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.