Gpr126 (Adgrg6) is expressed in cell types known to be exposed to mechanical stimuli.

GPR126 (ADGRG6) is an adhesion G protein-coupled receptor that plays an important role in a variety of tissues/organs, such as heart, sciatic nerve, cartilage, and ear. Moreover, GPR126 (ADGRG6) mutations are associated with human diseases, like adolescent idiopathic scoliosis, lung disease, bladder cancer, and intellectual disability. Despite its clinical importance, it remains elusive how GPR126 is activated and mediates signal transduction and what cellular processes depend on GPR126 signaling. Here, we generated a lacZ reporter mouse line to determine endogenous Gpr126 (Adgrg6) expression in a cell type-specific manner during embryonic development, at postnatal day 5 and in adult animals. Our results confirm Gpr126 expression data previously obtained utilizing antibodies and in situ hybridization in embryonic heart and sciatic nerve. In addition, we provide data with cellular resolution for previously described RT-PCR-based data, including lung and bladder. Moreover, new Gpr126-expressing tissues and cell types were identified, such as ureter and acinar secretory cells. Collectively, our data demonstrate that the newly generated lacZ reporter mouse is a suitable model to study Gpr126 expression during development and adulthood, provide detailed insight into Gpr126 expression at the cellular level, and reveal that all identified Gpr126-expressing cells are known to be exposed to mechanical stimuli.

Organ-specific function of adhesion G protein-coupled receptor GPR126 is domain-dependent.

Despite their abundance and multiple functions in a variety of organ systems, the function and signaling mechanisms of adhesion G protein-coupled receptors (GPCRs) are poorly understood. Adhesion GPCRs possess large N termini containing various functional domains. In addition, many of them are autoproteolytically cleaved at their GPS sites into an N-terminal fragment (NTF) and C-terminal fragment. Here we demonstrate that Gpr126 is expressed in the endocardium during early mouse heart development. Gpr126 knockout in mice and knockdown in zebrafish caused hypotrabeculation and affected mitochondrial function. Ectopic expression of Gpr126-NTF that lacks the GPS motif (NTF(ΔGPS)) in zebrafish rescued the trabeculation but not the previously described myelination phenotype in the peripheral nervous system. These data support a model in which the NTF of Gpr126, in contrast to the C-terminal fragment, plays an important role in heart development. Collectively, our analysis provides a unique example of the versatile function and signaling properties of adhesion GPCRs in vertebrates.

Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish.

The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.

Cardiac deletion of Smyd2 is dispensable for mouse heart development.

Chromatin modifying enzymes play a critical role in cardiac differentiation. Previously, it has been shown that the targeted deletion of the histone methyltransferase, Smyd1, the founding member of the SET and MYND domain containing (Smyd) family, interferes with cardiomyocyte maturation and proper formation of the right heart ventricle. The highly related paralogue, Smyd2 is a histone 3 lysine 4- and lysine 36-specific methyltransferase expressed in heart and brain. Here, we report that Smyd2 is differentially expressed during cardiac development with highest expression in the neonatal heart. To elucidate the functional role of Smyd2 in the heart, we generated conditional knockout (cKO) mice harboring a cardiomyocyte-specific deletion of Smyd2 and performed histological, functional and molecular analyses. Unexpectedly, cardiac deletion of Smyd2 was dispensable for proper morphological and functional development of the murine heart and had no effect on global histone 3 lysine 4 or 36 methylation. However, we provide evidence for a potential role of Smyd2 in the transcriptional regulation of genes associated with translation and reveal that Smyd2, similar to Smyd3, interacts with RNA Polymerase II as well as to the RNA helicase, HELZ.

TWEAK is a positive regulator of cardiomyocyte proliferation.

