Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells.

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

Complementary and Inducible creERT2 Mouse Models for Functional Evaluation of Endothelial Cell Subtypes in the Bone Marrow.

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis.

Dbh+ catecholaminergic cardiomyocytes contribute to the structure and function of the cardiac conduction system in murine heart.

The heterogeneity of functional cardiomyocytes arises during heart development, which is essential to the complex and highly coordinated cardiac physiological function. Yet the biological and physiological identities and the origin of the specialized cardiomyocyte populations have not been fully comprehended. Here we report a previously unrecognised population of cardiomyocytes expressing Dbhgene encoding dopamine beta-hydroxylase in murine heart. We determined how these myocytes are distributed across the heart by utilising advanced single-cell and spatial transcriptomic analyses, genetic fate mapping and molecular imaging with computational reconstruction. We demonstrated that they form the key functional components of the cardiac conduction system by using optogenetic electrophysiology and conditional cardiomyocyte Dbh gene deletion models. We revealed their close relationship with sympathetic innervation during cardiac conduction system formation. Our study thus provides new insights into the development and heterogeneity of the mammalian cardiac conduction system by revealing a new cardiomyocyte population with potential catecholaminergic endocrine function.

Nkx2-5 Loss of Function in the His-Purkinje System Hampers Its Maturation and Leads to Mechanical Dysfunction.

The ventricular conduction or His-Purkinje system (VCS) mediates the rapid propagation and precise delivery of electrical activity essential for the synchronization of heartbeats. Mutations in the transcription factor Nkx2-5 have been implicated in a high prevalence of developing ventricular conduction defects or arrhythmias with age. Nkx2-5 heterozygous mutant mice reproduce human phenotypes associated with a hypoplastic His-Purkinje system resulting from defective patterning of the Purkinje fiber network during development. Here, we investigated the role of Nkx2-5 in the mature VCS and the consequences of its loss on cardiac function. Neonatal deletion of Nkx2-5 in the VCS using a Cx40-CreERT2 mouse line provoked apical hypoplasia and maturation defects of the Purkinje fiber network. Genetic tracing analysis demonstrated that neonatal Cx40-positive cells fail to maintain a conductive phenotype after Nkx2-5 deletion. Moreover, we observed a progressive loss of expression of fast-conduction markers in persistent Purkinje fibers. Consequently, Nkx2-5-deleted mice developed conduction defects with progressively reduced QRS amplitude and RSR' complex associated with higher duration. Cardiac function recorded by MRI revealed a reduction in the ejection fraction in the absence of morphological changes. With age, these mice develop a ventricular diastolic dysfunction associated with dyssynchrony and wall-motion abnormalities without indication of fibrosis. These results highlight the requirement of postnatal expression of Nkx2-5 in the maturation and maintenance of a functional Purkinje fiber network to preserve contraction synchrony and cardiac function.

Optogenetic termination of atrial tachyarrhythmias by brief pulsed light stimulation.

AIMS: The most efficient way to acutely restore sinus rhythm from atrial fibrillation (AF) is electrical cardioversion, which is painful without adequate sedation. Recent studies in various experimental models have indicated that optogenetic termination of AF using light-gated ion channels may provide a myocardium-specific and potentially painless alternative future therapy. However, its underlying mechanism(s) remain(s) incompletely understood. As brief pulsed light stimulation, even without global illumination, can achieve optogenetic AF termination, besides direct conduction block also modulation of action potential (AP) properties may be involved in the termination mechanism. We studied the relationship between optogenetic AP duration (APD) and effective refractory period (ERP) prolongation by brief pulsed light stimulation and termination of atrial tachyarrhythmia (AT). METHODS AND RESULTS: Hearts from transgenic mice expressing the H134R variant of channelrhodopsin-2 in atrial myocytes were explanted and perfused retrogradely. AT induced by electrical stimulation was terminated by brief pulsed blue light stimulation (470 nm, 10 ms, 16 mW/mm2) with 68% efficacy. The termination rate was dependent on pulse duration and light intensity. Optogenetically imposed APD and ERP changes were systematically examined and optically monitored. Brief pulsed light stimulation (10 ms, 6 mW/mm2) consistently prolonged APD and ERP when light was applied at different phases of the cardiac action potential. Optical tracing showed light-induced APD prolongation during the termination of AT. CONCLUSION: Our results directly demonstrate that cationic channelrhodopsin activation by brief pulsed light stimulation prolongs the atrial refractory period suggesting that this is one of the key mechanisms of optogenetic termination of AT.

