Inhibition of NOX2 or NLRP3 inflammasome prevents cardiac remote ischemic preconditioning.

Introduction: Short episodes of ischemia-reperfusion (IR) in the heart (classical ischemic preconditioning, IPC) or in a limb (remote ischemic preconditioning, RIPC) before a prolonged ischemic episode, reduce the size of the infarct. It is unknown whether IPC and RIPC share common mechanisms of protection. Animals KO for NOX2, a superoxide-producing enzyme, or KO for NLRP3, a protein component of inflammasome, are not protected by IPC. The aim of this study was to investigate if NOX2 or NLRP3 inflammasome are involved in the protection induced by RIPC. Methods: We preconditioned rats using 4 × 5 min periods of IR in the limb with or without a NOX2 inhibitor (apocynin) or an NLRP3 inhibitor (Bay117082). In isolated hearts, we measured the infarct size after 30 min of ischemia and 60 min of reperfusion. In hearts from preconditioned rats we measured the activity of NOX2; the mRNA of Nrf2, gamma-glutamylcysteine ligase, glutathione dehydrogenase, thioredoxin reductase and sulfiredoxin by RT-qPCR; the content of glutathione; the activation of the NLRP3 inflammasome and the content of IL-1ß and IL-10 in cardiac tissue. In exosomes isolated from plasma, we quantified NOX2 activity. Results: The infarct size after IR decreased from 40% in controls to 9% of the heart volume after RIPC. This protective effect was lost in the presence of both inhibitors. RIPC increased NOX2 activity in the heart and exosomes, as indicated by the increased association of p47phox to the membrane and by the increased oxidation rate of NADPH. RIPC also increased the mRNA of Nrf2 and antioxidant enzymes. Also, RIPC increased the content of glutathione and the GSH/GSSG ratio. The inflammasome proteins NLRP3, procaspase-1, and caspase-1 were all increased in the hearts of RIPC rats. At the end of RIPC protocol, IL-1ß increased in plasma but decreased in cardiac tissue. At the same time, IL-10 did not change in cardiac tissue but increased by 70% during the next 50 min of perfusion. Conclusion: RIPC activates NOX2 which upregulates the heart's antioxidant defenses and activates the NLRP3 inflammasome which stimulates a cardiac anti-inflammatory response. These changes may underlie the decrease in the infarct size induced by RIPC.

Zebrafish Neural Crest: Lessons and Tools to Study In Vivo Cell Migration.

The study of cell migration has been greatly enhanced by the development of new model systems and analysis protocols to study this process in vivo. Zebrafish embryos have been a principal protagonist because they are easily accessible, genetically tractable, and optically transparent. Neural crest cells, on the other hand, are the ideal system to study cell migration. These cells migrate extensively, using different modalities of movement and sharing many traits with metastatic cancer cells. In this chapter, we present new tools and protocols that allow the study of NC development and migration in vivo.

Inhibition of chymotrypsin-like activity of the proteasome by ixazomib prevents mitochondrial dysfunction during myocardial ischemia.

The heart is critically dependent on mitochondrial respiration for energy supply. Ischemia decreases oxygen availability, with catastrophic consequences for cellular energy systems. After a few minutes of ischemia, the mitochondrial respiratory chain halts, ATP levels drop and ion gradients across cell membranes collapse. Activation of cellular proteases and generation of reactive oxygen species by mitochondria during ischemia alter mitochondrial membrane permeability, causing mitochondrial swelling and fragmentation and eventually cell death. The mitochondria, therefore, are important targets of cardioprotection against ischemic injury. We have previously shown that ixazomib (IXA), a proteasome inhibitor used for treating multiple myeloma, effectively reduced the size of the infarct produced by global ischemia in isolated rat hearts and prevented degradation of the sarcoplasmic reticulum calcium release channel RyR2. The aim of this work was to further characterize the protective effect of IXA by determining its effect on mitochondrial morphology and function after ischemia. We also quantified the effect of IXA on levels of mitofusin-2, a protein involved in maintaining mitochondrial morphology and mitochondria-SR communication. We found that mitochondria were significantly preserved and functional parameters such as oxygen consumption, the ability to generate a membrane potential, and glutathione content were improved in mitochondria isolated from hearts perfused with IXA prior to ischemia. IXA also blocked the release of cytochrome c observed in ischemia and significantly preserved mitofusin-2 integrity. These beneficial effects resulted in a significant decrease in the left ventricular end diastolic pressure upon reperfusion and a smaller infarct in isolated hearts.

