The endocannabinoid system:no longer anonymous in the control of nitrergic signalling?

The endocannabinoid system (ECS) is a key cellular signalling system which has been implicated in the regulation of diverse cellular functions. Importantly, growing evidence suggests that the biological actions of the ECS may, in part, be mediated through its ability to regulate the production and/or release of nitric oxide, a ubiquitous bioactive molecule which functions as a versatile signalling intermediate. Herein, we review and discuss evidence pertaining to ECS mediated regulation of nitric oxide production, the involvement of reactive nitrogen species in regulating ECS induced signal transduction, as well as highlighting emerging work supporting nitrergic modulation of ECS function. Importantly, the studies outlined reveal that interactions between the ECS and nitrergic signalling systems can be both stimulatory and inhibitory in nature, depending on cellular context. Moreover, such crosstalk may act to maintain proper cell function, whereas abnormalities in either system can undermine cellular homeostasis and contribute to various pathologies associated with their dysregulation. Consequently, future studies targeting these signalling systems may provide new insights into the potential role of the ECS - nitric oxide signalling axis in disease development, and/or lead to the identification of novel therapeutic targets for the treatment of nitrosative stress-related neurological, cardiovascular and metabolic disorders

A Novel Micropeptide, \u3cem\u3eSlitharin\u3c/em\u3e, Exerts Cardioprotective Effects in Myocardial Infarction

Purpose: Micropeptides are an emerging class of proteins that play critical roles in cell signaling. Here, we describe the discovery of a novel micropeptide, dubbed slitharin (Slt), in conditioned media from Cardiosphere-derived cells (CDCs), a therapeutic cardiac stromal cell type. Experimental design: We performed mass spectrometry of peptide-enriched fractions from the conditioned media of CDCs and a therapeutically inert cell type (human dermal fibrobasts). We then evaluated the therapeutic capacity of the candidate peptide using an in vitro model of cardiomyocyte injury and a rat model of myocardial infarction. Results: We identified a novel 24-amino acid micropeptide (dubbed Slitharin [Slt]) with a non-canonical leucine start codon, arising from long intergenic non-coding (LINC) RNA 2099. Neonatal rat ventricular myocytes (NRVMs) exposed to Slt were protected from hypoxic injury in vitro compared to a vehicle or scrambled control. Transcriptomic analysis of cardiomyocytes exposed to Slt reveals cytoprotective capacity, putatively through regulation of stress-induced MAPK-ERK. Slt also exerted cardioprotective effects in rats with myocardial infarction as shown by reduced infarct size 48 h post-injury. Conclusions and clinical relavance: Thus, Slt is a non-coding RNA-derived micropeptide, identified in the extracellular space, with a potential cardioprotective function

Interaction between Connexin 43 and nitric oxide synthase in mice heart mitochondria

Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down-regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild-type and Cx43-deficient (Cx43Cre-ER(T)/fl) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine-nucleotide-translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co-localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild-type mice. In contrast, iNOS expression was increased in Cx43Cre-ER(T)/fl mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43Cre-ER(T)/fl mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild-type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation. © 2015 The Authors

S-Nitrosierung von mitochondrialem Connexin 43 reguliert die mitochondriale Funktion und spielt eine Rolle bei der Kardioprotektion