New Insights into the Development and Morphogenesis of the Cardiac Purkinje Fiber Network: Linking Architecture and Function.

The rapid propagation of electrical activity through the ventricular conduction system (VCS) controls spatiotemporal contraction of the ventricles. Cardiac conduction defects or arrhythmias in humans are often associated with mutations in key cardiac transcription factors that have been shown to play important roles in VCS morphogenesis in mice. Understanding of the mechanisms of VCS development is thus crucial to decipher the etiology of conduction disturbances in adults. During embryogenesis, the VCS, consisting of the His bundle, bundle branches, and the distal Purkinje network, originates from two independent progenitor populations in the primary ring and the ventricular trabeculae. Differentiation into fast-conducting cardiomyocytes occurs progressively as ventricles develop to form a unique electrical pathway at late fetal stages. The objectives of this review are to highlight the structure-function relationship between VCS morphogenesis and conduction defects and to discuss recent data on the origin and development of the VCS with a focus on the distal Purkinje fiber network.

Nkx2-5 defines distinct scaffold and recruitment phases during formation of the murine cardiac Purkinje fiber network.

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PFs share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and network morphogenesis are poorly understood. Here, we apply genetic fate mapping and temporal clonal analysis to identify murine cardiomyocytes committed to the PF lineage as early as E7.5. We find that a polyclonal PF network emerges by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors. At late fetal stages, the segregation of conductive cells increases during a phase of rapid recruitment to build the definitive PF network through a non-cell autonomous mechanism. We also show that PF differentiation is impaired in Nkx2-5 haploinsufficient embryos leading to failure to extend the scaffold. In particular, late fetal recruitment fails, resulting in PF hypoplasia and persistence of bipotent progenitors. Our results identify how transcription factor dosage regulates cell fate divergence during distinct phases of PF network morphogenesis.

A Notch3-Marked Subpopulation of Vascular Smooth Muscle Cells Is the Cell of Origin for Occlusive Pulmonary Vascular Lesions.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by profound vascular remodeling in which pulmonary arteries narrow because of medial thickening and occlusion by neointimal lesions, resulting in elevated pulmonary vascular resistance and right heart failure. Therapies targeting the neointima would represent a significant advance in PAH treatment; however, our understanding of the cellular events driving neointima formation, and the molecular pathways that control them, remains limited. METHODS: We comprehensively map the stepwise remodeling of pulmonary arteries in a robust, chronic inflammatory mouse model of pulmonary hypertension. This model demonstrates pathological features of the human disease, including increased right ventricular pressures, medial thickening, neointimal lesion formation, elastin breakdown, increased anastomosis within the bronchial circulation, and perivascular inflammation. Using genetic lineage tracing, clonal analysis, multiplexed in situ hybridization, immunostaining, deep confocal imaging, and staged pharmacological inhibition, we define the cell behaviors underlying each stage of vascular remodeling and identify a pathway required for neointima formation. RESULTS: Neointima arises from smooth muscle cells (SMCs) and not endothelium. Medial SMCs proliferate broadly to thicken the media, after which a small number of SMCs are selected to establish the neointima. These neointimal founder cells subsequently undergoing massive clonal expansion to form occlusive neointimal lesions. The normal pulmonary artery SMC population is heterogeneous, and we identify a Notch3-marked minority subset of SMCs as the major neointimal cell of origin. Notch signaling is specifically required for the selection of neointimal founder cells, and Notch inhibition significantly improves pulmonary artery pressure in animals with pulmonary hypertension. CONCLUSIONS: This work describes the first nongenetically driven murine model of pulmonary hypertension (PH) that generates robust and diffuse occlusive neointimal lesions across the pulmonary vascular bed and does so in a stereotyped timeframe. We uncover distinct cellular and molecular mechanisms underlying medial thickening and neointima formation and highlight novel transcriptional, behavioral, and pathogenic heterogeneity within pulmonary artery SMCs. In this model, inflammation is sufficient to generate characteristic vascular pathologies and physiological measures of human PAH. We hope that identifying the molecular cues regulating each stage of vascular remodeling will open new avenues for therapeutic advancements in the treatment of PAH.

