Cardiac responses to β‐adrenoceptor stimulation is partly dependent on mitochondrial calcium uniporter activity

Background and Purpose: Despite the importance of mitochondrial Ca2+ to metabolic regulation and cell physiology, little is known about the mechanisms that regulate Ca2+ entry into the mitochondria. Accordingly, we established a system to determine the role of the mitochondrial Ca2+ uniporter in an isolated heart model, at baseline and during increased workload following β-adrenoceptor stimulation. Experimental Approach: Cardiac contractility, oxygen consumption and intracellular Ca2+ transients were measured in ex vivo perfused murine hearts. Ru360 and spermine were used to modify mitochondrial Ca2+ uniporter activity. Changes in mitochondrial Ca2+ content and energetic phosphate metabolite levels were determined. Key Results: The addition of Ru360, a selective inhibitor of the mitochondrial Ca2+ uniporter, induced progressively and sustained negative inotropic effects that were dose-dependent with an EC50 of 7 μM. Treatment with spermine, a uniporter agonist, showed a positive inotropic effect that was blocked by Ru360. Inotropic stimulation with isoprenaline elevated oxygen consumption (2.7-fold), Ca2+-dependent activation of pyruvate dehydrogenase (5-fold) and mitochondrial Ca2+ content (2.5-fold). However, in Ru360-treated hearts, this parameter was attenuated. In addition, β-adrenoceptor stimulation in the presence of Ru360 did not affect intracellular Ca2+ handling, PKA or Ca2+/calmodulin-dependent PK signalling. Conclusions and Implications: Inhibition of the mitochondrial Ca2+ uniporter decreases β-adrenoceptor response, uncoupling between workload and production of energetic metabolites. Our results support the hypothesis that the coupling of workload and energy supply is partly dependent on mitochondrial Ca2+ uniporter activity.Centro de Investigaciones Cardiovasculare

An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy

Role of Mitochondrial ROS for Calcium Alternans in Atrial Myocytes

Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternations in CaT amplitude and is causally linked to atrial fibrillation (AF). Mitochondria play a significant role in cardiac excitation–contraction coupling and Ca signaling through redox environment regulation. In isolated rabbit atrial myocytes, ROS production is enhanced during CaT alternans, measured by fluorescence microscopy. Exogenous ROS (tert-butyl hydroperoxide) enhanced CaT alternans, whereas ROS scavengers (dithiothreitol, MnTBAP, quercetin, tempol) alleviated CaT alternans. While the inhibition of cellular NADPH oxidases had no effect on CaT alternans, interference with mitochondrial ROS (ROSm) production had profound effects: (1) the superoxide dismutase mimetic MitoTempo diminished CaT alternans and shifted the pacing threshold to higher frequencies; (2) the inhibition of cyt c peroxidase by SS-31, and inhibitors of ROSm production by complexes of the electron transport chain S1QEL1.1 and S3QEL2, decreased the severity of CaT alternans; however (3) the impairment of mitochondrial antioxidant defense by the inhibition of nicotinamide nucleotide transhydrogenase with NBD-Cl and thioredoxin reductase-2 with auranofin enhanced CaT alternans. Our results suggest that intact mitochondrial antioxidant defense provides crucial protection against pro-arrhythmic CaT alternans. Thus, modulating the mitochondrial redox state represents a potential therapeutic approach for alternans-associated arrhythmias, including AF

Small Interfering RNA Targeting Mitochondrial Calcium Uniporter Improves Cardiomyocyte Cell Viability in Hypoxia/Reoxygenation Injury by Reducing Calcium Overload

Intracellular Ca2+ mishandling is an underlying mechanism in hypoxia/reoxygenation (H/R) injury that results in mitochondrial dysfunction and cardiomyocytes death. These events are mediated by mitochondrial Ca2+ (mCa2+) overload that is facilitated by the mitochondrial calcium uniporter (MCU) channel. Along this line, we evaluated the effect of siRNA-targeting MCU in cardiomyocytes subjected to H/R injury. First, cardiomyocytes treated with siRNA demonstrated a reduction of MCU expression by 67%, which resulted in significant decrease in mitochondrial Ca2+ transport. siRNA treated cardiomyocytes showed decreased mitochondrial permeability pore opening and oxidative stress trigger by Ca2+ overload. Furthermore, after H/R injury MCU silencing decreased necrosis and apoptosis levels by 30% and 50%, respectively, and resulted in reduction in caspases 3/7, 9, and 8 activity. Our findings are consistent with previous conclusions that demonstrate that MCU activity is partly responsible for cellular injury induced by H/R and support the concept of utilizing siRNA-targeting MCU as a potential therapeutic strategy. © 2017 Yuriana Oropeza-Almazán et al

