S-nitrosation of mitochondrial Connexin 43 regulates mitochondrial function: implication for cardioprotection

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

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

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

Newt cells secrete extracellular vesicles with therapeutic bioactivity in mammalian cardiomyocytes

Newts can regenerate amputated limbs and cardiac tissue, unlike mammals which lack broad regenerative capacity. Several signaling pathways involved in cell proliferation, differentiation and survival during newt tissue regeneration have been elucidated, however the factors that coordinate signaling between cells, as well as the conservation of these factors in other animals, are not well defined. Here we report that media conditioned by newt limb explant cells (A1 cells) protect mammalian cardiomyocytes from oxidative stress-induced apoptosis. The cytoprotective effect of A1-conditioned media was negated by exposing A1 cells to GW4869, which suppresses the generation of extracellular vesicles (EVs). A1-EVs are similar in diameter (~100–150 nm), structure, and share several membrane surface and cargo proteins with mammalian exosomes. However, isolated A1-EVs contain significantly higher levels of both RNA and protein per particle than mammalian EVs. Additionally, numerous cargo RNAs and proteins are unique to A1-EVs. Of particular note, A1-EVs contain numerous mRNAs encoding nuclear receptors, membrane ligands, as well as transcription factors. Mammalian cardiomyocytes treated with A1-EVs showed increased expression of genes in the PI3K/AKT pathway, a pivotal player in survival signaling. We conclude that newt cells secrete EVs with diverse, distinctive RNA and protein contents. Despite ~300 million years of evolutionary divergence between newts and mammals, newt EVs confer cytoprotective effects on mammalian cardiomyocytes

Sperm storage and copulation duration in a sexually cannibalistic spider

Female St Andrew’s Cross spiders control copulation duration by timing sexual cannibalism and may thereby control paternity if cannibalism affects sperm transfer. We have investigated the effect of copulation duration on sperm transfer and documented sperm storage patterns when we experimentally reduced the ability of females to attack and cannibalise the male. Virgin males and females were paired and randomly allocated either to a control treatment, where females were allowed to attack and cannibalise the male during copulation, or to an experimental treatment, where females were unable to cannibalise the male. The latter was achieved by placing a paintbrush against her chelicerae during copulation. Our experimental manipulation did not affect copulation duration or sperm storage. However, the number of sperm stored by the female increased with copulation duration only if the male was cannibalised, suggesting that cannibalism increases relative paternity not only through prolonged copulation duration following a fair raffle model but also through the cannibalism act itself. Future studies should explore whether cannibalised males ejaculate more sperm or whether females selectively store the sperm of cannibalised males.7 page(s

S-nitrosoglutathione reductase deficiency causes aberrant placental S-nitrosylation and preeclampsia

ABSTRACT Preeclampsia (PE), a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S-nitrosylated (SNO) proteins and reactive oxygen species (ROS), suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Here we show that mice lacking S-nitrosoglutathione reductase (GSNOR−/−), a denitrosylase regulating protein S-nitrosylation, exhibit a PE phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. ROS, nitric oxide (NO) and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental SNO-amino acid residues in GSNOR−/−mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S-nitrosoproteome, and identifies a unique set of SNO-proteins in GSNOR−/−mice. Importantly, the human preeclamptic placenta exhibits decreased GSNOR activity and increased nitrosative stress. Therefore, deficiency of GSNOR creates dysregulation of placental S-nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for PE. Competing Interest Statement The authors have declared no competing interest. Footnotes * This versions includes a full research article whereas the older version was written in a Report Form

S‐Nitrosoglutathione Reductase Deficiency Causes Aberrant Placental S‐Nitrosylation and Preeclampsia

Background Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S‐nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results Here, we show that mice lacking S‐nitrosoglutathione reductase (GSNOR−⁄−), a denitrosylase regulating protein S‐nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S‐nitrosylated amino acid residues in GSNOR−⁄− mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S‐nitrosoproteome, and identifies a unique set of S‐nitrosylated proteins in GSNOR−⁄− mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions Therefore, deficiency of GSNOR creates dysregulation of placental S‐nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia

Biological substrate modification suppresses ventricular arrhythmias in a porcine model of chronic ischaemic cardiomyopathy.

AimsCardiomyopathy patients are prone to ventricular arrhythmias (VA) and sudden cardiac death. Current therapies to prevent VA include radiofrequency ablation to destroy slowly conducting pathways of viable myocardium which support re-entry. Here, we tested the reverse concept, namely that boosting local tissue viability in zones of slow conduction might eliminate slow conduction and suppress VA in ischaemic cardiomyopathy.Methods and resultsExosomes are extracellular vesicles laden with bioactive cargo. Exosomes secreted by cardiosphere-derived cells (CDCEXO) reduce scar and improve heart function after intramyocardial delivery. In a VA-prone porcine model of ischaemic cardiomyopathy, we injected CDCEXO or vehicle into zones of delayed conduction defined by electroanatomic mapping. Up to 1-month post-injection, CDCEXO, but not the vehicle, decreased myocardial scar, suppressed slowly conducting electrical pathways, and inhibited VA induction by programmed electrical stimulation. In silico reconstruction of electrical activity based on magnetic resonance images accurately reproduced the suppression of VA inducibility by CDCEXO. Strong anti-fibrotic effects of CDCEXO, evident histologically and by proteomic analysis from pig hearts, were confirmed in a co-culture assay of cardiomyocytes and fibroblasts.ConclusionBiological substrate modification by exosome injection may be worth developing as a non-destructive alternative to conventional ablation for the prevention of recurrent ventricular tachyarrhythmias