Straightjacket/α2δ3 deregulation is associated with cardiac conduction defects in myotonic dystrophy type 1.

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1.

Defects in Trabecular Development Contribute to Left Ventricular Noncompaction.

Left ventricular noncompaction (LVNC) is a genetically heterogeneous disorder the etiology of which is still debated. During fetal development, trabecular cardiomyocytes contribute extensively to the working myocardium and the ventricular conduction system. The impact of developmental defects in trabecular myocardium in the etiology of LVNC has been debated. Recently we generated new mouse models of LVNC by the conditional deletion of the key cardiac transcription factor encoding gene Nkx2-5 in trabecular myocardium at critical steps of trabecular development. These conditional mutant mice recapitulate pathological features similar to those observed in LVNC patients, including a hypertrabeculated left ventricle with deep endocardial recesses, subendocardial fibrosis, conduction defects, strain defects, and progressive heart failure. After discussing recent findings describing the respective contribution of trabecular and compact myocardium during ventricular morphogenesis, this review will focus on new data reflecting the link between trabecular development and LVNC.

Right coronary artery ligation in mice: a novel method to investigate right ventricular dysfunction and biventricular interaction.

Right ventricular (RV) dysfunction can lead to complications after acute inferior myocardial infarction (MI). However, it is unclear how RV failure after MI contributes to left-sided dysfunction. The aim of the present study was to investigate the consequences of right coronary artery (RCA) ligation in mice. RCA ligation was performed in C57BL/6JRj mice ( n = 38). The cardiac phenotypes were characterized using high-resolution echocardiography performed up to 4 wk post-RCA ligation. Infarct size was measured using 2,3,5-triphenyltetrazolium chloride staining 24 h post-RCA ligation, and the extent of the fibrotic area was determined 4 wk after MI. RV dysfunction was confirmed 24 h post-RCA ligation by a decrease in the tricuspid annular plane systolic excursion ( P < 0.001) and RV longitudinal strain analysis ( P < 0.001). Infarct size measured ex vivo represented 45.1 ± 9.1% of the RV free wall. RCA permanent ligation increased the RV-to-left ventricular (LV) area ratio ( P < 0.01). Septum hypertrophy ( P < 0.01) was associated with diastolic septal flattening. During the 4-wk post-RCA ligation, LV ejection fraction was preserved, yet it was associated with impaired LV diastolic parameters ( E/ E', global strain rate during early diastole). Histological staining after 4 wk confirmed the remodeling process with a thin and fibrotic RV. This study validates that RCA ligation in mice is feasible and induces RV heart failure associated with the development of LV diastolic dysfunction. Our model offers a new opportunity to study mechanisms and treatments of RV/LV dysfunction after MI. NEW & NOTEWORTHY Right ventricular (RV) dysfunction frequently causes complications after acute inferior myocardial infarction. How RV failure contributes to left-sided dysfunction is elusive because of the lack of models to study molecular mechanisms. Here, we created a new model of myocardial infarction by permanently tying the right coronary artery in mice. This model offers a new opportunity to unravel mechanisms underlying RV/left ventricular dysfunction and evaluate drug therapy.

Semi-automatic detection of myocardial trabeculation using cardiovascular magnetic resonance: correlation with histology and reproducibility in a mouse model of non-compaction.