AIMS: Proliferation of mammalian cardiomyocytes stops during the first weeks after birth, preventing the heart from regenerating after injury. Recently, several studies have indicated that induction of cardiomyocyte proliferation can be utilized to regenerate the mammalian heart. Thus, it is important to identify novel factors that can induce proliferation of cardiomyocytes. Here, we determine the effect of TNF-related weak inducer of apoptosis (TWEAK) on cardiomyocytes, a cytokine known to regulate proliferation in several other cell types. METHODS AND RESULTS: Stimulation of neonatal rat cardiomyocytes with TWEAK resulted in increased DNA synthesis, increased expression of the proliferative markers Cyclin D2 and Ki67, and downregulation of the cell cycle inhibitor p27KIP1. Importantly, TWEAK stimulation resulted also in mitosis (H3P), cytokinesis (Aurora B), and increased cardiomyocyte numbers. Loss of function experiments revealed that re-induction of proliferation was dependent on tumour necrosis factor receptor superfamily member 12A (FN14) signalling. Downstream signalling was mediated through activation of extracellular signal-regulated kinases and phosphatidylinositol 3-kinase as well as inhibition of glycogen synthase kinase-3beta. In contrast to neonatal cardiomyocytes, TWEAK had no effect on adult rat cardiomyocytes due to developmental downregulation of its receptor FN14. However, adenoviral expression of FN14 enabled efficient induction of cell cycle re-entry in adult cardiomyocytes after TWEAK stimulation. CONCLUSION: Our data establish TWEAK as a positive regulator of cardiomyocyte proliferation.

E2F4 is required for cardiomyocyte proliferation.

AIMS: Although the fundamental role of the E2F transcription factor family in cell proliferation is well established, the specific function of E2F4 is unclear. On the basis of findings from cell culture experiments, E2F4 is generally considered as an inhibitor of cell proliferation. Accumulating evidence suggests, however, that E2F4 acts as an activator of cell proliferation in certain contexts. Here, we have investigated the role of E2F4 during heart development and in proliferating cardiomyocytes. METHODS AND RESULTS: Nuclear E2F4 expression in cardiomyocytes declined during mouse heart development, which correlates with the loss of proliferative capacity of cardiomyocytes. Re-induction of proliferation in postnatal cardiomyocytes increased nuclear E2F4 expression. E2F4 accumulated in the nucleus at the end of the S phase, remained nuclear during mitosis, and disappeared at the end of cytokinesis. siRNA-mediated inhibition of E2F4 in proliferating postnatal cardiomyocytes resulted in a significant reduction in mitosis, but not in DNA synthesis. Co-staining of E2F4 and Crest revealed that E2F4 co-localizes with kinetochores. Moreover, chromatin immunoprecipitation showed that E2F4 binds to centromeric alpha-satellite DNA during mitosis. CONCLUSION: Our data indicate that E2F4 is required for cardiomyocyte proliferation and suggest a function for E2F4 in mitosis.

Features of cardiomyocyte proliferation and its potential for cardiac regeneration.

The human heart does not regenerate. Instead, following injury, human hearts scar. The loss of contractile tissue contributes significantly to morbidity and mortality. In contrast to humans, zebrafish and newts faithfully regenerate their hearts. Interestingly, regeneration is in both cases based on cardiomyocyte proliferation. In addition, mammalian cardiomyocytes proliferate during foetal development. Their proliferation reaches its maximum around chamber formation, stops shortly after birth, and subsequent heart growth is mostly achieved by an increase in cardiomyocyte size (hypertrophy). The underlying mechanisms that regulate cell cycle arrest and the switch from proliferation to hypertrophy are unclear. In this review, we highlight features of dividing cardiomyocytes, summarize the attempts to induce mammalian cardiomyocyte proliferation, critically discuss methods commonly used for its detection, and explore the potential and problems of inducing cardiomyocyte proliferation to improve function in diseased hearts.

Cryoinjury: a model of myocardial regeneration.

INTRODUCTION: Although traditionally adult cardiomyocytes are thought to be unable to divide, recent observations provide evidence for cardiomyocyte proliferation after myocardial injury. Myocardial cryoinjury has been shown to be followed by neovascularization. We hypothesize that, in addition to neovascularization, cardiomyocyte proliferation after myocardial cryoinjury contributes to regeneration. METHOD: Cryolesions were applied to the left ventricle of mouse hearts. Inflammatory cell infiltration (F4/80, neutrophils), neovascularization (CD31), and cardiomyocyte proliferation (5-bromo-2-deoxyuridine, Ki-67, mitotic spindle) were determined at different time points (2-70 days) after cryoinjury. RESULTS: Between Days 7 and 14 after injury, a 150- and 280-fold increase in number of proliferating cardiomyocytes was observed, as compared to controls. At the same time, numerous proliferating capillaries were found in between the proliferating cardiomyocytes. Presence of high numbers of macrophages in the cryolesion preceded and coincided with this proliferation. The area of cryolesion decreased significantly between Days 7 (23+/-5%) and 14 (8+/-2%) after cryoinjury. Moreover, regeneration of viable, nonhypertrophied myocardium was observed. After 14 days, cardiomyocyte proliferation decreased to numbers observed in controls, and concomitantly, the number of macrophages strongly decreased. CONCLUSION: Our data show that adult cardiomyocytes proliferate in sufficiently high numbers to effectuate myocardial regeneration after left ventricular cryoinjury in mice.