What Is Next in This "Age" of Heme-Driven Pathology and Protection by Hemopexin? An Update and Links with Iron.

This review provides a synopsis of the published literature over the past two years on the heme-binding protein hemopexin (HPX), with some background information on the biochemistry of the HPX system. One focus is on the mechanisms of heme-driven pathology in the context of heme and iron homeostasis in human health and disease. The heme-binding protein hemopexin is a multi-functional protectant against hemoglobin (Hb)-derived heme toxicity as well as mitigating heme-mediated effects on immune cells, endothelial cells, and stem cells that collectively contribute to driving inflammation, perturbing vascular hemostasis and blood-brain barrier function. Heme toxicity, which may lead to iron toxicity, is recognized increasingly in a wide range of conditions involving hemolysis and immune system activation and, in this review, we highlight some newly identified actions of heme and hemopexin especially in situations where normal processes fail to maintain heme and iron homeostasis. Finally, we present preliminary data showing that the cytokine IL-6 cross talks with activation of the c-Jun N-terminal kinase pathway in response to heme-hemopexin in models of hepatocytes. This indicates another level of complexity in the cell responses to elevated heme via the HPX system when the immune system is activated and/or in the presence of inflammation.

N-Acetylcysteine Prevents the Spatial Memory Deficits and the Redox-Dependent RyR2 Decrease Displayed by an Alzheimer's Disease Rat Model.

We have previously reported that primary hippocampal neurons exposed to synaptotoxic amyloid beta oligomers (AßOs), which are likely causative agents of Alzheimer's disease (AD), exhibit abnormal Ca2+ signals, mitochondrial dysfunction and defective structural plasticity. Additionally, AßOs-exposed neurons exhibit a decrease in the protein content of type-2 ryanodine receptor (RyR2) Ca2+ channels, which exert critical roles in hippocampal synaptic plasticity and spatial memory processes. The antioxidant N-acetylcysteine (NAC) prevents these deleterious effects of AßOs in vitro. The main contribution of the present work is to show that AßOs injections directly into the hippocampus, by engaging oxidation-mediated reversible pathways significantly decreased RyR2 protein content but increased single RyR2 channel activation by Ca2+ and caused considerable spatial memory deficits. AßOs injections into the CA3 hippocampal region impaired rat performance in the Oasis maze spatial memory task, decreased hippocampal glutathione levels and overall content of plasticity-related proteins (c-Fos, Arc, and RyR2) and increased ERK1/2 phosphorylation. In contrast, in hippocampus-derived mitochondria-associated membranes (MAM) AßOs injections increased RyR2 levels. Rats fed with NAC for 3-weeks prior to AßOs injections displayed comparable redox potential, RyR2 and Arc protein contents, similar ERK1/2 phosphorylation and RyR2 single channel activation by Ca2+ as saline-injected (control) rats. NAC-fed rats subsequently injected with AßOs displayed the same behavior in the spatial memory task as control rats. Based on the present in vivo results, we propose that redox-sensitive neuronal RyR2 channels partake in the mechanism underlying AßOs-induced memory disruption in rodents.

High-Fat-Diet-Induced Obesity Produces Spontaneous Ventricular Arrhythmias and Increases the Activity of Ryanodine Receptors in Mice.

Ventricular arrhythmias are a common cause of sudden cardiac death, and their occurrence is higher in obese subjects. Abnormal gating of ryanodine receptors (RyR2), the calcium release channels of the sarcoplasmic reticulum, can produce ventricular arrhythmias. Since obesity promotes oxidative stress and RyR2 are redox-sensitive channels, we investigated whether the RyR2 activity was altered in obese mice. Mice fed a high fat diet (HFD) became obese after eight weeks and exhibited a significant increase in the occurrence of ventricular arrhythmias. Single RyR2 channels isolated from the hearts of obese mice were more active in planar bilayers than those isolated from the hearts of the control mice. At the molecular level, RyR2 channels from HFD-fed mice had substantially fewer free thiol residues, suggesting that redox modifications were responsible for the higher activity. Apocynin, provided in the drinking water, completely prevented the appearance of ventricular arrhythmias in HFD-fed mice, and normalized the activity and content of the free thiol residues of the protein. HFD increased the expression of NOX4, an isoform of NADPH oxidase, in the heart. Our results suggest that HFD increases the activity of RyR2 channels via a redox-dependent mechanism, favoring the appearance of ventricular arrhythmias.

Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death.

Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion.

Leader Cells Define Directionality of Trunk, but Not Cranial, Neural Crest Cell Migration.

Collective cell migration is fundamental for life and a hallmark of cancer. Neural crest (NC) cells migrate collectively, but the mechanisms governing this process remain controversial. Previous analyses in Xenopus indicate that cranial NC (CNC) cells are a homogeneous population relying on cell-cell interactions for directional migration, while chick embryo analyses suggest a heterogeneous population with leader cells instructing directionality. Our data in chick and zebrafish embryos show that CNC cells do not require leader cells for migration and all cells present similar migratory capacities. In contrast, laser ablation of trunk NC (TNC) cells shows that leader cells direct movement and cell-cell contacts are required for migration. Moreover, leader and follower identities are acquired before the initiation of migration and remain fixed thereafter. Thus, two distinct mechanisms establish the directionality of CNC cells and TNC cells. This implies the existence of multiple molecular mechanisms for collective cell migration.

Stimulation of NOX2 in isolated hearts reversibly sensitizes RyR2 channels to activation by cytoplasmic calcium.

The response of ryanodine receptor (RyR) channels to cytoplasmic free calcium concentration ([Ca(2+)]) is redox sensitive. Here, we report the effects of a mild oxidative stress on cardiac RyR (RyR2) channels in Langendorff perfused rat hearts. Single RyR2 channels from control ventricles displayed the same three responses to Ca(2+) reported in other mammalian tissues, characterized by low, moderate, or high maximal activation. A single episode of 5 min of global ischemia, followed by 1 min of reperfusion, enhanced 2.3-fold the activity of NOX2 compared to controls and changed the frequency distribution of the different responses of RyR2 channels to calcium, favoring the more active ones: high activity response increased and low activity response decreased with respect to controls. This change was fully prevented by perfusion with apocynin or VAS 2870 before ischemia and totally reversed by the extension of the reperfusion period to 15 min. In vitro activation of NOX2 in control SR vesicles mimicked the effect of the ischemia/reperfusion episode on the frequencies of emergence of single RyR2 channel responses to [Ca(2+)] and increased 2.2-fold the rate of calcium release in Ca(2+)-loaded SR vesicles. In vitro changes were reversed at the single channel level by DTT and in isolated SR vesicles by glutaredoxin. Our results indicate that in whole hearts a mild oxidative stress enhances the response of cardiac RyR2 channels to calcium via NOX2 activation, probably by S-glutathionylation of RyR2 protein. This change is transitory and fully reversible, suggesting a possible role of redox modification in the physiological response of cardiac RyR2 to cellular calcium influx.

Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine.

The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca²âº release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca²âº uptake completion prevented this response. Maurocalcine enhanced equilibrium [³H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent Ki for Mg²âº inhibition of [³H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca²âº] from 10 µM to 0.1 µM or at cytoplasmic [Mg²âº]≥30 µM. At 0.1 µM [Ca²âº], only channels that displayed poor activation by Ca²âº were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.

Preconditioning tachycardia decreases the activity of the mitochondrial permeability transition pore in the dog heart.

Cardioprotection by preconditioning is a central issue of current research on heart function. Several reports indicate that preventing the assembly and opening of the mitochondrial permeability transition pore (mPTP) protects the heart against ischemia-reperfusion injury. We have previously reported that brief episodes of tachycardia decrease the infarct size produced by subsequent prolonged occlusion of a coronary artery, indicating that controlled tachycardia is an effective preconditioning manoeuvre. The effects of preconditioning tachycardia on mPTP activity have not been reported. Therefore, in this work we investigated if preconditioning tachycardia protects against calcium-induced mitochondrial swelling, a measure of mPTP activity. We found that tachycardia decreased by 2.5-fold the rate of mitochondrial calcium-induced swelling, a factor that presumably contributes to the cardioprotective effects of tachycardia. The oxidative status of the cell increased after tachycardia, as evidenced by the decrease in the cellular and mitochondrial GSH/GSSG ratio. We also observed increased S-glutathionylation of cyclophilin-D, an essential mPTP component, after tachycardia. This reversible redox modification of cyclophilin-D may account, al least in part, for the decreased mPTP activity produced by preconditioning tachycardia.