S-nitrosation of connexin 43 formed channels alters dye uptake in astrocytes and gap junctional communication in endothelial cells. Apart from forming channels in the cell surface membrane of several cell types, connexin 43 is also located at the inner membrane of myocardial subsarcolemmal mitochondria, but not in interfibrillar mitochondria. The absence or pharmacological blockade of mitochondrial connexin 43 decreases mitochondrial dye and potassium uptake. A lack of mitochondrial connexin 43 is associated with the loss of cardioprotection by ischemic preconditioning, which is mediated by formation of reactive oxygen species. Whether or not mitochondrial Lucifer Yellow, ion uptake, or reactive oxygen generation are affected by S-nitrosation of mitochondrial connexin 43 and whether or not cardioprotective interventions influence S-nitrosation of mitochondrial connexin 43 remains unknown.Subsarcolemmal mitochondria from rat hearts showed an increased Lucifer Yellow uptake in response to nitric oxide donors (S-nitroso-N-acetyl-DL-penicillamine (SNAP): 38.4 ± 7.1%, p<0.05; S-nitrosoglutathione (GSNO): 28.1 ± 7.4%, p<0.05) and an increased refilling rate of potassium (SNAP: 227.9 ± 30.1%, p<0.05; GSNO: 122.6 ± 28.1%, p<0.05). These effects were abolished following blockade of connexin 43 hemichannel by carbenoxolone as well as in interfibrillar mitochondria, which lack connexin 43. Unlike potassium, the sodium permeability was not affected by application of nitric oxide. Furthermore, mitochondrial reactive oxygen species formation was enhanced in response to nitric oxide application compared to control treatment group (SNAP: 22.9 ± 1.8%, p<0.05; GSNO: 40.6 ± 7.1%, p<0.05), but decreased following nitric oxide treatment in interfibrillar mitochondria compared to control treated interfibrillar mitochondria (SNAP: 14.4 ± 4%, p<0.05; GSNO: 13.8 ± 4%, p<0.05). Administration of nitric oxide donors to isolated subsarcolemmal mitochondria or nitrite application into the cavity of left ventricles in mice in vivo enhanced S-nitrosation of mitochondrial connexin 43 by 109.2 ± 15.8% and by 59.3 ± 18.2%, respectively (p<0.05). Ischemic preconditioning by 4 cycles of ischemia and reperfusion, enhanced S-nitrosation of mitochondrial connexin 43 by 41.6 ± 1.7% (p<0.05) in comparison to subsarcolemmal mitochondria from control perfused rat hearts. These data suggest that S-nitrosation of mitochondrial connexin 43 increases mitochondrial permeability, especially for potassium and leads to increased formation of reactive oxygen species. The increased amount of S-nitrosated mitochondrial connexin 43 by ischemic preconditioning or nitrite administration may link nitric oxide and connexin 43 in the signal transduction cascade of cardioprotection by preconditioning.Neben der Bildung von transmembranen Kanälen an der Zelloberfläche, ist Connexin 43 auch in der inneren Membran von subsarkolemmalen Mitochondrien lokalisiert. In interfibrillären Mitochondrien ist Connexin 43 jedoch nicht nachweisbar. Die Abwesenheit oder pharmakologische Inhibierung von mitochondrialem Connexin 43 verringert die mitochondriale Farbstoff- und Kaliumaufnahme und führt zum Verlust von Kardioprotektion durch ischämische Präkonditionierung, welche durch die moderate Produktion von reaktiven Sauerstoffspezies ausgelöst wird. Die S-Nitrosierung von Connexin 43 gebildeten Membrankanälen führt zu einer veränderten Farbstoffaufnahme in Astrozyten und beeinflusst die auf Gap Junctions basierende Kommunikation zwischen den Zellen des Endothels. Gegenstand der vorliegenden Untersuchung ist die Analyse der S-Nitrosierung von mitochondrialem Connexin 43 und dessen Einfluss auf die mitochondriale Farbstoffaufnahme, mitochondriale Ioneneinströme, und Formierung reaktiver Sauerstoffspezies. Zusätzlich wurde die S-Nitrosierung vom mitochondrialem Connexin 43 nach kardioprotektiven Interventionen quantifiziert. In subsarkolemmalen Mitochondrien von Rattenherzen, die mit den Stickstoffmonoxid-Donatoren S-nitrosoglutathione (GSNO) und S-nitroso-N-acetyl-DL-penicillamine (SNAP) behandelt wurden, war die Lucifer Yellow Farbstoffaufnahme (SNAP: 38.4 ± 7.1%, p<0.05; GSNO: 28.1 ± 7.4%, p<0.05) und die Geschwindigkeit des Kaliumstroms erhöht (SNAP: 227.9 ± 30.1%, p<0.05; GSNO: 122.6 ± 28.1%, p<0.05). Die Wirkung der Stickstoffmonoxid-Donatoren wurde durch Inhibierung der Connexin 43 Hemikanäle aufgehoben und war in interfibrilären Mitochondrien, die kein Connexin 43 enthalten, nicht nachweisbar. Im Gegensatz zu Kalium, war die Natrium-Permeabilität durch die Verabreichung von Stickstoffmonoxid nicht beeinflussbar. Außerdem wurde die mitochondriale Produktion von reaktiven Sauerstoffspezies durch die Zugabe von Sticksfoffmonoxid-Donatoren (SNAP und GSNO) in subsarkolemmalen Mitochondrien gesteigert (SNAP: 22.9 ± 1.8%, p<0.05; GSNO: 40.6 ± 7.1%, p<0.05). Im Gegensatz dazu führte die Zugabe von Stickstoffmonoxid in interfibrillären Mitochondrien zu einer Reduktion der Produktion von reaktiven Sauerstoffspezies (SNAP: 14.4 ± 4%, p<0.05; GSNO: 13.8 ± 4%, p<0.05). Die Verabreichung von Stickstoffmonoxid-Donatoren oder die Injektion von Nitrit in den linken Ventrikel von Mäusen in vivo führte zu einer Erhöhung der S-Nitrosierung von Connexin 43 in subsarkolemmalen Mitochondrien um 109.2 ± 15.8% bzw. 59.3 ± 18.2%, (p<0.05). Ischämische Präkonditionierung, hervorgerufen durch vier Zyklen von Ischämie und Reperfusion, erhöhte die S-Nitrosierung vom mitochondrialem Connexin 43 um 41.6 ± 1.7% (p<0.05) im Vergleich zu subsarkolemmalen Mitochondrien von kontroll-perfundierten Rattenherzen.Die im Rahmen dieser Arbeit erfassten Daten zeigen, dass S-Nitrosierung von mitochondrialem Connexin 43 die mitochondriale Permeabilität für Farbstoff und besonders für Kalium-Ionen erhöht. Die S-Nitrosierung von mitochondrialem Connexin 43 führt zu einer erhöhten Produktion von reaktiven Sauerstoffspezies. Ischämische Präkonditionierung als auch die der Verabreichung von Nitrit führte zu einer erhöhten Menge an S-nitrosiertem mitochondrialen Connexin 43. Schlussfolgernd liegt es nahe, dass die S-Nitrosierung von mitochondrialem Connexin 43 für die Vermittlung des kardioprotektiven Signals von Bedeutung ist