Macrophage depletion impairs wound healing and increases left ventricular remodeling after myocardial injury in mice.

Macrophages have been suggested to be beneficial for myocardial wound healing. We investigated the role of macrophages in myocardial wound healing by inhibition of macrophage infiltration after myocardial injury. We used a murine cryoinjury model to induce left ventricular damage. Infiltrating macrophages were depleted during the 1st week after cryoinjury by serial intravenous injections of clodronate-containing liposomes. After injury, the presence of macrophages, which secreted high levels of transforming growth factor-beta and vascular endothelial growth factor-A, led to rapid removal of cell debris and replacement by granulation tissue containing inflammatory cells and blood vessels, followed by myofibroblast infiltration and collagen deposition. In macrophage-depleted hearts, nonresorbed cell debris was still observed 4 weeks after injury. Secretion of transforming growth factor-beta and vascular endothelial growth factor-A as well as neovascularization, myofibroblast infiltration, and collagen deposition decreased. Moreover, macrophage depletion resulted in a high mortality rate accompanied by increased left ventricular dilatation and wall thinning. In conclusion, infiltrating macrophage depletion markedly impairs wound healing and increases remodeling and mortality after myocardial injury, identifying the macrophage as a key player in myocardial wound healing. Based on these findings, we propose that increasing macrophage numbers early after myocardial infarction could be a clinically relevant option to promote myocardial wound healing and subsequently to reduce remodeling and heart failure.

The correlation between difference in foreign body reaction between implant locations and cytokine and MMP expression.

The foreign body reaction (FBR) differs between subcutaneously and supra-epicardially implanted materials. We hypothesize that this is a result of differences in cytokine, chemokine and matrix metalloproteinase (MMP) dynamics. Therefore we applied collagen disks subcutaneously and on the epicardium in mice and analyzed the FBR from day 1 to 21. Both the influx of leukocytes and implant degradation were higher in supra-epicardially implanted collagen than in subcutaneously implanted material. This correlated with a higher gene expression of pro-inflammatory cytokines such as IL-1 and IL-6, and a lower expression of the anti-inflammatory cytokine IL-10. Furthermore, the higher supra-epicardial expression of PMN attractants CXCL1/KC and CXCL2/MIP2 correlated with a higher and prolonged PMN influx. The gene expression levels of collagen degrading MMPs, i.e. MMP8, MMP13 and MMP14 were similar in subcutaneous and supra-epicardial disks. However, the activity of these enzymes was markedly higher supra-epicardially. In addition, the MMP9 expression was higher supra-epicardially, suggesting a role for this enzyme in the degradation process. In conclusion, a strong pro-inflammatory milieu is generated after supra-epicardial implantation that enables prolonged PMN presence and activation. This, together with the high supra-epicardial MMP9 level, could explain the observed difference in Col-I degradation between locations.

Increased inflammatory response and neovascularization in reperfused vs. non-reperfused murine myocardial infarction.

