Precondicionamiento Isquémico Remoto: bases de la protección y resultados clínicos

El precondicionamiento isquémico remoto es una manera eficaz de disminuir el daño por isquemia y reperfusión en el corazón y otros órganos como cerebro o riñón, en modelos experimentales. Este consiste en realizar entre 3 y 5 ciclos de 5 minutos de isquemia seguidos del mismo tiempo de reperfusión, en un tejido alejado del que se quiere proteger, normalmente una extremidad. Estudios preclínicos en animales indican que la isquemia precondicionante inicia señales nerviosas y humorales en el tejido isquémico remoto, que en el corazón activan mecanismos de protección. La señal nerviosa se origina en fibras sensoriales que a nivel cerebral producen una activación del sistema parasimpático. El nervio vago activa ganglios cardíacos intrínsecos del corazón lo que induce protección. Además, desde el tejido isquémico se liberan a la circulación diferentes mediadores que viajan en forma libre o en vesículas lipídicas (exosomas) que inician vías de señalización protectoras en el corazón. A pesar del éxito del precondicionamiento isquémico remoto en animales de experimentación, su aplicación en seres humanos no ha tenido resultados claros. Esta discrepancia puede deberse a una diversidad de factores tales como la edad, la existencia de otras patologías, uso de fármacos u otros tratamientos que afectan la respuesta de los pacientes. Se requiere un mayor conocimiento de las bases moleculares de este mecanismo de protección para que su aplicación en clínica sea exitosa.

In experimental models, remote ischemic preconditioning effectively decreases ischemia reperfusion injury to the heart and other organs such as the brain or kidney. It consists of 3 to 5 cycles of 5 minutes of ischemia followed by 5 minutes of reperfusion, in a remote tissue, usually a limb. Preclinical studies in animals indicate that preconditioning ischemia initiates neural and humoral signals in the remote ischemic tissue, which activate protective mechanisms in the heart. The nervous signal originates in sensory fibers that activate the parasympathetic system in the brain. The vagus nerve activates the intrinsic cardiac ganglia of the heart, leading to protection from ischemic injury. Furthermore, mediators are released from the ischemic tissue into the circulation that travels freely or in lipid vesicles (exosomes) to the heart where they initiate protective signaling pathways. Despite the success of remote ischemic preconditioning in experimental animals, its application in humans has not produced clear results. This discrepancy may be due to a variety of factors such as age, the existence of other pathologic processes, or the use of drugs or other treatments that affect the patient´s response. An increased knowledge of the molecular bases of this protective mechanism is required for its clinical application to be successful.

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.

Polycystin-1 Is a Crucial Regulator of BIN1 Expression and T-Tubule Remodeling Associated with the Development of Dilated Cardiomyopathy.

Cardiomyopathy is commonly observed in patients with autosomal dominant polycystic kidney disease (ADPKD), even when they have normal renal function and arterial pressure. The role of cardiomyocyte polycystin-1 (PC1) in cardiovascular pathophysiology remains unknown. PC1 is a potential regulator of BIN1 that maintains T-tubule structure, and alterations in BIN1 expression induce cardiac pathologies. We used a cardiomyocyte-specific PC1-silenced (PC1-KO) mouse model to explore the relevance of cardiomyocyte PC1 in the development of heart failure (HF), considering reduced BIN1 expression induced T-tubule remodeling as a potential mechanism. PC1-KO mice exhibited an impairment of cardiac function, as measured by echocardiography, but no signs of HF until 7-9 months of age. Of the PC1-KO mice, 43% died suddenly at 7 months of age, and 100% died after 9 months with dilated cardiomyopathy. Total BIN1 mRNA, protein levels, and its localization in plasma membrane-enriched fractions decreased in PC1-KO mice. Moreover, the BIN1 + 13 isoform decreased while the BIN1 + 13 + 17 isoform was overexpressed in mice without signs of HF. However, BIN1 + 13 + 17 overexpression was not observed in mice with HF. T-tubule remodeling and BIN1 score measured in plasma samples were associated with decreased PC1-BIN1 expression and HF development. Our results show that decreased PC1 expression in cardiomyocytes induces dilated cardiomyopathy associated with diminished BIN1 expression and T-tubule remodeling. In conclusion, positive modulation of BIN1 expression by PC1 suggests a novel pathway that may be relevant to understanding the pathophysiological mechanisms leading to cardiomyopathy in ADPKD patients.