Myofilament Phosphorylation in Stem Cell Treated Diastolic Heart Failure

RationalePhosphorylation of sarcomeric proteins has been implicated in heart failure with preserved ejection fraction (HFpEF); such changes may contribute to diastolic dysfunction by altering contractility, cardiac stiffness, Ca2+-sensitivity, and mechanosensing. Treatment with cardiosphere-derived cells (CDCs) restores normal diastolic function, attenuates fibrosis and inflammation, and improves survival in a rat HFpEF model.ObjectivePhosphorylation changes that underlie HFpEF and those reversed by CDC therapy, with a focus on the sarcomeric subproteome were analyzed.Methods and resultsDahl salt-sensitive rats fed a high-salt diet, with echocardiographically verified diastolic dysfunction, were randomly assigned to either intracoronary CDCs or placebo. Dahl salt-sensitive rats receiving low salt diet served as controls. Protein and phosphorylated Ser, Thr, and Tyr residues from left ventricular tissue were quantified by mass spectrometry. HFpEF hearts exhibited extensive hyperphosphorylation with 98% of the 529 significantly changed phospho-sites increased compared with control. Of those, 39% were located within the sarcomeric subproteome, with a large group of proteins located or associated with the Z-disk. CDC treatment partially reverted the hyperphosphorylation, with 85% of the significantly altered 76 residues hypophosphorylated. Bioinformatic upstream analysis of the differentially phosphorylated protein residues revealed PKC as the dominant putative regulatory kinase. PKC isoform analysis indicated increases in PKC α, β, and δ concentration, whereas CDC treatment led to a reversion of PKCβ. Use of PKC isoform specific inhibition and overexpression of various PKC isoforms strongly suggests that PKCβ is the dominant kinase involved in hyperphosphorylation in HFpEF and is altered with CDC treatment.ConclusionsIncreased protein phosphorylation at the Z-disk is associated with diastolic dysfunction, with PKC isoforms driving most quantified phosphorylation changes. Because CDCs reverse the key abnormalities in HFpEF and selectively reverse PKCβ upregulation, PKCβ merits being classified as a potential therapeutic target in HFpEF, a disease notoriously refractory to medical intervention