Lucile Miquerol

Macrophages Facilitate Electrical Conduction in the Heart

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Thymosin β4 facilitates epicardial neovascularization of the injured adult heart

Ischemic heart disease complicated by coronary artery occlusion causes myocardial infarction (MI), which is the major cause of morbidity and mortality in humans (http://www.who.int/cardiovascular_diseases/resources/atlas/en/index.html). After MI the human heart has an impaired capacity to regenerate and, despite the high prevalence of cardiovascular disease worldwide, there is currently only limited insight into how to stimulate repair of the injured adult heart from its component parts. Efficient cardiac regeneration requires the replacement of lost cardiomyocytes, formation of new coronary blood vessels, and appropriate modulation of inflammation to prevent maladaptive remodeling, fibrosis/scarring, and consequent cardiac dysfunction. Here we show that thymosin β4 (Tβ4) promotes new vasculature in both the intact and injured mammalian heart. We demonstrate that limited EPDC‐derived endothelial‐restricted neovascularization constitutes suboptimal “endogenous repair,” following injury, which is significantly augmented by Tβ4 to increase and stabilize the vascular plexus via collateral vessel growth. As such, we identify Tβ4 as a facilitator of cardiac neovascularization and highlight adult EPDCs as resident progenitors which, when instructed by Tβ4, have the capacity to sustain the myocardium after ischemic damage.

Thymosins in Health and Disease: 2nd International Symposium

Ischemic heart disease complicated by coronary artery occlusion causes myocardial infarction (MI), which is the major cause of morbidity and mortality in humans (http://www.who.int/cardiovascular_diseases/resources/atlas/en/index.html). After MI the human heart has an impaired capacity to regenerate and, despite the high prevalence of cardiovascular disease worldwide, there is currently only limited insight into how to stimulate repair of the injured adult heart from its component parts. Efficient cardiac regeneration requires the replacement of lost cardiomyocytes, formation of new coronary blood vessels, and appropriate modulation of inflammation to prevent maladaptive remodeling, fibrosis/scarring, and consequent cardiac dysfunction. Here we show that thymosin β4 (Tβ4) promotes new vasculature in both the intact and injured mammalian heart. We demonstrate that limited EPDC‐derived endothelial‐restricted neovascularization constitutes suboptimal “endogenous repair,” following injury, which is significantly augmented by Tβ4 to increase and stabilize the vascular plexus via collateral vessel growth. As such, we identify Tβ4 as a facilitator of cardiac neovascularization and highlight adult EPDCs as resident progenitors which, when instructed by Tβ4, have the capacity to sustain the myocardium after ischemic damage.

Organogenesis of the vertebrate heart

Organogenesis of the vertebrate heart involves a complex sequence of events initiating with specification and differentiation of myocardial and endocardial cells in anterior lateral mesoderm shortly after gastrulation, followed by formation and rightward looping of the early heart tube. During looping, the heart tube elongates by addition of second heart field progenitor cells from adjacent pharyngeal mesoderm at the arterial and venous poles. Progressive differentiation is controlled by intercellular signaling events between pharyngeal mesoderm, foregut endoderm, and neural crest‐derived mesenchyme. Regulated patterns of myocardial gene expression and proliferation within the embryonic heart drive morphogenesis of atrial and ventricular chambers, while cardiac cushions, precursors of the definitive valves, form in the atrioventricular and outflow regions. In amniotes, separate systemic and pulmonary circulatory systems arise by septation and remodeling events that divide the atria and ventricles into left and right chambers. Cardiac neural crest cells play a key role in dividing the arterial pole of the heart into the ascending aorta and pulmonary trunk. During the remodeling phase the definitive cardiac conduction system, that coordinates the heartbeat, is established. In addition, the epicardium, critical for regulated ventricular growth and development of the coronary vasculature, spreads over the surface of the heart as an epithelium from which cells invade the myocardium to give rise to diverse cell types including fibroblasts and smooth muscle cells. Cardiogenesis thus involves highly coordinated development of multiple cell types and insight into the different lineage contributions and molecular regulation of each of these steps is expanding rapidly. WIREs Dev Biol 2013, 2:17–29. doi: 10.1002/wdev.68

The effect of connexin40 deficiency on ventricular conduction system function during development