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.

Inhibition of the proteasome preserves Mitofusin-2 and mitochondrial integrity, protecting cardiomyocytes during ischemia-reperfusion injury.

Cardiomyocyte loss is the main cause of myocardial dysfunction following an ischemia-reperfusion (IR) injury. Mitochondrial dysfunction and altered mitochondrial network dynamics play central roles in cardiomyocyte death. Proteasome inhibition is cardioprotective in the setting of IR; however, the mechanisms underlying this protection are not well-understood. Several proteins that regulate mitochondrial dynamics and energy metabolism, including Mitofusin-2 (Mfn2), are degraded by the proteasome. The aim of this study was to evaluate whether proteasome inhibition can protect cardiomyocytes from IR damage by maintaining Mfn2 levels and preserving mitochondrial network integrity. Using ex vivo Langendorff-perfused rat hearts and in vitro neonatal rat ventricular myocytes, we showed that the proteasome inhibitor MG132 reduced IR-induced cardiomyocyte death. Moreover, MG132 preserved mitochondrial mass, prevented mitochondrial network fragmentation, and abolished IR-induced reductions in Mfn2 levels in heart tissue and cultured cardiomyocytes. Interestingly, Mfn2 overexpression also prevented cardiomyocyte death. This effect was apparently specific to Mfn2, as overexpression of Miro1, another protein implicated in mitochondrial dynamics, did not confer the same protection. Our results suggest that proteasome inhibition protects cardiomyocytes from IR damage. This effect could be partly mediated by preservation of Mfn2 and therefore mitochondrial integrity.

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.

Reversible redox modifications of ryanodine receptor ameliorate ventricular arrhythmias in the ischemic-reperfused heart.

Previous results from our laboratory showed that phosphorylation of ryanodine receptor 2 (RyR2) by Ca(2+) calmodulin-dependent kinase II (CaMKII) was a critical but not the unique event responsible for the production of reperfusion-induced arrhythmogenesis, suggesting the existence of other mechanisms cooperating in an additive way to produce these rhythm alterations. Oxidative stress is a prominent feature of ischemia/reperfusion injury. Both CaMKII and RyR2 are proteins susceptible to alteration by redox modifications. This study was designed to elucidate whether CaMKII and RyR2 redox changes occur during reperfusion and whether these changes are involved in the genesis of arrhythmias. Langendorff-perfused hearts from rats or transgenic mice with genetic ablation of CaMKII phosphorylation site on RyR2 (S2814A) were subjected to ischemia-reperfusion in the presence or absence of a free radical scavenger (mercaptopropionylglycine, MPG) or inhibitors of NADPH oxidase and nitric oxide synthase. Left ventricular contractile parameters and monophasic action potentials were recorded. Oxidation and phosphorylation of CaMKII and RyR2 were assessed. Increased oxidation of CaMKII during reperfusion had no consequences on the level of RyR2 phosphorylation. Avoiding the reperfusion-induced thiol oxidation of RyR2 with MPG produced a reduction in the number of arrhythmias and did not modify the contractile recovery. Conversely, selective prevention of S-nitrosylation and S-glutathionylation of RyR2 was associated with higher numbers of arrhythmias and impaired contractility. In S2814A mice, treatment with MPG further reduced the incidence of arrhythmias. Taken together, the results suggest that redox modification of RyR2 synergistically with CaMKII phosphorylation modulates reperfusion arrhythmias.

Exercise preconditioning of myocardial infarct size in dogs is triggered by calcium.

We showed that exercise induces early and late myocardial preconditioning in dogs and that these effects are mediated through nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activation. As the intracoronary administration of calcium induces preconditioning and exercise enhances the calcium inflow to the cell, we studied if this effect of exercise triggers exercise preconditioning independently of its hemodynamic effects. We analyzed in 81 dogs the effect of blocking sarcolemmal L-type Ca channels with a low dose of verapamil on early and late preconditioning by exercise, and in other 50 dogs, we studied the effect of verapamil on NADPH oxidase activation in early exercise preconditioning. Exercise reduced myocardial infarct size by 76% and 52% (early and late windows respectively; P < 0.001 both), and these effects were abolished by a single low dose of verapamil given before exercise. This dose of verapamil did not modify the effect of exercise on metabolic and hemodynamic parameters. In addition, verapamil blocked the activation of NADPH oxidase during early preconditioning. The protective effect of exercise preconditioning on myocardial infarct size is triggered, at least in part, by calcium inflow increase to the cell during exercise and, during the early window, is mediated by NADPH oxidase activation.