INTRODUCTION: Fundamental knowledge of the inflammatory response after myocardial infarction (MI) is indispensable for intervention toward cardiac regeneration. Although reperfusion is preferred as clinical therapy, for basic research, also permanent ligation MI models are widely used. METHODS: In this report, we pathohistologically compared the kinetics of the inflammatory and angiogenic response after MI induced by permanent ligation or ligation followed by reperfusion of the left anterior descending coronary artery in mice. RESULTS: Permanent ligation resulted in a higher mortality rate accompanied by increased left ventricular dilatation and more progressive wall thinning. However, reperfused infarcts showed higher inflammatory cell influx. Neutrophil numbers were higher after reperfusion post-MI, although their presence was prolonged after ligation. Also, the number of macrophages after reperfusion was continuously higher, but the course of macrophage influx was comparable in both models. The number of lymphocytes was low in both models. Only the peak in myofibroblast numbers at 7 days was higher after ligation than after reperfusion. Moreover, cardiomyocyte remnants were cleared faster, and collagen deposition started earlier after reperfusion. In addition, reperfusion resulted in an increased angiogenic response, as was reflected in increased numbers of medium-sized and large vessels at 7 and 14 days post-MI. CONCLUSION: We show less adverse remodeling together with a higher presence of inflammatory cells and enhanced neovascularization in reperfused MI. These differences between non-reperfused and reperfused MI should be taken into consideration for experimental use of MI models.

The enzymatic degradation of scaffolds and their replacement by vascularized extracellular matrix in the murine myocardium.

Replacement of injured myocardium by cell-based degradable scaffolds is a novel approach to regenerate myocardium. Understanding the foreign body reaction (FBR) induced by the scaffold is requisite to predict unwanted site effects or implant failure. We evaluated the FBR against a biodegradable scaffold applied on injured myocardium in mice. Cryolesions and collagen type I scaffolds (Col-I) were applied to the left ventricle of mice. Cell infiltration, neovascularization, collagen deposition, matrix metalloproteinase (MMP-8) expression, enzymatic activity and scaffold degradation were determined at different time points (2-70 days). Infiltration of mainly macrophages, neutrophils and blood vessels was completed within 14 days. High numbers of neutrophils accumulated around the Col-I fibers and degradation of Col-I fibers into small fragments was observed on day 14. Active MMP-8 co-localized with the neutrophils on day 14, indicating enzymatic degradation of Col-I by neutrophil collagenase. Highly vascularized extracellular matrix remained at day 70. No differences were observed in the FBR to Col-I after application on healthy or injured myocardium. The FBR had no adverse effects on the adjacent myocardial tissue. In conclusion, cardiac scaffolds are degraded by MMP-8 and replaced by vascularized extracellular matrix during the FBR on injured myocardium.

Vesicle-mediated transport and release of CCL21 in endangered neurons: a possible explanation for microglia activation remote from a primary lesion.

Whenever neurons in the CNS are injured, microglia become activated. In addition to local activation, microglia remote from the primary lesion site are stimulated. Because this so-called secondary activation of microglia is instrumental for long-term changes after neuronal injury, it is important to understand how microglia activity is controlled. The remote activation of microglia implies that the activating signals are transported along neuronal projections. However, the identity of these signals has not yet been identified. It is shown here that glutamate-treated neurons rapidly express and release the chemokine CCL21. We also provide evidence that neuronal CCL21 is packed in vesicles and transported throughout neuronal processes to reach presynaptic structures. Chemotaxis assays show that functional CCL21 is released from endangered neurons and activate microglia via the chemokine receptor CXCR3. Based on these findings, we suggest that neuronal CCL21 is important in directed neuron-microglia signaling and that this communication could account for the remote activation of microglia, far distant from a primary lesion.

Neovascularization and vascular markers in a foreign body reaction to subcutaneously implanted degradable biomaterial in mice.

To study the spatiotemporal processes of angiogenesis during a foreign body reaction (FBR), biodegradable bovine collagen type-1 (COL-I) discs were implanted in mice for a period up to 28 days. The cellular infiltration (consisting mainly of macrophages, giant cells and fibroblasts), and the extent of neovascularization into the discs were determined. Also the expression levels and/or distribution of the endothelial cell markers von Willebrand factor (vWF), platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, MECA-32 antigens and endomucin, and of the basal lamina marker collagen type IV (Coll IV) were analysed. In time, a strong neovascularization of the discs was observed, with frequently occurring vascular sprouting, and intussusceptive growth of vessels. In this model, vWF, MECA-32 and endomucin antibodies often failed to stain neovessels in the COL-I discs. In contrast, staining for collagen IV basal lamina component in combination with CD31 covered the complete range of neo-vessels. We conclude that the model described in this study is a useful model to study FBR induced angiogenesis because of the active neovascularization taking place during prolonged periods of time.