SUMMARY 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, while 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.

Rationale Macrophages populate the steady-state myocardium. Previously, all macrophages were thought to arise from monocytes; however, it emerged that in several organs tissue-resident macrophages may self-maintain through local proliferation. Objective To study the contribution of monocytes to cardiac resident macrophages in steady-state, after macrophage depletion in CD11bDTR/+ mice and in myocardial infarction. Methods and Results Using in vivo fate mapping and flow cytometry, we estimated that during steady-state the heart macrophage population turns over in about one month. To explore the source of cardiac resident macrophages, we joined the circulation of mice using parabiosis. After 6 weeks, we observed blood monocyte chimerism of 35.3±3.4% while heart macrophages showed a much lower chimerism of 2.7±0.5% (p<0.01). Macrophages self renewed locally through proliferation: 2.1±0.3% incorporated BrdU 2 hours after a single injection and 13.7±1.4% heart macrophages stained positive for the cell cycle marker Ki67. The cells likely participate in defense against infection, as we found them to ingest fluorescently labeled bacteria. In ischemic myocardium, we observed that tissue resident macrophages died locally while some also migrated to hematopoietic organs. If the steady-state was perturbed by coronary ligation or diphtheria toxin-induced macrophage depletion in CD11bDTR/+ mice, blood monocytes replenished heart macrophages. However, in the chronic phase after myocardial infarction, macrophages residing in the infarct were again independent from the blood monocyte pool, returning to the steady-state situation. Conclusions In this study we show differential contribution of monocytes to heart macrophages during steady-state, after macrophage depletion or in the acute and chronic phase after myocardial infarction. We found that macrophages participate in the immunosurveillance of myocardial tissue. These data correspond with previous studies on tissue-resident macrophages and raise important questions on the fate and function of macrophages during the development of heart failure.

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H ؉ ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.We recently identified bicarbonate-activated soluble adenylyl cyclase (sAC) 1 as a chemosensor mediating bicarbonate-dependent elevation of cAMP (1), defining a potential transduction pathway for cells to sense variations in bicarbonate, as well as the closely related parameters, pCO 2 and pH (1-3). sAC is distinct from transmembrane adenylyl cyclases. It is insensitive to regulation by forskolin or heterotrimeric G proteins (2) but is directly activated by bicarbonate ions. It does not have predicted transmembrane domains and is present in both soluble and particulate fractions of cellular extracts (4 -6). Mammalian sAC is similar to bicarbonate-regulated adenylyl cyclases present in cyanobacteria (1, 2), suggesting there may be a unifying mechanism for the bicarbonate regulation of cAMP signaling in many biological systems.sAC is highly expressed in spermatozoa (7) where it is proposed to mediate the bicarbonate-dependent cAMP elevation that precedes capacitation, hyperactivated motility, and acrosome reaction needed for fertilization (1). While spermatozoa mature and are stored along the epididymal lumen, they are kept in a quiescent state by an acidic pH of 6.5-6.8 and a low bicarbonate concentration of 2-7 mM (8). We have previously shown (9, 10) that a sub-population of epithelial cells, the so-called clear cells, are important players in the acidification capacity of the epididymis. Clear cells express high levels of the V-ATPase in their apical pole, and are responsible for the bulk of proton secretion in the vas deferens. Proton secretion by clear cells occurs in a chloride-independent but bicarbonate-dependent manner (11). Similarly to kidney intercalated cells, epididymal clear cells regulate their rate of proton secretion via V-ATPase recycling between intracellular vesicles and the apical plasma membrane (12). In these cells, as well a...

Basal cells are by definition located on the basolateral side of several epithelia, and they have never been observed reaching the lumen. Using high-resolution 3D confocal imaging, we report that basal cells extend long and slender cytoplasmic projections that not only reach towards the lumen but can cross the tight junction barrier in some epithelia of the male reproductive and respiratory tracts. In this way, the basal cell plasma membrane is exposed to the luminal environment. In the epididymis, in which luminal acidification is crucial for sperm maturation and storage, these projections contain the angiotensin II type 2 receptor (AGTR2). Activation of AGTR2 by luminal angiotensin II, increases proton secretion by adjacent clear cells, which are devoid of AGTR2. We propose a new paradigm in which basal cells scan and sense the luminal environment of pseudostratified epithelia, and modulate epithelial function by a mechanism involving cross-talk with other epithelial cells.

One of the most intriguing aspects of male reproductive physiology is the ability to generate spermatogenic cells - which are “foreign” to the host - without triggering immune activation. After leaving the testis, spermatozoa enter the epididymis where they mature and are stored. We report here a previously unrecognized dense network of dendritic cells located at the base of the epididymal epithelium. This network was detected in transgenic mice expressing CD11c-EYFP and CX3CR1-GFP reporters. Epididymal dendritic cells (eDCs) establish intimate interactions with the epithelium and project long dendrites between epithelial cells toward the lumen. We show that isolated eDCs express numerous leukocyte markers described previously in other organs that are in contact with the external environment, and present and cross-present ovalbumin to T cells in vitro. eDCs are, therefore, strategically positioned to regulate the complex interplay between immune tolerance and activation, a balance that is fundamental to male fertility.

Dutta et al. show that targeting VACM-1 expression in splenic macrophages impairs extramedullary hematopoiesis, thus reducing inflammation in mouse ischemic heart and atherosclerotic plaques.

The role of the actin cytoskeleton in regulating membrane protein trafficking is complex and depends on the cell type and protein being examined. Using the epididymis as a model system in which luminal acidification is crucial for sperm maturation and storage, we now report that modulation of the actin cytoskeleton by the calcium-activated actin-capping and -severing protein gelsolin plays a key role in regulating vacuolar