AIMS The aim of this study was to characterize ventricular activation patterns in normal and connexin40-deficient mice in order to dissect the role of connexin40 in developing the conduction system. METHODS AND RESULTS We performed optical mapping of epicardial activation between ED9.5-18.5 and analysed ventricular activation patterns and times of left ventricular activation. Mouse embryos deficient for connexin40 were compared with normal and heterozygous littermates. Morphology of the primary interventricular ring (PIR) was delineated with the help of T3-LacZ transgene. Four major types of ventricular activation patterns characterized by primary breakthrough in different parts of the heart were detected during development: PIR, left ventricular apex, right ventricular apex, and dual right and left ventricular apices. Activation through PIR was frequently present at the early stages until ED12.5. From ED14.5, the majority of hearts showed dual left and right apical breakthrough, suggesting functionality of both bundle branches. Connexin40-deficient embryos showed initially a delay in left bundle branch function, but the right bundle branch block, previously described in the adults, was not detected in ED14.5 embryos and appeared only gradually with 80% penetrance at ED18.5. CONCLUSION The switch of function from the early PIR conduction pathway to the mature apex to base activation is dependent upon upregulation of connexin40 expression in the ventricular trabeculae. The early function of right bundle branch does not depend on connexin40. Quantitative analysis of normal mouse embryonic ventricular conduction patterns will be useful for interpretation of effects of mutations affecting the function of the cardiac conduction system.

Epistatic Rescue of Nkx2.5 Adult Cardiac Conduction Disease Phenotypes by Prospero-Related Homeobox Protein 1 and HDAC3

Rationale: Nkx2.5 is one of the most widely studied cardiac-specific transcription factors, conserved from flies to man, with multiple essential roles in both the developing and adult heart. Specific dominant mutations in NKX2.5 have been identified in adult congenital heart disease patients presenting with conduction system anomalies and recent genome-wide association studies implicate the NKX2.5 locus, as causative for lethal arrhythmias (“sudden cardiac death”) that occur at a frequency in the population of 1 in 1000 per annum worldwide. Haploinsufficiency for Nkx2.5 in the mouse phenocopies human conduction disease pathology yet the phenotypes, described in both mouse and man, are highly pleiotropic, implicit of unknown modifiers and/or factors acting in epistasis with Nkx2.5/NKX2.5. Objective: To identify bone fide upstream genetic modifier(s) of Nkx2.5/NKX2.5 function and to determine epistatic effects relevant to the manifestation of NKX2.5-dependent adult congenital heart disease. Methods and Results: A study of cardiac function in prospero-related homeobox protein 1 (Prox1) heterozygous mice, using pressure-volume loop and micromannometry, revealed rescue of hemodynamic parameters in Nkx2.5Cre/+; Prox1loxP/+ animals versus Nkx2.5Cre/+ controls. Anatomic studies, on a Cx40EGFP background, revealed Cre-mediated knock-down of Prox1 restored the anatomy of the atrioventricular node and His-Purkinje network both of which were severely hypoplastic in Nkx2.5Cre/+ littermates. Steady state surface electrocardiography recordings and high-speed multiphoton imaging, to assess Ca2+ handling, revealed atrioventricular conduction and excitation-contraction were also normalized by Prox1 haploinsufficiency, as was expression of conduction genes thought to act downstream of Nkx2.5. Chromatin immunoprecipitation on adult hearts, in combination with both gain and loss-of-function reporter assays in vitro, revealed that Prox1 recruits the corepressor HDAC3 to directly repress Nkx2.5 via a proximal upstream enhancer as a mechanism for regulating Nkx2.5 function in adult cardiac conduction. Conclusions: Here we identify Prox1 as a direct upstream modifier of Nkx2.5 in the maintenance of the adult conduction system and rescue of Nkx2.5 conduction disease phenotypes. This study is the first example of rescue of Nkx2.5 function and establishes a model for ensuring electrophysiological function within the adult heart alongside insight into a novel Prox1-HDAC3-Nkx2.5 signaling pathway for therapeutic targeting in conduction disease.

Endothelial Plasticity Drives Arterial Remodeling Within the Endocardium After Myocardial Infarction

RATIONALE Revascularization of injured, ischemic, and regenerating organs is essential to restore organ function. In the postinfarct heart, however, the mechanisms underlying the formation of new coronary arteries are poorly understood. OBJECTIVE To study vascular remodeling of coronary arteries after infarction. METHODS AND RESULTS We performed permanent left coronary ligation on Connexin40-GFP mice expressing green fluorescent protein (GFP) in endothelial cells of coronary arteries but not veins, capillaries, or endocardium. GFP(+) endothelial foci were identified within the endocardium in the infarct zone. These previously undescribed structures, termed endocardial flowers, have a distinct endothelial phenotype (Cx40(+), VEGFR2(+), and endoglin(-)) to the surrounding endocardium (Cx40(-), VEGFR2(-), and endoglin(+)). Endocardial flowers are contiguous with coronary vessels and associated with subendocardial smooth muscle cell accumulation. Genetic lineage tracing reveals extensive endothelial plasticity in the postinfarct heart, showing that endocardial flowers develop by arteriogenesis of Cx40(-) cells and by outgrowth of pre-existing coronary arteries. Finally, endocardial flowers exhibit angiogenic features, including early VEGFR2 expression and active proliferation of adjacent endocardial and smooth muscle cells. CONCLUSIONS Arterial endothelial foci within the endocardium reveal extensive endothelial cell plasticity in the infarct zone and identify the endocardium as a site of endogenous arteriogenesis and source of endothelial cells to promote vascularization in regenerative strategies.