BACKGROUND: The definition of left ventricular (LV) non-compaction is controversial, and discriminating between normal and excessive LV trabeculation remains challenging. Our goal was to quantify LV trabeculation on cardiovascular magnetic resonance (CMR) images in a genetic mouse model of non-compaction using a dedicated semi-automatic software package and to compare our results to the histology used as a gold standard. METHODS: Adult mice with ventricular non-compaction were generated by conditional trabecular deletion of Nkx2-5. Thirteen mice (5 controls, 8 Nkx2-5 mutants) were included in the study. Cine CMR series were acquired in the mid LV short axis plane (resolution 0.086 × 0.086x1mm3) (11.75 T). In a sub set of 6 mice, 5 to 7 cine CMR were acquired in LV short axis to cover the whole LV with a lower resolution (0.172 × 0.172x1mm3). We used semi-automatic software to quantify the compacted mass (Mc), the trabeculated mass (Mt) and the percentage of trabeculation (Mt/Mc) on all cine acquisitions. After CMR all hearts were sliced along the short axis and stained with eosin, and histological LV contouring was performed manually, blinded from the CMR results, and Mt, Mc and Mt/Mc were quantified. Intra and interobserver reproducibility was evaluated by computing the intra class correlation coefficient (ICC). RESULTS: Whole heart acquisition showed no statistical significant difference between trabeculation measured at the basal, midventricular and apical parts of the LV. On the mid-LV cine CMR slice, the median Mt was 0.92 mg (range 0.07-2.56 mg), Mc was 12.24 mg (9.58-17.51 mg), Mt/Mc was 6.74% (0.66-17.33%). There was a strong correlation between CMR and the histology for Mt, Mc and Mt/ Mc with respectively: r2 = 0.94 (p < 0.001), r2 = 0.91 (p < 0.001), r2 = 0.83 (p < 0.001). Intra- and interobserver reproducibility was 0.97 and 0.8 for Mt; 0.98 and 0.97 for Mc; 0.96 and 0.72 for Mt/Mc, respectively and significantly more trabeculation was observed in the Mc Mutant mice than the controls. CONCLUSION: The proposed semi-automatic quantification software is accurate in comparison to the histology and reproducible in evaluating Mc, Mt and Mt/ Mc on cine CMR.

Correction: Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy.

[This corrects the article DOI: 10.1371/journal.pgen.1007502.].

Embryonic Tbx3+ cardiomyocytes form the mature cardiac conduction system by progressive fate restriction.

A small network of spontaneously active Tbx3+ cardiomyocytes forms the cardiac conduction system (CCS) in adults. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and the development of biological pacemakers to treat arrhythmias. We found that Tbx3 expression in the embryonic mouse heart is associated with automaticity. Genetic inducible fate mapping revealed that Tbx3+ cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages, contribution of Tbx3+ cells was restricted to the definitive CCS. We identified a Tbx3+ population in the outflow tract of the early heart tube that formed the atrioventricular bundle. Whereas Tbx3+ cardiomyocytes also contributed to the adjacent Gja5+ atrial and ventricular chamber myocardium, embryonic Gja5+ chamber cardiomyocytes did not contribute to the Tbx3+ sinus node or to atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate restriction of a Tbx3+ cell population in the early developing heart, which implicates Tbx3 as a useful tool for developing strategies to study and treat CCS diseases.

Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy.

Left ventricular non-compaction (LVNC) is a rare cardiomyopathy associated with a hypertrabeculated phenotype and a large spectrum of symptoms. It is still unclear whether LVNC results from a defect of ventricular trabeculae development and the mechanistic basis that underlies the varying severity of this pathology is unknown. To investigate these issues, we inactivated the cardiac transcription factor Nkx2-5 in trabecular myocardium at different stages of trabecular morphogenesis using an inducible Cx40-creERT2 allele. Conditional deletion of Nkx2-5 at embryonic stages, during trabecular formation, provokes a severe hypertrabeculated phenotype associated with subendocardial fibrosis and Purkinje fiber hypoplasia. A milder phenotype was observed after Nkx2-5 deletion at fetal stages, during trabecular compaction. A longitudinal study of cardiac function in adult Nkx2-5 conditional mutant mice demonstrates that excessive trabeculation is associated with complex ventricular conduction defects, progressively leading to strain defects, and, in 50% of mutant mice, to heart failure. Progressive impaired cardiac function correlates with conduction and strain defects independently of the degree of hypertrabeculation. Transcriptomic analysis of molecular pathways reflects myocardial remodeling with a larger number of differentially expressed genes in the severe versus mild phenotype and identifies Six1 as being upregulated in hypertrabeculated hearts. Our results provide insights into the etiology of LVNC and link its pathogenicity with compromised trabecular development including compaction defects and ventricular conduction system hypoplasia.