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.

Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy.

AIMS: Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation-contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. METHODS AND RESULTS: To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. CONCLUSION: These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels.

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.

Modulation of cardiac ryanodine receptor activity by ROS and RNS.

Calcium release through cardiac ryanodine receptors (RyR2) triggers heart muscle contraction. Reactive oxygen/nitrogen species (ROS/RNS), normally produced in the heart, promote endogenous RyR2 S-nitrosylation and S-glutathionylation. These reversible redox modifications increase RyR2 activity in vitro, and presumably also in vivo. RyR2 S-glutathionylation increases under physiologically relevant conditions (tachycardia and exercise), suggesting that cardiac cells utilize this redox modification to increase RyR2 activity under increased demand. In contrast, in vivo changes in RyR2 S-nitrosylation in response to physiological stimuli remain uncharacterized. The number and identity of the highly reactive RyR2 cysteine residues and the nature of the redox modification they undergo are presently unknown. Likewise, the physiological sources of ROS/RNS responsible for functionally relevant RyR2 redox modifications have not been completely identified. The redox state of RyR2 is altered in heart failure leading to enhanced RyR2 activity, which presumably contributes to decrease SR calcium content and induce other calcium release abnormalities observed in heart failure. Greater understanding of RyR2 redox modulation is necessary to counteract the deleterious consequences of RyR2 activity deregulation caused by oxidative stress.

The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury.

Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) plays an important role mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr(17) site of phospholamban, without changes in total protein, consistent with an increase in CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of ryanodine receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/RyR2 did not change. Inhibition of the reverse Na(+)/Ca(2+)exchanger (NCX) mode (KBR7943) diminished phospholamban phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery. CaMKII-inhibition (KN-93), significantly decreased phospholamban phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis), TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca(2+)-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog, KN-92. Blocking SR Ca(2+) loading and release (thapsigargin/dantrolene), mitochondrial Ca(2+) uniporter (ruthenium red/RU360), or mitochondrial permeability transition pore (cyclosporine A), significantly decreased infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr(17) of phospholamban at the onset of reflow and exhibited a significant decrease in infarct size, apoptosis and necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.

Calpains and proteasomes mediate degradation of ryanodine receptors in a model of cardiac ischemic reperfusion.

Type-2 ryanodine receptors (RyR2)--the calcium release channels of cardiac sarcoplasmic reticulum--have a central role in cardiac excitation-contraction coupling. In the heart, ischemia/reperfusion causes a rapid and significant decrease in RyR2 content but the mechanisms responsible for this effect are not fully understood. We have studied the involvement of three proteolytic systems--calpains, the proteasome and autophagy--on the degradation of RyR2 in rat neonatal cardiomyocyte cultures subjected to simulated ischemia/reperfusion (sI/R). We found that 8h of ischemia followed by 16h of reperfusion decreased RyR2 content by 50% without any changes in RyR2 mRNA. Specific inhibitors of calpains and the proteasome prevented the decrease of RyR2 caused by sI/R, implicating both pathways in its degradation. Proteasome inhibitors also prevented the degradation of calpastatin, the endogenous calpain inhibitor, hindering the activation of calpain induced by calpastatin degradation. Autophagy was activated during sI/R as evidenced by the increase in LC3-II and beclin-1, two proteins involved in autophagosome generation, and in the emergence of GFP-LC3 containing vacuoles in adenovirus GFP-LC3 transduced cardiomyocytes. Selective autophagy inhibition, however, induced even further RyR2 degradation, making unlikely the participation of autophagy in sI/R-induced RyR2 degradation. Our results suggest that calpain activation as a result of proteasome-induced degradation of calpastatin initiates RyR2 proteolysis, which is followed by proteasome-dependent degradation of the resulting RyR2 fragments. The decrease in RyR2 content during ischemia/reperfusion may be relevant to the decrease of heart contractility after ischemia.

Ischemia enhances activation by Ca2+ and redox modification of ryanodine receptor channels from rat brain cortex.

