Oxidation Of Ryanodine Receptor Research Articles

Pharmacological Modulation of Mitochondrial Ca2+ Uptake Regulates Sarcoplasmic Reticulum Ca2+ Release via Oxidation of Ryanodine Receptor by Reactive Oxygen Species

Pharmacological Modulation of Mitochondrial Ca2+ Uptake Regulates Sarcoplasmic Reticulum Ca2+ Release via Oxidation of Ryanodine Receptor by Reactive Oxygen Species

The Role of Myofibroblast Senescence in Arrhythmogenesis of the Aged Infarcted Heart

Aging increases risk of Sudden Cardiac Death (SCD) by more than 10-20 fold. Prior myocardial infarction (MI) increases short- and long-term SCD risk. Cellular senescence is an irreversible cell cycle arrest process which regulates tumor suppression and wound healing, however, accumulation of senescent cells in various tissues is thought to drive aging pathology. We hypothesize that with age, persistent cardiac myofibroblast (CMF) senescence promotes persistent inflammation in the scar and thereby facilitating arrhythmogenesis. Our laboratory has established that the infarcted New Zealand White rabbit model of cardiac aging recapitulates pathology observed in aging humans, namely cardiac fibrosis, abnormal conduction, ryanodine receptor oxidation, and spontaneous and inducible ventricular arrhythmias post-MI. Here, we show that aged infarcted rabbits display increased peri-procedural arrhythmic death, prolonged action potential duration and slowed conduction at the IBZ compared to young, which correlates with anchoring of higher frequency ventricular fibrillation in the IBZ of aged rabbits. We show no change in scar geometry between young and aged rabbits in the first 3 weeks post-MI. However, we observed delayed and persistent CMF senescence in the aged scar, even until 12 weeks post-MI. We also demonstrate increased expression of senescence-associated markers in the aged IBZ and scar compared to young, and colocalization of senescent CMFs and inflammatory macrophages in the aged scar and IBZ. In conclusion, we demonstrate the infarcted aged heart heals differently than young, including delayed and persistent scar CMF senescence, increased VT/VF, and electrophysiological remodeling. Our data support our hypothesis that altered CMF senescence with age induces pathological tissue remodeling post-MI, underlying the increased risk of SCD.

Pharmacological Modulation of Mitochondrial Ca2+ Content Regulates Sarcoplasmic Reticulum Ca2+ Release via Oxidation of the Ryanodine Receptor by Mitochondria-Derived Reactive Oxygen Species.

In a physiological setting, mitochondria increase oxidative phosphorylation during periods of stress to meet increased metabolic demand. This in part is mediated via enhanced mitochondrial Ca2+ uptake, an important regulator of cellular ATP homeostasis. In a pathophysiological setting pharmacological modulation of mitochondrial Ca2+ uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca2+-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca2+ accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca2+ uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na+/Ca2+ exchanger, and MCU inhibitor Ru360 in rat ventricular myocytes (VMs) from control rats and rats with hypertrophy induced by thoracic aortic banding (TAB). In periodically paced VMs under β-adrenergic stimulation, treatment with kaempferol (10 μmol/L) or CGP-37157 (1 μmol/L) enhanced mitochondrial Ca2+ accumulation monitored by mitochondrial-targeted Ca2+ biosensor mtRCamp1h. Experiments with mitochondrial membrane potential-sensitive dye TMRM revealed this was accompanied by depolarization of the mitochondrial matrix. Using redox-sensitive OMM-HyPer and ERroGFP_iE biosensors, we found treatment with kaempferol or CGP-37157 increased the levels of reactive oxygen species (ROS) in mitochondria and the sarcoplasmic reticulum (SR), respectively. Confocal Ca2+ imaging showed that accelerated Ca2+ accumulation reduced Ca2+ transient amplitude and promoted generation of spontaneous Ca2+ waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca2+ release channel ryanodine receptor (RyR). Western blot analyses showed increased RyR oxidation after treatment with kaempferol or CGP-37157 vs. controls. Furthermore, in freshly isolated TAB VMs, confocal Ca2+ imaging demonstrated that enhancement of mitochondrial Ca2+ accumulation further perturbed global Ca2+ handling, increasing the number of cells exhibiting spontaneous Ca2+ waves, shortening RyR refractoriness and decreasing SR Ca2+ content. In ex vivo optically mapped TAB hearts, kaempferol exacerbated proarrhythmic phenotype. On the contrary, incubation of cells with MCU inhibitor Ru360 (2 μmol/L, 30 min) normalized RyR oxidation state, improved intracellular Ca2+ homeostasis and reduced triggered activity in ex vivo TAB hearts. These findings suggest facilitation of mitochondrial Ca2+ uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca2+ homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca2+ accumulation can be protective.