Resolving cell lineage contributions to the ventricular conduction system with a Cx40-GFP allele: A dual contribution of the first and second heart fields

Background: The ventricular conduction system (VCS) coordinates the heartbeat and is composed of central components (the atrioventricular node, bundle, and right and left bundle branches) and a peripheral Purkinje fiber network. Conductive myocytes develop from common progenitor cells with working myocytes in a bimodal process of lineage restriction followed by limited outgrowth. The lineage relationship between progenitor cells giving rise to different components of the VCS is unclear. Results: Cell lineage contributions to different components of the VCS were analysed by a combination of retrospective clonal analysis, regionalized transgene expression studies, and genetic tracing experiments using Connexin40‐GFP mice that precisely delineate the VCS. Analysis of a library of hearts containing rare large clusters of clonally related myocytes identifies two VCS lineages encompassing either the right Purkinje fiber network or left bundle branch. Both lineages contribute to the atrioventricular bundle and right bundle branch that segregate early from working myocytes. Right and left VCS lineages share the transcriptional program of the respective ventricular working myocytes and genetic tracing experiments discount alternate progenitor cell contributions to the VCS. Conclusions: The mammalian VCS is comprised of cells derived from two lineages, supporting a dual contribution of first and second heart field progenitor cells. Developmental Dynamics 242:665–677, 2013.

Inducible glomerular erythropoietin production in the adult kidney

Hypoxia-inducible factor (HIF)-2-triggered erythropoietin production in renal interstitial fibroblast-like cells is the physiologically relevant source of erythropoietin for regulating erythropoiesis. During renal fibrosis, these cells transform into myofibroblasts and lose their ability to produce sufficient erythropoietin leading to anemia. To find if other cells for erythropoietin production might exist in the kidney we tested for the capability of nonepithelial glomerular cells to elaborate erythropoietin. Therefore, HIF transcription factors were stabilized by cell-specific deletion of the von Hippel-Lindau (VHL) gene. Inducible deletion of VHL in glomerular connexin40-expressing cells (endothelial, renin-expressing, and mesangial cells) markedly increased glomerular erythropoietin mRNA expression levels, plasma erythropoietin concentrations, and hematocrit values. These changes were mimicked by inducible cell-specific VHL deletion in renin-expressing and in mesangial cells but not in endothelial cells. The increases of erythropoietin production were absent, when VHL was co-deleted with HIF-2. The induction of glomerular erythropoietin expression was associated with the downregulation of juxtaglomerular renin expression, again in a HIF-2-dependent manner. Thus, VHL deletion in renin-expressing and in mesangial cells induces the capability to produce relevant amounts of erythropoietin and to suppress renin expression in the adult kidney if HIF-2 is stabilized.

Coronary stem development in wild-type and Tbx1 null mouse hearts

Background: Coronary artery (CA) stems connect the ventricular coronary tree with the aorta. Defects in proximal CA patterning are a cause of sudden cardiac death. In mice lacking Tbx1, common arterial trunk is associated with an abnormal trajectory of the proximal left CA. Here we investigate CA stem development in wild‐type and Tbx1 null embryos. Results: Genetic lineage tracing reveals that limited outgrowth of aortic endothelium contributes to proximal CA stems. Immunohistochemistry and fluorescent tracer injections identify a periarterial vascular plexus present at the onset of CA stem development. Transplantation experiments in avian embryos indicate that the periarterial plexus originates in mesenchyme distal to the outflow tract. Tbx1 is required for the patterning but not timing of CA stem development and a Tbx1 reporter allele is expressed in myocardium adjacent to the left but not right CA stem. This expression domain is maintained in Sema3c−/− hearts with a common arterial trunk and leftward positioned CA. Ectopic myocardial differentiation is observed on the left side of the Tbx1−/− common arterial trunk. Conclusions: A periarterial plexus bridges limited outgrowth of the aortic endothelium with the ventricular plexus during CA stem development. Molecular differences associated with left and right CA stems provide new insights into the etiology of CA patterning defects. Developmental Dynamics 245:445–459, 2016.