Stefan R. Mazurek

Ca handling during excitation–contraction coupling in heart failure

In the heart, coupling between excitation of the surface membrane and activation of contractile apparatus is mediated by Ca released from the sarcoplasmic reticulum (SR). Several components of Ca machinery are perfectly arranged within the SR network and the T-tubular system to generate a regular Ca cycling and thereby rhythmic beating activity of the heart. Among these components, ryanodine receptor (RyR) and SR Ca ATPase (SERCA) complexes play a particularly important role and their dysfunction largely underlies abnormal Ca homeostasis in diseased hearts such as in heart failure. The abnormalities in Ca regulation occur at practically all main steps of Ca cycling in the failing heart, including activation and termination of SR Ca release, diastolic SR Ca leak, and SR Ca uptake. The contributions of these different mechanisms to depressed contractile function and enhanced arrhythmogenesis may vary in different HF models. This brief review will therefore focus on modifications in RyR and SERCA structure that occur in the failing heart and how these molecular modifications affect SR Ca regulation and excitation–contraction coupling.

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

Non‐technical summary  Contraction and relaxation of the heart strongly depend on calcium (Ca2+) stored in the sarcoplasmic reticulum (SR). Ca2+ stored within the SR is determined by the balance between Ca2+ uptake and Ca2+ leak that occurs mainly via Ca2+ release channels, called ryanodine receptors (RyRs). Alterations in the RyR activity can lead to enhanced SR Ca2+ leak and arrhythmias. Ca2+ tightly regulates the RyR activity from both sides of the SR (cytosolic and luminal). In this work, we studied the effects of cytosolic Ca2+ on SR Ca2+ leak in isolated ventricular myocytes. Elevation of cytosolic Ca2+ increased SR Ca2+ leak by a direct activation of RyRs. In intact myocytes, at the end of contraction and at the beginning of the relaxation phase, SR Ca2+ leak remains relatively constant due to the coordinated regulation of RyRs by cytosolic and luminal Ca2+. Thus, this dual regulation of the RyR contributes to the control of the SR Ca2+ content, preventing excessive loss of Ca2+ that could lead to pathological conditions such as cardiac arrhythmia.

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 Ca(2+)-induced Ca(2+) release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca(2+) release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca(2+) 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 Ca(2+) release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca(2+)]SR, increased SR Ca(2+) fractional release, and increased spark- and non-spark-mediated SR Ca(2+) leak. Single-channel recordings from RyRs 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 Ca(2+) load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca(2+) leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca(2+) handling during periods of oxidative stress.

Functional Impact of Ryanodine Receptor Oxidation on Intracellular Calcium Regulation in the Heart

Type 2 ryanodine receptor (RyR2) serves as the major intracellular Ca2+ release channel that drives heart contraction. RyR2 is activated by cytosolic Ca2+ via the process of Ca2+-induced Ca2+ release (CICR). To ensure stability of Ca2+ dynamics, the self-reinforcing CICR must be tightly controlled. Defects in this control cause sarcoplasmic reticulum (SR) Ca2+ mishandling, which manifests in a variety of cardiac pathologies that include myocardial infarction and heart failure. These pathologies are also associated with oxidative stress. Given that RyR2 contains a large number of cysteine residues, it is no surprise that RyR2 plays a key role in the cellular response to oxidative stress. RyRs many cysteine residues pose an experimental limitation in defining a specific target or mechanism of action for oxidative stress. As a result, the current understanding of redox-mediated RyR2 dysfunction remains incomplete. Several oxidative modifications, including S-glutathionylation and S-nitrosylation, have been suggested playing an important role in the regulation of RyR2 activity. Moreover, oxidative stress can increase RyR2 activity by forming disulfide bonds between two neighboring subunits (intersubunit cross-linking). Since intersubunit interactions within the RyR2 homotetramer complex dictate the channel gating, such posttranslational modification of RyR2 would have a significant impact on RyR2 function and Ca2+ regulation. This review summarizes recent findings on oxidative modifications of RyR2 and discusses contributions of these RyR2 modifications to SR Ca2+ mishandling during cardiac pathologies.