The Receptor-Binding Domain of Influenza Virus Hemagglutinin Produced in Escherichia coli Folds into Its Native, Immunogenic Structure ▿

The hemagglutinin (HA) surface glycoprotein promotes influenza virus entry and is the key protective antigen in natural immunity and vaccines. The HA protein is a trimeric envelope glycoprotein consisting of a globular receptor-binding domain (HA-RBD) that is inserted into a membrane fusion-mediating stalk domain. Similar to other class I viral fusion proteins, the fusogenic stalk domain spontaneously refolds into its postfusion conformation when expressed in isolation, consistent with this domain being trapped in a metastable conformation. Using X-ray crystallography, we show that the influenza virus HA-RBD refolds spontaneously into its native, immunogenic structure even when expressed in an unglycosylated form in Escherichia coli. In the 2.10-Å structure of the HA-RBD, the receptor-binding pocket is intact and its conformational epitopes are preserved. Recombinant HA-RBD is immunogenic and protective in ferrets, and the protein also binds with specificity to sera from influenza virus-infected humans. Overall, the data provide a structural basis for the rapid production of influenza vaccines in E. coli. From an evolutionary standpoint, the ability of the HA-RBD to refold spontaneously into its native conformation suggests that influenza virus acquired this domain as an insertion into an ancestral membrane-fusion domain. The insertion of independently folding domains into fusogenic stalk domains may be a common feature of class I viral fusion proteins

Proinflammatory Cytokines Are Soluble Mediators Linked with Ventricular Arrhythmias and Contractile Dysfunction in a Rat Model of Metabolic Syndrome

Metabolic syndrome (MS) increases cardiovascular risk and is associated with cardiac dysfunction and arrhythmias, although the precise mechanisms are still under study. Chronic inflammation in MS has emerged as a possible cause of adverse cardiac events. Male Wistar rats fed with 30% sucrose in drinking water and standard chow for 25–27 weeks were compared to a control group. The MS group showed increased weight, visceral fat, blood pressure, and serum triglycerides. The most important increases in serum cytokines included IL-1β (7-fold), TNF-α (84%), IL-6 (41%), and leptin (2-fold), the latter also showing increased gene expression in heart tissue (35-fold). Heart function ex vivo in MS group showed a decreased mechanical performance response to isoproterenol challenge (ISO). Importantly, MS hearts under ISO showed nearly twofold the incidence of ventricular fibrillation. Healthy rat cardiomyocytes exposed to MS group serum displayed impaired contractile function and Ca2+ handling during ISO treatment, showing slightly decreased cell shortening and Ca2+ transient amplitude (23%), slower cytosolic calcium removal (17%), and more frequent spontaneous Ca2+ release events (7.5-fold). As spontaneous Ca2+ releases provide a substrate for ventricular arrhythmias, our study highlights the possible role of serum proinflammatory mediators in the development of arrhythmic events during MS

Ex Vivo Cardiotoxicity of Antineoplastic Casiopeinas Is Mediated through Energetic Dysfunction and Triggered Mitochondrial-Dependent Apoptosis

Casiopeinas are a group of copper-based antineoplastic molecules designed as a less toxic and more therapeutic alternative to cisplatin or Doxorubicin; however, there is scarce evidence about their toxic effects on the whole heart and cardiomyocytes. Given this, rat hearts were perfused with Casiopeinas or Doxorubicin and the effects on mechanical performance, energetics, and mitochondrial function were measured. As well, the effects of Casiopeinas-triggered cell death were explored in isolated cardiomyocytes. Casiopeinas III-Ea, II-gly, and III-ia induced a progressive and sustained inhibition of heart contractile function that was dose- and time-dependent with an IC50 of 1.3 ± 0.2, 5.5 ± 0.5, and 10 ± 0.7 μM, correspondingly. Myocardial oxygen consumption was not modified at their respective IC50, although ATP levels were significantly reduced, indicating energy impairment. Isolated mitochondria from Casiopeinas-treated hearts showed a significant loss of membrane potential and reduction of mitochondrial Ca2+ retention capacity. Interestingly, Cyclosporine A inhibited Casiopeinas-induced mitochondrial Ca2+ release, which suggests the involvement of the mitochondrial permeability transition pore opening. In addition, Casiopeinas reduced the viability of cardiomyocytes and stimulated the activation of caspases 3, 7, and 9, demonstrating a cell death mitochondrial-dependent mechanism. Finally, the early perfusion of Cyclosporine A in isolated hearts decreased Casiopeinas-induced dysfunction with reduction of their toxic effect. Our results suggest that heart cardiotoxicity of Casiopeinas is similar to that of Doxorubicin, involving heart mitochondrial dysfunction, loss of membrane potential, changes in energetic metabolites, and apoptosis triggered by mitochondrial permeability