Oxidation of ryanodine receptor after ischemia-reperfusion increases propensity of Ca2+ waves during β-adrenergic receptor stimulation.

β-Adrenergic receptor (β-AR) activation produces the main positive inotropic response of the heart. During ischemia-reperfusion (I/R), however, β-AR activation can trigger life-threatening arrhythmias. Because I/R is frequently associated with oxidative stress, we investigated whether ryanodine receptor (RyR) oxidation contributes to proarrythmogenic Ca2+ waves during β-AR activation. Measurements of contractile and electrical activity from Langendorff-perfused rabbit hearts revealed that I/R produces tachyarrhythmias. Ventricular myocytes isolated from I/R hearts had an increased level of oxidized glutathione (i.e., oxidative stress) and a decreased level of free thiols in RyRs (i.e., RyR oxidation). Furthermore, myocytes from I/R hearts were characterized by increased sarcoplasmic reticulum (SR) Ca2+ leak and enhanced fractional SR Ca2+ release. In myocytes from nonischemic hearts, β-AR activation with isoproterenol (10 nM) produced only a positive inotropic effect, whereas in myocytes from ischemic hearts, isoproterenol at the same concentration triggered spontaneous Ca2+ waves. β-AR activation produced a similar effect on RyR phosphorylation in control and I/R myocytes. Treatment of myocytes from I/R hearts with the reducing agent mercaptopropionylglycine (100 μM) attenuated RyR oxidization and decreased Ca2+ wave frequency during β-AR activation. On the other hand, treatment of myocytes from nonischemic hearts with H2O2 (50 μM) increased SR Ca2+ leak and triggered Ca2+ waves during β-AR activation. Collectively, these results suggest that RyR oxidation after I/R plays a critical role in the transition from positive inotropic to arrhythmogenic effects during β-AR stimulation. Prevention of RyR oxidation can be a promising strategy to inhibit arrhythmias and preserve positive inotropic effect of β-AR activation during myocardial infarction. NEW & NOTEWORTHY Oxidative stress induced by ischemia plays a critical role in triggering arrhythmias during adrenergic stimulation. The combined increase in sarcoplasmic reticulum Ca2+ leak (because of ryanodine receptor oxidation) and sarcoplasmic reticulum Ca2+ load (because of adrenergic stimulation) can trigger proarrythmogenic Ca2+ waves. Restoring normal ryanodine receptor redox status can be a promising strategy to prevent arrhythmias and preserve positive inotropic effect of adrenergic stimulation during myocardial infarction.

The role of RyR2 oxidation in the blunted frequency-dependent facilitation of Ca2+ transient amplitude in rabbit failing myocytes.

Defective Ca2+ regulation plays a key role in the blunted force-frequency response in heart failure (HF). Since HF is commonly associated with oxidative stress, we studied whether oxidation of ryanodine receptor (RyR2) contributes to this defect. In control ventricular myocytes, oxidative stress induced formation of disulfide bonds between RyR2 subunits: intersubunit cross-linking (XL). Western blot analysis and Ca2+ imaging revealed a strong positive correlation between RyR2 XL and sarcoplasmic reticulum (SR) Ca2+ leak. These results illustrate that RyR2 XL can be used as a sensitive indicator of RyR2 dysfunction during oxidative stress. HF myocytes were in a state of oxidative stress since they exhibited an increase in reactive oxygen species (ROS) level, a decrease in ROS defense and an overall protein oxidation. These myocytes were also characterized by RyR2 XL and increased SR Ca2+ leak. Moreover, the frequency-dependent increase of Ca2+ transient amplitude was suppressed due to the inability of the SR to maintain Ca2+ load at high pacing rates. Because SR Ca2+ load is determined by the balance between SR Ca2+ uptake and leak, the blunted frequency-dependent inotropy in HF can be mediated by ROS-induced SR Ca2+ leak. Preventing RyR2 XL in HF myocytes decreased SR Ca2+ leak and increased Ca2+ transients at high pacing rate. We also studied whether RyR2 oxidation alone can cause the blunted frequency-dependent facilitation of Ca2+ transient amplitude in control myocytes. When RyR2 XL was induced in control myocytes to a similar level seen in HF, an increase of Ca2+ transient amplitude at high pacing rate was significantly suppressed. These results suggest that SR Ca2+ leak induced by RyR2 oxidation can play an important role in the blunted frequency-dependent inotropy of HF.

