Teresa M. Ribeiro-Rodrigues

Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells

Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, mediate the transfer of biological information between donor and acceptor cells. Although the biological effects of exosomes in target cells have been intensively studied, the mechanisms that govern exosomal uptake are not fully understood. Here, we show that Connexin 43 (Cx43), the most widely expressed GJ protein, is present in exosomes in the form of hexameric channels and, more importantly, that exosomal Cx43 is able to modulate the interaction and transfer of information between exosomes and acceptor cells. This study envisions a new paradigm where Cx43-containing channels mediate the release of exosomal content into cells, which constitutes a novel and unanticipated mechanism to modulate intercellular communication.

AMSH-mediated deubiquitination of Cx43 regulates internalization and degradation of gap junctions

Gap junctions (GJs) are specialized cell‐cell contacts formed by connexins (Cxs), which provide direct intercellular communication between eukaryotic cells. Although Cx43 has long been known to be a substrate for ubiquitination, the reversal of this modification by deubiquitylases (DUBs) has never been described. Here we report that the DUB‐associated molecule with the SH3 domain of STAM (AMSH) interacts with Cx43 and mediates its deubiquitination. In this study, we demonstrate that Cx43 is modified with lysine 63‐linked polyubiquitin chains and that these increase the interaction between Cx43 and AMSH. We also show that AMSH is recruited to GJ plaque sites at the plasma membrane, where it mediates the deubiquitination of Cx43. Using siRNA depletion or overexpression of a catalytically inactive mutant of AMSH, we show that by decreasing Cx43 deubiquitination, both the internalization and degradation rate of Cx43 are increased. Overall, these data strongly suggest that AMSH‐mediated deubiquitination of Cx43 protects GJs from degradation.—Ribeiro‐Rodrigues, T. M., Catarino, S., Marques, C., Ferreira, J. V., Martins‐Marques, T., Pereira, P., Girão, H., AMSH‐mediated deubiquitination of Cx43 regulates internalization and degradation of gap junctions. FASEB J. 28, 4629–4641 (2014). www.fasebj.org

Autophagy and Ubiquitination in Cardiovascular Diseases

A main function of the heart is to pump blood to the tissues and organs of the body. Although formed by different types of cells, the cardiomyocytes are the ones responsible for the coordinated and synchronized heart contraction. Given their low mitotic activity, cardiomyocytes largely depend on protein degradation mechanisms to maintain proteostasis and energetic balance. Autophagy, one of the main pathways whereby cells eliminate damaged, nonfunctional, or obsolete proteins, and organelles, is vital to ensure cell function, including in cardiomyocytes, both in rest and stress conditions. However, the impact of autophagy activation in the heart, being either protective or harmful, is not consensual and likely depends upon the severity of the stimuli and consequently the autophagy players involved. One of the signals that direct proteins for autophagy degradation, namely in the context of heart disorders, is ubiquitin. Indeed, the attachment of ubiquitin moieties to a target substrate and further recognition by autophagy adaptors constitute a main regulatory pathway that directs proteins to the lysosome. Therefore, a better understanding of the mechanisms and signals that regulate the autophagy process in the heart, including substrates targeting, is of utmost importance to design new approaches directed to this degradation pathway. We have previously shown that ubiquitination of the gap junction (GJ) protein Connexin43 (Cx43) triggers its degradation by autophagy through a process that requires the ubiquitin adaptors epidermal growth factor receptor substrate 15 (Eps15) and p62. This is particularly relevant in the heart because GJs, that form intercellular channels, are responsible for the rapid and efficient anisotropic propagation of the electrical impulse through the cardiomyocytes, essential for synchronized contraction of the cardiac muscle. In this review, we present recent studies devoted to the involvement of autophagy in heart homeostasis, with a particular focus on ubiquitin and GJs.

Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis

Aims Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). Methods and results Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. Conclusions This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply.

The Force at the Tip - Modelling Tension and Proliferation in Sprouting Angiogenesis

Sprouting angiogenesis, where new blood vessels grow from pre-existing ones, is a complex process where biochemical and mechanical signals regulate endothelial cell proliferation and movement. Therefore, a mathematical description of sprouting angiogenesis has to take into consideration biological signals as well as relevant physical processes, in particular the mechanical interplay between adjacent endothelial cells and the extracellular microenvironment. In this work, we introduce the first phase-field continuous model of sprouting angiogenesis capable of predicting sprout morphology as a function of the elastic properties of the tissues and the traction forces exerted by the cells. The model is very compact, only consisting of three coupled partial differential equations, and has the clear advantage of a reduced number of parameters. This model allows us to describe sprout growth as a function of the cell-cell adhesion forces and the traction force exerted by the sprout tip cell. In the absence of proliferation, we observe that the sprout either achieves a maximum length or, when the traction and adhesion are very large, it breaks. Endothelial cell proliferation alters significantly sprout morphology, and we explore how different types of endothelial cell proliferation regulation are able to determine the shape of the growing sprout. The largest region in parameter space with well formed long and straight sprouts is obtained always when the proliferation is triggered by endothelial cell strain and its rate grows with angiogenic factor concentration. We conclude that in this scenario the tip cell has the role of creating a tension in the cells that follow its lead. On those first stalk cells, this tension produces strain and/or empty spaces, inevitably triggering cell proliferation. The new cells occupy the space behind the tip, the tension decreases, and the process restarts. Our results highlight the ability of mathematical models to suggest relevant hypotheses with respect to the role of forces in sprouting, hence underlining the necessary collaboration between modelling and molecular biology techniques to improve the current state-of-the-art.

