Connexin 43 (Cx43) is present at the sarcolemma and the inner membrane of cardiomyocyte subsarcolemmal mitochondria (SSM). Lack or inhibition of mitochondrial Cx43 is associated with reduced mitochondrial potassium influx, which might affect mitochondrial respiration. Therefore, we analysed the importance of mitochondrial Cx43 for oxygen consumption. Acute inhibition of Cx43 in rat left ventricular (LV) SSM by 18α glycyrrhetinic acid (GA) or Cx43 mimetic peptides (Cx43-MP) reduced ADP-stimulated complex I respiration and ATP generation. Chronic reduction of Cx43 in conditional knockout mice (Cx43Cre-ER(T)/fl + 4-OHT, 5–10% of Cx43 protein compared with control Cx43fl/fl mitochondria) reduced ADP-stimulated complex I respiration of LV SSM to 47.8 ± 2.4 nmol O2/min.*mg protein (n = 8) from 61.9 ± 7.4 nmol O2/min.*mg protein in Cx43fl/fl mitochondria (n = 10, P < 0.05), while complex II respiration remained unchanged. The LV complex I activities (% of citrate synthase activity) of Cx43Cre-ER(T)/fl+4-OHT mice (16.1 ± 0.9%, n = 9) were lower than in Cx43fl/fl mice (19.8 ± 1.3%, n = 8, P < 0.05); complex II activities were similar between genotypes. Supporting the importance of Cx43 for respiration, in Cx43-overexpressing HL-1 cardiomyocytes complex I respiration was increased, whereas complex II respiration remained unaffected. Taken together, mitochondrial Cx43 is required for optimal complex I activity and respiration and thus mitochondrial ATP-production.

S-nitrosation (SNO) of connexin 43 (Cx43)-formed channels modifies dye uptake in astrocytes and gap junctional communication in endothelial cells. Apart from forming channels at the plasma membrane of several cell types, Cx43 is also located at the inner membrane of myocardial subsarcolemmal mitochondria (SSM), but not in interfibrillar mitochondria (IFM). The absence or pharmacological blockade of mitochondrial Cx43 (mtCx43) reduces dye and potassium uptake. Lack of mtCx43 is associated with loss of endogenous cardioprotection by ischemic preconditioning (IPC), which is mediated by formation of reactive oxygen species (ROS). Whether or not mitochondrial Lucifer Yellow (LY), ion uptake, or ROS generation are affected by SNO of mtCx43 and whether or not cardioprotective interventions affect SNO of mtCx43 remains unknown. In SSM from rat hearts, application of NO donors (48 nmol to 1 mmol) increased LY uptake (0.5 mmol SNAP 38.4 ± 7.1 %, p < 0.05; 1 mmol GSNO 28.1 ± 7.4 %, p < 0.05) and the refilling rate of potassium (SNAP 227.9 ± 30.1 %, p < 0.05; GSNO 122.6 ± 28.1 %, p < 0.05). These effects were absent following blockade of Cx43 hemichannels by carbenoxolone as well as in IFM lacking Cx43. Unlike potassium, the sodium permeability was not affected by application of NO. Furthermore, mitochondrial ROS formation was increased following NO application compared to control SSM (0.5 mmol SNAP 22.9 ± 1.8 %, p < 0.05; 1 mmol GSNO 40.6 ± 7.1 %, p < 0.05), but decreased in NO treated IFM compared to control (0.5 mmol SNAP 14.4 ± 4 %, p < 0.05; 1 mmol GSNO 13.8 ± 4 %, p < 0.05). NO donor administration to isolated SSM increased SNO of mtCx43 by 109.2 ± 15.8 %. Nitrite application (48 nmol) to mice was also associated with elevated SNO of mtCx43 by 59.3 ± 18.2 % (p < 0.05). IPC by four cycles of 5 min of ischemia and 5 min of reperfusion increased SNO of mtCx43 by 41.6 ± 1.7 % (p < 0.05) when compared to control perfused rat hearts. These data suggest that SNO of mtCx43 increases mitochondrial permeability, especially for potassium and leads to increased ROS formation. The increased amount of SNO mtCx43 by IPC or nitrite administration may link NO and Cx43 in the signal transduction cascade of cardioprotective interventions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-014-0433-x) contains supplementary material, which is available to authorized users.

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.

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.

Introduction: The troponin complex consists of three proteins that fundamentally couple excitation with contraction. Circulating cardiac-specific Troponin I (cTnI) serves as diagnostic biomarker tools for risk stratification of acute coronary syndromes and acute myocardial infarction (MI). Within the heart, cTnI oscillates between inactive and active conformations to either block or disinhibit actinomyosin formation. This molecular mechanism is fine-tuned through extensive protein modifications whose profiles are maladaptively altered with co-morbidities including hypertrophic cardiomyopathy, diabetes, and heart failure. Technological advances in analytical platforms over the last decade enable routine baseline cTnI analysis in patients without cardiovascular complications, and hold potential to expand cTnI readouts that include modified cTnI proteoforms. Areas covered: This review covers the current state, advances, and prospects of analytical platforms that now enable routine baseline cTnI analysis in patients. In parallel, improved mass spectrometry instrumentation and workflows already reveal an array of modified cTnI proteoforms with promising diagnostic implications. Expert commentary: New analytical capabilities provide clinicians and researchers with an opportunity to address important questions surrounding circulating cTnI in the improved diagnosis of specific patient cohorts. These techniques also hold considerable promise for new predictive and prescriptive applications for individualized profiling and improve patient care.

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.

Rationale: Phosphorylation 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, Ca 2+ -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. Objective: Phosphorylation changes that underlie HFpEF and those reversed by CDC therapy, with a focus on the sarcomeric subproteome were analyzed. Methods and Results: Dahl 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 to 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 PKCβ is the dominant kinase involved in hyperphosphorylation in HFpEF and is altered with CDC treatment. Conclusions: Increased 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,

Astrocytes protect neurons during cerebral injury through several postulated mechanisms. Recent therapeutic attention has focused on enhancing or augmenting the neuroprotective actions of astrocytes but in some instances astrocytes can assume a neurotoxic phenotype. The signaling mechanisms that drive astrocytes toward a protective versus toxic phenotype are not fully known but cell–cell signaling via proteases acting on cell‐specific receptors underlies critical mechanistic steps in neurodevelopment and disease. The protease activated receptor (PAR), resides in multiple brain cell types, and most PARs are found on astrocytes. We asked whether neuron‐generated thrombin constituted an important astrocyte activation signal because our previous studies have shown that neurons contain prothrombin gene and transcribed protein. We used neuron and astrocyte mono‐cell cultures exposed to oxygen‐glucose deprivation and a model of middle cerebral artery occlusion. We found that ischemic neurons secrete thrombin into culture media, which leads to astrocyte activation; such astrocyte activation can be reproduced with low doses of thrombin. Media from prothrombin‐deficient neurons failed to activate astrocytes and adding thrombin to such media restored activation. Astrocytes lacking PAR1 did not respond to neuron‐generated thrombin. Induced astrocyte activation was antagonized dose‐dependently with thrombin inhibitors or PAR1 antagonists. Ischemia‐induced astrocyte activation in vivo was inhibited after neuronal prothrombin knockout, resulting in larger strokes. Restoring prothrombin to neurons with a lentiviral gene vector restored astrocyte activation and reduced stroke damage. We conclude that neuron‐generated thrombin, released during ischemia, acts via PAR1 and may cause astrocyte activation and paracrine neuroprotection.