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The lymphatic vasculature is a blind-ended network crucial for tissue-fluid homeostasis, immune surveillance and lipid absorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. By contrast, here we show that cardiac lymphatic vessels in mice have a heterogeneous cellular origin, whereby formation of at least part of the cardiac lymphatic network is independent of sprouting from veins. Multiple Cre– lox -based lineage tracing revealed a potential contribution from the putative haemogenic endothelium during development, and discrete lymphatic endothelial progenitor populations were confirmed by conditional knockout of Prox1 in Tie2 + and Vav1 + compartments. In the adult heart, myocardial infarction promoted a significant lymphangiogenic response, which was augmented by treatment with VEGF-C, resulting in improved cardiac function. These data prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and suggest that lymphangiogenesis may represent a therapeutic target to promote cardiac repair following injury. The lymphatic system is thought to be derived by transdifferentiation of venous endothelium; this study shows that the origin of cardiac lymphatics is in fact more heterogeneous, including both venous and non-venous origins and that lymphangiogenesis occurs in the adult heart following myocardial infarction and can be enhanced to improve heart function. Embryonic origins of lymphatic vessels The lymphatic endothelium wass thought to arise entirely from transdifferentiation of the venous endothelium. Two studies published in this issue of Nature demonstrate that the origins of this vasculature are more varied than anticipated. Karina Yaniv and colleagues use live imaging of transgenic zebrafish embryos to trace the lineage of individual lymphatic endothelial cells and their progeny. They find that lymphatic progenitors emerge from a previously uncharacterized pool of multipotent angioblasts located in the floor of the cardinal vein, which bears the potential to generate not only lymphatic, but also arterial and venous fates. Paul Riley and colleagues used genetic fate-mapping techniques in mice to demonstrate a dual origin of cardiac lymphatic endothelial cells, involving both venous endothelium and a separate non-venous progenitor source arising from yolk sac cells. They also show that the lymphangiogenesis in the heart can be influenced by myocardial infarction, and they suggest that promoting lymphangiogenesis with VEGF-C improves cardiac function after infarction

Myocardial infarction (MI) arising from obstruction of the coronary circulation engenders massive cardiomyocyte loss and replacement by non-contractile scar tissue, leading to pathological remodeling, dysfunction, and ultimately heart failure. This is presently a global health problem for which there is no effective cure. Following MI, the innate immune system directs the phagocytosis of dead cell debris in an effort to stimulate cell repopulation and tissue renewal. In the mammalian adult heart, however, the persistent influx of immune cells, coupled with the lack of an inherent regenerative capacity, results in cardiac fibrosis. Here, we reveal that stimulation of cardiac lymphangiogenesis with VEGF-C improves clearance of the acute inflammatory response after MI by trafficking immune cells to draining mediastinal lymph nodes (MLNs) in a process dependent on lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Deletion of Lyve1 in mice, preventing docking and transit of leukocytes through the lymphatic endothelium, results in exacerbation of chronic inflammation and long-term deterioration of cardiac function. Our findings support targeting of the lymphatic/immune cell axis as a therapeutic paradigm to promote immune modulation and heart repair.

Iron deficiency augments hypoxic pulmonary arterial pressure in healthy individuals and exacerbates pulmonary arterial hypertension (PAH) in patients, even without anemia. Conversely, iron supplementation has been shown to be beneficial in both settings. The mechanisms underlying the effects of iron availability are not known, due to lack of understanding of how cells of the pulmonary vasculature respond to changes in iron levels. The iron export protein ferroportin (FPN) and its antagonist peptide hepcidin control systemic iron levels by regulating release from the gut and spleen, the sites of absorption and recycling, respectively. We found FPN to be present in pulmonary arterial smooth muscle cells (PASMCs) and regulated by hepcidin cell autonomously. To interrogate the importance of this regulation, we generated mice with smooth muscle-specific knock in of the hepcidin-resistant isoform fpn C326Y. While retaining normal systemic iron levels, this model developed PAH and right heart failure as a consequence of intracellular iron deficiency and increased expression of the vasoconstrictor endothelin-1 (ET-1) within PASMCs. PAH was prevented and reversed by i.v. iron and by the ET receptor antagonist BQ-123. The regulation of ET-1 by iron was also demonstrated in healthy humans exposed to hypoxia and in PASMCs from PAH patients with mutations in bone morphogenetic protein receptor type II. Such mutations were further associated with dysregulation of the HAMP/FPN axis in PASMCs. This study presents evidence that intracellular iron deficiency specifically within PASMCs alters pulmonary vascular function. It offers a mechanistic underpinning for the known effects of iron availability in humans.

Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion ofFpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes ofFpnknockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.

The mdx mouse has proven to be useful in understanding the cardiomyopathy that frequently occurs in muscular dystrophy patients. Here we employed a comprehensive array of clinically relevant in vivo MRI techniques to identify early markers of cardiac dysfunction and follow disease progression in the hearts of mdx mice. Serial measurements of cardiac morphology and function were made in the same group of mdx mice and controls (housed in a non-SPF facility) using MRI at 1, 3, 6, 9 and 12 months after birth. Left ventricular (LV) and right ventricular (RV) systolic and diastolic function, response to dobutamine stress and myocardial fibrosis were assessed. RV dysfunction preceded LV dysfunction, with RV end systolic volumes increased and RV ejection fractions reduced at 3 months of age. LV ejection fractions were reduced at 12 months, compared with controls. An abnormal response to dobutamine stress was identified in the RV of mdx mice as early as 1 month. Late-gadolinium-enhanced MRI identified increased levels of myocardial fibrosis in 6, 9 and 12-month-old mdx mice, the extent of fibrosis correlating with the degree of cardiac remodeling and hypertrophy. MRI could identify cardiac abnormalities in the RV of mdx mice as young as 1 month, and detected myocardial fibrosis at 6 months. We believe these to be the earliest MRI measurements of cardiac function reported for any mice, and the first use of late-gadolinium-enhancement in a mouse model of congenital cardiomyopathy. These techniques offer a sensitive and clinically relevant in vivo method for assessment of cardiomyopathy caused by muscular dystrophy and other diseases.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable human cardiac cells to be studied in vitro, although they use glucose as their primary metabolic substrate and do not recapitulate the properties of adult cardiomyocytes. Here, we have explored the interplay between maturation by stimulation of fatty acid oxidation and by culture in 3D. We have investigated substrate metabolism in hiPSC-CMs grown as a monolayer and in 3D, in porous collagen-derived scaffolds and in engineered heart tissue (EHT), by measuring rates of glycolysis and glucose and fatty acid oxidation (FAO), and changes in gene expression and mitochondrial oxygen consumption. FAO was stimulated by activation of peroxisome proliferator-activated receptor alpha (PPARα), using oleate and the agonist WY-14643, which induced an increase in FAO in monolayer hiPSC-CMs. hiPSC-CMs grown in 3D on collagen-derived scaffolds showed reduced glycolysis and increased FAO compared with monolayer cells. Activation of PPARα further increased FAO in cells on collagen/elastin scaffolds but not collagen or collagen/chondroitin-4-sulphate scaffolds. In EHT, FAO was significantly higher than in monolayer cells or those on static scaffolds and could be further increased by culture with oleate and WY-14643. In conclusion, a more mature metabolic phenotype can be induced by culture in 3D and FAO can be incremented by pharmacological stimulation.

In humans, new-born infants can regenerate their heart during early life. This is modelled in the mouse, where regenerative capacity is maintained for the first week after birth but lost thereafter. Reactivation of this process holds great therapeutic potential; however, the molecular pathways that might be targeted to extend neonatal regeneration remain elusive. Here, we explored a role for hypoxia and HIF signalling on the regulation of epicardial activity in the developing mouse heart and in modulating the response to injury. Hypoxic regions were found in the epicardium from mid-gestation, associating with HIF-1α and HIF-2α, and expression of the epicardial master regulator Wilms’ tumour 1 (WT1). Epicardial deletion of Hif1α reduced WT1 levels, leading to impaired coronary vasculature. Targeting of the HIF degradation enzyme PHD, through pharmacological inhibition with a clinically approved drug or epicardial-specific genetic deletion of Egln1 , stabilised HIF and promoted WT1 activity ex vivo. Finally, a combination of genetic and pharmacological stabilisation of HIF during neonatal heart injury led to prolonged epicardial activation, preservation of myocardium, augmented infarct resolution and preserved function beyond the 7-day regenerative window. These findings suggest modulation of HIF signalling extends epicardial activation to maintain myocardial survival beyond the neonatal regenerative window and may represent a viable strategy for treating ischaemic heart disease.