Single-cell analysis of early progenitor cells that build coronary arteries.

Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.

Characterizing the role of atrial natriuretic peptide signaling in the development of embryonic ventricular conduction system.

Patients born with congenital heart defects frequently encounter arrhythmias due to defects in the ventricular conduction system (VCS) development. Although recent studies identified transcriptional networks essential for the heart development, there is scant information on the mechanisms regulating VCS development. Based on the association of atrial natriuretic peptide (ANP) expression with VCS forming regions, it was reasoned that ANP could play a critical role in differentiation of cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) toward a VCS cell lineage. The present study showed that treatment of embryonic ventricular cells with ANP or cell permeable 8-Br-cGMP can induce gene expression of important VCS markers such as hyperpolarization-activated cyclic nucleotide-gated channel-4 (HCN4) and connexin 40 (Cx40). Inhibition of protein kinase G (PKG) via Rp-8-pCPT-cGMPS further confirmed the role of ANP/NPRA/cGMP/PKG pathway in the regulation of HCN4 and Cx40 gene expression. Additional experiments indicated that ANP may regulate VCS marker gene expression by modulating levels of miRNAs that are known to control the stability of transcripts encoding HCN4 and Cx40. Genetic ablation of NPRA revealed significant decreases in VCS marker gene expression and defects in Purkinje fiber arborisation. These results provide mechanistic insights into the role of ANP/NPRA signaling in VCS formation.

Genetic targeting of Purkinje fibres by Sema3a-CreERT2.

The maintenance of the heart rhythm and the conduction of excitatory signals require changing excitatory signals via electrical activity and coordination by communication between working and conductive cardiomyocytes. Understanding how the ventricular conduction system is established provides novel insights into the pathophysiological progress of cardiac arrhythmias. However, the major hurdle in this field is the lack of a specific genetic tool that targets the Purkinje fibres of the ventricular conduction system and no other types of cardiomyocytes or coronary vessels. Here, we generated a Sema3a-CreERT2 knock-in mouse line to test its specificity for genetically labelled Purkinje fibres. We found that Sema3a was expressed in the subendocardial layer of the trabecular myocardium in the embryonic heart and was restricted to the Purkinje fibres in the adult heart. A fate mapping study based on the Sema3a-CreERT2 line revealed that the Sema3a+ cardiomyocytes were restricted to the fate of Purkinje fibres in the perinatal but not the embryonic stage. Collectively, our study provides a new genetic tool, i.e., Sema3a-CreERT2, for studying the molecular mechanisms that regulate the function of Purkinje fibres.

Connexin40 controls endothelial activation by dampening NFκB activation.

Connexins are proteins forming gap junction channels for intercellular communication. Connexin40 (Cx40) is highly expressed by endothelial cells (ECs) of healthy arteries but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is atherogenic partly through activation of the pro-inflammatory NFκB pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NFκB. Shear stress-modifying casts were placed around carotid arteries of mice expressing eGFP under the Cx40 promoter (Cx40+/eGFP ). We found that Cx40 expression is decreased in carotid regions of oscillatory shear stress but conserved in high and low laminar shear stress regions. These results were confirmed in vitro. Using phage display, we retrieved a binding motif for the intracellular regulatory Cx40 C-terminus (Cx40CT), i.e. HS[I, L, V][K, R]. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5-to-16 of IκBα, a member of the protein complex inhibiting NFκB activation. Binding of IκBα (peptide) and Cx40 was confirmed by crosslinking and en face proximity ligation assay on carotid arteries. TNFα-induced nuclear translocation of NFκB in ECs was enhanced after reducing Cx40 with siRNA. Transfection of HeLa cells with either full-length Cx40 or Cx40CT demonstrated that Cx40CT was sufficient for inhibition of TNFα-induced NFκB phosphorylation. Finally, Tie2CreTgCx40fl/flApoe-/- mice showed exaggerated shear stress-induced atherosclerosis and enhanced NFκB nuclear translocation. Our data show a novel functional IκBα-Cx40 interaction that may be relevant for the control of NFκB activation by shear stress in atherogenesis.

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.