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain–domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM–RyR2 and FK506-binding protein (FKBP12.6)–RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca2+ sparks and spontaneous Ca2+ waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM–RyR2 binding, but had no effect on FKBP12.6–RyR2 binding. Dantrolene restored CaM–RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10–RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM–RyR2 affinity, has no effect on FKBP12.6–RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM–RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca2+-induced Ca2+ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca2+ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca2+ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca2+ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca2+]SR, increased SR Ca2+ fractional release, and increased spark- and non-spark-mediated SR Ca2+ leak. Single-channel recordings from RyR’s incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca2+ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca2+ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca2+ handling during periods of oxidative stress.

Oxidation of Ryanodine Receptor following Ischemia/Reperfusion Increases the Propensity of Ca Waves during Beta-Adrenergic Receptor Stimulation

Beta-Adrenergic receptor (beta-AR) stimulation generates the main positive inotropic response in the heart. However, after ischemia/reperfusion (I/R), beta-AR stimulation can generate arrhythmias. I/R also increased intracellular reactive oxygen species (ROS) production. We investigated whether ryanodine receptor (RyR) oxidation by ROS contributes to the transition from positive inotropic to arrythmogenic effect in ventricular myocytes after I/R. Measurements of contractile and electrical activity from ex vivo rabbit hearts revealed that global I/R produces severe tachi-arrhythmias. Ventricular myocytes isolated from ischemic hearts were characterized by increased both SR Ca leak rates and fractional SR Ca release compared to cells from non-ischemic hearts. Furthermore, myocytes from ischemic hearts showed increased ROS production, decreased level of free thiols in RyRs (RyR oxidation), and increased level of oxidized glutathione (GSSG). Pretreatment of myocytes from ischemic hearts with the reducing agent mercaptopropionylglycine attenuated the oxidization of free thiols in RyRs and normalized systolic SR Ca release and diastolic Ca leak. In myocytes from ischemic hearts, isoproterenol (ISO; 10 nM) led to the occurrence of spontaneous Ca waves, whereas in cells from non-ischemic hearts the same dose of ISO only caused a positive inotropic effect. Treatment of myocytes from non-ischemic hearts with H2O2 (0.1 mM) increased SR Ca leak and fractional SR Ca release to similar levels observed in myocytes from ischemic hearts. Moreover, application of ISO (10 nM) to myocytes from non-ischemic hearts pretreated with H2O2 increased the propensity of Ca waves. These results indicate that augmentation of ROS production following I/R causes RyR oxidation. This post-translational modification of the RyR plays a critical role in the transition from positive inotropic to arrhythmogenic effect during beta-AR stimulation.

Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy

Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak. Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.

Ryanodine Receptor Oxidation Causes Intracellular Calcium Leak and Muscle Weakness in Aging

Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24months) rodents was oxidized, cysteine-nitrosylated, and depleted of the channel-stabilizing subunit calstabin1, compared to RyR1 from younger (3-6months) adults. This RyR1 channel complex remodeling resulted in "leaky" channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Similarly, 6-month-old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month-old wild-type mice. Treating aged mice with S107 stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca(2+) release, muscle-specific force, and exercise capacity. Taken together, these data indicate that leaky RyR1 contributes to age-related loss of muscle function.

Redox modification of ryanodine receptors underlies calcium alternans in a canine model of sudden cardiac death

Although cardiac alternans is a known predictor of lethal arrhythmias, its underlying causes remain largely undefined in disease settings. The potential role of, and mechanisms responsible for, beat-to-beat alternations in the amplitude of systolic Ca(2+) transients (Ca(2+) alternans) was investigated in a canine post-myocardial infarction (MI) model of sudden cardiac death (SCD). Post-MI dogs had preserved left ventricular (LV) function and susceptibility to ventricular fibrillation (VF) during exercise. LV wedge preparations from VF dogs were more susceptible to action potential (AP) alternans and the frequency-dependence of Ca(2+) alternans was shifted towards slower rates in myocytes isolated from VF dogs relative to controls. In both groups of cells, cytosolic Ca(2+) transients ([Ca(2+)](c)) alternated in phase with changes in diastolic Ca(2+) in sarcoplasmic reticulum ([Ca(2+)](SR)), but the dependence of [Ca(2+)](c) amplitude on [Ca(2+)](SR) was steeper in VF cells. Abnormal ryanodine receptor (RyR) function in VF cells was indicated by increased fractional Ca(2+) release for a given amplitude of Ca(2+) current and elevated diastolic RyR-mediated SR Ca(2+) leak. SR Ca(2+) uptake activity did not differ between VF and control cells. VF myocytes had an increased rate of reactive oxygen species production and increased RyR oxidation. Treatment of VF myocytes with reducing agents normalized parameters of Ca(2+) handling and shifted the threshold of Ca(2+) alternans to higher frequencies. Redox modulation of RyRs promotes generation of Ca(2+) alternans by enhancing the steepness of the Ca(2+) release-load relationship and thereby providing a substrate for post-MI arrhythmias.