To a) achieve cardiac 19F-Magnetic Resonance Imaging (MRI) of perfluoro-crown-ether (PFCE) labeled cardiac progenitor stem cells (CPCs) and bone-derived bone marrow macrophages, b) determine label concentration and cellular load limits, and c) achieve spectroscopic and image-based quantification. Theoretical simulations and experimental comparisons of spoiled-gradient echo (SPGR), rapid acquisition with relaxation enhancement (RARE), and steady state at free precession (SSFP) pulse sequences, and phantom validations, were conducted using 19F MRI/Magnetic Resonance Spectroscopy (MRS) at 9.4 T. Successful cell labeling was confirmed using flow cytometry and confocal microscopy. For CPC and macrophage concentration quantification, in vitro and post-mortem cardiac validations were pursued with the use of the transfection agent FuGENE. Feasibility of fast imaging is demonstrated in murine cardiac acquisitions in vivo, and in post-mortem murine skeletal and cardiac applications. SPGR/SSFP proved favorable imaging sequences yielding good signal-to-noise ratio values. Confocal microscopy confirmed heterogeneity of cellular label uptake in CPCs. 19F MRI indicated lack of additional benefits upon label concentrations above 7.5-10 mg/ml/million cells. The minimum detectable CPC load was ~500k (~10k/voxel) in two-dimensional (2D) acquisitions (3-5 min) using the butterfly coil. Additionally, absolute 19F based concentration and intensity estimates (trifluoroacetic-acid solutions, macrophages, and labeled CPCs in vitro and post-CPC injections in the post-mortem state) scaled linearly with fluorine concentrations. Fast, quantitative cardiac 19F-MRI was demonstrated with SPGR/SSFP and MRS acquisitions spanning 3-5 min, using a butterfly coil. The developed methodologies achieved in vivo cardiac 19F of exogenously injected labeled CPCs for the first time, accelerating imaging to a total acquisition of a few minutes, providing evidence for their potential for possible translational work.

Diabetes is a global epidemic, with cardiovascular disease being the leading cause of death in diabetic patients. There is a pressing need for an in vitro model to aid understanding of the mechanisms driving diabetic heart disease, and to provide an accurate, reliable tool for drug testing. Human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have potential as a disease modelling tool. There are several factors that drive molecular changes inside cardiomyocytes contributing to diabetic cardiomyopathy, including hyperglycaemia, lipotoxicity and hyperinsulinemia. Here we discuss these factors and how they can be seen in animal models and utilised in cell culture to mimic the diabetic heart. The use of human iPSC-CMs will allow for a greater understanding of disease pathogenesis and open up new avenues for drug testing.

19F MRI-based quantification in solutions and CPCs and determination of cellular detectability limit. 19F MR spectroscopy, image-based quantification, and sensitivity detection limits: (A, B) Axial 1H and 19F images from TFA phantoms of different concentrations (25–100 mM), and images of a multivial sensitivity phantom containing 0.25, 0.5, 0.75, and 1 million labeled/transfected CT cells suspended in media for sensitivity limit detection (cell pellets resided at the bottom of the Eppendorf tubes) using the butterfly coil. The linearity of the evoked fully relaxed spectral area versus cell number was independently confirmed using fast, direct, image-based SPGR using CPCs (butterfly coil) (results not shown). https://doi.org/10.1371/journal.pone.0225786.g002 S1 Appendix erroneously contains annotated comments. In accordance to basic MRI principles, SNR can be formulated as ) , , , , ( 2 1 ,  TE TR T T S T V SNT total acq   where Δ V is the sample volume, and T acq,total is the total data acquisition time considered as the time during which the data acquisition window is open during each readout (equal to the number of frequency encoding steps, N freq , divided by the receiver bandwidth (BW)) [1], BW N T N T freq sampling freq total acq   . , T 1 and T 2 the respective relaxation values, and θ is the flip angle. ) , , , , ( 2 1  TE TR T T S is a composite function that determines the dependence of the evoked signal on relaxation times and pulse sequence acquisition parameters.