Cerebral ischemia stimulates Ca2+ influx and thus increases neuronal intracellular free [Ca2+]. Using a rat model of cerebral ischemia without recirculation, we tested whether ischemia enhances the activation by Ca2+ of ryanodine receptor (RyR) channels, a requisite feature of RyR-mediated Ca2+-induced Ca2+ release (CICR). To this aim, we evaluated how single RyR channels from endoplasmic reticulum vesicles, fused into planar lipid bilayers, responded to cytoplasmic [Ca2+] changes. Endoplasmic reticulum vesicles were isolated from the cortex of rat brains incubated without blood flow for 5 min at 37 degrees C (ischemic) or at 4 degrees C (control). Ischemic brains displayed increased oxidative intracellular conditions, as evidenced by a lower ratio (approximately 130:1) of reduced/oxidized glutathione than controls (approximately 200:1). Single RyR channels from ischemic or control brains displayed the same three responses to Ca2+ reported previously, characterized by low, moderate, or high maximal activity. Relative to controls, RyR channels from ischemic brains displayed with increased frequency the high activity response and with lower frequency the low activity response. Both control and ischemic cortical vesicles contained the RyR2 and RyR3 isoforms in a 3:1 proportion, with undetectable amounts of RyR1. Ischemia reduced [3H]ryanodine binding and total RyR protein content by 35%, and increased at least twofold endogenous RyR2 S-nitrosylation and S-glutathionylation without affecting the corresponding RyR3 endogenous levels. In vitro RyR S-glutathionylation but not S-nitrosylation favored the emergence of high activity channels. We propose that ischemia, by enhancing RyR2 S-glutathionylation, allows RyR2 to sustain CICR; the resulting amplification of Ca2+ entry signals may contribute to cortical neuronal death.

Crosstalk between calcium and redox signaling: from molecular mechanisms to health implications.

Studies done many years ago established unequivocally the key role of calcium as a universal second messenger. In contrast, the second messenger roles of reactive oxygen and nitrogen species have emerged only recently. Therefore, their contributions to physiological cell signaling pathways have not yet become universally accepted, and many biological researchers still regard them only as cellular noxious agents. Furthermore, it is becoming increasingly apparent that there are significant interactions between calcium and redox species, and that these interactions modify a variety of proteins that participate in signaling transduction pathways and in other fundamental cellular functions that determine cell life or death. This review article addresses first the central aspects of calcium and redox signaling pathways in animal cells, and continues with the molecular mechanisms that underlie crosstalk between calcium and redox signals under a number of physiological or pathological conditions. To conclude, the review focuses on conditions that, by promoting cellular oxidative stress, lead to the generation of abnormal calcium signals, and how this calcium imbalance may cause a variety of human diseases including, in particular, degenerative diseases of the central nervous system and cardiac pathologies.

Exercise and tachycardia increase NADPH oxidase and ryanodine receptor-2 activity: possible role in cardioprotection.

AIM: Our objective was to investigate in cardiac muscle the contribution of NADPH oxidase to (a) ryanodine receptor-2 (RyR2) S-glutathionylation and (b) the preconditioning effects of exercise and tachycardia on infarct size following coronary artery occlusion. METHODS AND RESULTS: We measured NADPH oxidase activity, RyR2 S-glutathionylation, and calcium release kinetics in sarcoplasmic reticulum (SR) vesicles isolated from dog ventricular muscle after exercise and tachycardia, plus or minus prior administration of the NADPH oxidase inhibitor apocynin. In ventricular muscle sections, we studied the colocalization of NADPH oxidase and RyR2 by confocal microscopy using fluorescent antibodies. We determined the effect of apocynin on the infarct size produced by occlusion of the descendent anterior coronary artery in animals preconditioned by exercise or tachycardia. Exercise and tachycardia increased NADPH oxidase activity, RyR2 S-glutathionylation, and calcium release rates in isolated SR vesicles. Cardiac muscle sections displayed significant colocalization of NADPH oxidase and RyR2, suggesting direct and specific effects of reactive oxygen species (ROS) produced by NADPH oxidase on RyR2 activation. The NADPH oxidase inhibitor apocynin prevented the increase in RyR2 S-glutathionylation, reduced calcium release activity, and completely prevented the protective effects of exercise and tachycardia on infarct size. CONCLUSIONS: The loss of cardioprotection induced by the NADPH oxidase inhibitor suggests that ROS generated by this enzyme are important mediators of the preconditioning response, which presumably involves NADPH oxidase-induced RyR2 S-glutathionylation.

Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation.

We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.