Connexin 43 ubiquitination determines the fate of gap junctions: restrict to survive

Connexins (Cxs) are transmembrane proteins that form channels which allow direct intercellular communication (IC) between neighbouring cells via gap junctions. Mechanisms that modulate the amount of channels at the plasma membrane have emerged as important regulators of IC and their de-regulation has been associated with various diseases. Although Cx-mediated IC can be modulated by different mechanisms, ubiquitination has been described as one of the major post-translational modifications involved in Cx regulation and consequently IC. In this review, we focus on the role of ubiquitin and its effect on gap junction intercellular communication.

Understanding the Dynamics of Tumor Angiogenesis: A Systems Biology Approach

The process of sprouting angiogenesis is extremely complex involving hundreds of proteins that regulate transcription and participate in signaling pathways controlling cellular movement, proliferation and phenotype alteration. Modeling has been attempted to understand all these mechanisms, and hence, in this chapter, we will focus on models that deal individually with each one of these mechanisms relevant to angiogenesis, as well as with platforms that integrate various models into multiscale models of the whole process.

Role of connexin 43 in different forms of intercellular communication – gap junctions, extracellular vesicles and tunnelling nanotubes

ABSTRACT Communication is important to ensure the correct and efficient flow of information, which is required to sustain active social networks. A fine-tuned communication between cells is vital to maintain the homeostasis and function of multicellular or unicellular organisms in a community environment. Although there are different levels of complexity, intercellular communication, in prokaryotes to mammalians, can occur through secreted molecules (either soluble or encapsulated in vesicles), tubular structures connecting close cells or intercellular channels that link the cytoplasm of adjacent cells. In mammals, these different types of communication serve different purposes, may involve distinct factors and are mediated by extracellular vesicles, tunnelling nanotubes or gap junctions. Recent studies have shown that connexin 43 (Cx43, also known as GJA1), a transmembrane protein initially described as a gap junction protein, participates in all these forms of communication; this emphasizes the concept of adopting strategies to maximize the potential of available resources by reutilizing the same factor in different scenarios. In this Review, we provide an overview of the most recent advances regarding the role of Cx43 in intercellular communication mediated by extracellular vesicles, tunnelling nanotubes and gap junctions. Summary: We give an overview of the intercellular communication mechanisms involving Cx43, describing the role played by Cx43 in gap junctions, tunnelling nanotubes and extracellular vesicles.

Characterization of phospholipid nitroxidation by LC-MS in biomimetic models and in H9c2 Myoblast using a lipidomic approach

Abstract Under nitroxidative stress conditions, lipids are prone to be modified by reaction with reactive nitrogen species (RNS) and different modifications were reported to occur in fatty acids. However, in the case of phospholipids (PL) studied under nitroxidative stress conditions, only nitroalkene derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were reported when using both in vitro biomimetic conditions and in vivo model system of type 1 diabetes mellitus. Therefore, in order to further explore other nitroxidative modifications of PL, a biomimetic model of nitroxidation combined with liquid chromatography mass spectrometry (MS) and MS/MS approaches were used to characterize the nitrated and nitroxidized derivatives of PCs and PEs. Single and multiple nitrated derivatives of phospholipids (PLs) such as nitroso and dinitroso, nitro, dinitro, and nitronitroso derivatives, together with nitroxidized derivatives were identified. Further, the specific MS/MS fragmentation pathways of these products were studied. Product ions arising from loss of HNO and HNO2, from the combined loss of HNO (or HNO2) and polar head groups, [NOn‐FA+On+H]+ and [NOn‐FA+On‐H]− (n=1–2) product ions corresponding to the modified fatty acyl chains were observed, depending on each modification. The knowledge obtained from the study of the MS/MS fragmentation pattern has allowed us to identify nitrated PCs, including NO2‐PC, (NO2)2‐PCs, (NO2)(NO)‐PC, NO‐PC; nitrated PEs, NO2‐PEs; and nitroxidized PCs, (NO2)(2O)‐PC in H9c2 cells under starvation, but not under ischemia or control conditions. The physiological relevance of this nitrated and nitroxidized PCs and PEs species observed exclusively in cardiomyoblast cells (H9c2) under starvation is still unknown but deserves to be explored. HighlightsFirst characterization by MS of nitroxidized phospholipids(PL) is reported.First time reported nitrated/nitroxidized PL in cardiomyoblast H9c2 in starvation.Nitrated PL were absent in cardiomyoblast H9c2 in ischemia conditions.

Proteostasis and SUMO in the heart.

Heart proteostasis relies on a complex and integrated network of molecular processes surveilling organ performance under physiological and pathological conditions. For this purpose, cardiac cells depend on the correct function of their proteolytic systems, such as the ubiquitin-proteasome system (UPS), autophagy and the calpain system. Recently, the role of protein SUMOylation (an ubiquitin-like modification), has emerged as important modulator of cardiac proteostasis, which will be the focus of this review.