNA isolated. The three samples from each group with the highest amount of RNA were spiked with external RNA controls consortium (ERCC) controls, sequenced and aligned to reference genome with gene ontology (GO) enrichment analysis performed. Gene expression of ctnnb1, ccnb1, ccnd1, cyp7a1a, dkk3, ifng1-2, igf2a, il1b, lef1, nos2b, saa1, stat3, tnfa and wnt5a were confirmed to be elevated in SBS by RT-qPCR. RESULTS: RNA-seq analysis identified 1346 significantly upregulated genes and 678 significantly downregulated genes in SBS zebrafish intestine compared to sham with Ingenuity analysis. The upregulated genes were involved in cell proliferation, acute phase response signaling, innate and adaptive immunity, bile acid regulation, production of nitric oxide and reactive oxygen species, cellular barrier and coagulation. The downregulated genes were involved in folate synthesis, gluconeogenesis, glycogenolysis, fatty-acid oxidation and activation and drug and steroid metabolism. RT-qPCR confirmed gene expression differences from RNA-Sequencing. CONCLUSION: Changes of gene expression after 2 weeks of SBS indicate complex and extensive alterations of multiple pathways, some previously implicated as effects of TPN. The systemic sequelae of SBS alone are significant and indicate multiple targets for investigating future therapies.

Epidermal growth factor suppresses intestinal epithelial cell shedding through a MAPK-dependent pathway.

Cell shedding from the intestinal villus is a key element of tissue turnover that is essential to maintain health and homeostasis. However, the signals regulating this process are not well understood. We asked whether shedding is controlled by epidermal growth factor receptor (EGFR), an important driver of intestinal growth and differentiation. In 3D ileal enteroid culture and cell culture models (MDCK, IEC-6 and IPEC-J2 cells), extrusion events were suppressed by EGF, as determined by direct counting of released cells or rhodamine-phalloidin labeling of condensed actin rings. Blockade of the MEK-ERK pathway, but not other downstream pathways such as phosphoinositide 3-kinase (PI3K) or protein kinase C (PKC), reversed EGF inhibition of shedding. These effects were not due to a change in cell viability. Furthermore, EGF-driven MAPK signaling inhibited both caspase-independent and -dependent shedding pathways. Similar results were found in vivo, in a novel zebrafish model for intestinal epithelial shedding. Taken together, the data show that EGF suppresses cell shedding in the intestinal epithelium through a selective MAPK-dependent pathway affecting multiple extrusion mechanisms. EGFR signaling might be a therapeutic target for disorders featuring excessive cell turnover, such as inflammatory bowel diseases.