Increased Energy Demand during Adrenergic Receptor Stimulation Contributes to Ca2+ Wave Generation

While β-adrenergic receptor (β-AR) stimulation ensures adequate cardiac output during stress, it can also trigger life-threatening cardiac arrhythmias. We have previously shown that proarrhythmic Ca(2+) waves during β-AR stimulation temporally coincide with augmentation of reactive oxygen species (ROS) production. In this study, we tested the hypothesis that increased energy demand during β-AR stimulation plays an important role in mitochondrial ROS production and Ca(2+)-wave generation in rabbit ventricular myocytes. We found that β-AR stimulation with isoproterenol (0.1 μM) decreased the mitochondrial redox potential and the ratio of reduced to oxidated glutathione. As a result, β-AR stimulation increased mitochondrial ROS production. These metabolic changes induced by isoproterenol were associated with increased sarcoplasmic reticulum (SR) Ca(2+) leak and frequent diastolic Ca(2+) waves. Inhibition of cell contraction with the myosin ATPase inhibitor blebbistatin attenuated oxidative stress as well as spontaneous SR Ca(2+) release events during β-AR stimulation. Furthermore, we found that oxidative stress induced by β-AR stimulation caused the formation of disulfide bonds between two ryanodine receptor (RyR) subunits, referred to as intersubunit cross-linking. Preventing RyR cross-linking with N-ethylmaleimide decreased the propensity of Ca(2+) waves induced by β-AR stimulation. These data suggest that increased energy demand during sustained β-AR stimulation weakens mitochondrial antioxidant defense, causing ROS release into the cytosol. By inducing RyR intersubunit cross-linking, ROS can increase SR Ca(2+) leak to the critical level that can trigger proarrhythmic Ca(2+) waves.

Cytosolic Ca2+ buffering determines the intra-SR Ca2+ concentration at which cardiac Ca2+ sparks terminate

Single ryanodine receptor (RyR) Ca(2+) flux amplitude (i(Ca-RyR)) decreases as intra-sarcoplasmic reticulum (SR) Ca(2+) levels fall during a cardiac Ca(2+) spark. Since i(Ca-RyR) drives the inter-RyR Ca(2+)-induced Ca(2+) release (CICR) that underlies the spark, decreasing i(Ca-RyR) may contribute to spark termination because RyRs that spontaneously close may stay closed. To test this possibility, we simultaneously measured local cytosolic and intra-SR ([Ca(2+)]cyto and [Ca(2+)]SR) during Ca(2+) sparks in permeabilized rabbit ventricular myocytes. Local cytosolic or intra-SR Ca(2+) dynamics were manipulated using Ca(2+) buffers. Buffer manipulations applied in cells had no effect on individual RyR channels reconstituted in planar lipid bilayers. Presence of a fast cytosolic Ca(2+) buffer (BAPTA) significantly suppressed Ca(2+) spark activity and sparks terminated earlier at a higher than usual [Ca(2+)]SR level (∼80% vs. ∼62%). When cytosolic Ca(2+) buffer power was reduced (i.e. cytosolic EGTA level decreased), sparks terminated later and at a lower than usual [Ca(2+)]SR level (∼45% vs. ∼62%). When intra-SR Ca(2+) buffer power was increased, sparks also terminated later and at a lower than usual [Ca(2+)]SR (∼48% vs. ∼62%). These results suggest that cytosolic local control of inter-RyR CICR by i(Ca-RyR) plays a substantial role during the spark termination process. Thus, alterations in local cytosolic Ca(2+) handling dynamics in the dyadic cleft (Ca(2+) buffering, extrusion, etc.) likely influence Ca(2+) spark termination.

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 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.

Nitrosylation of RyR2 Prevents Activation of Ca Waves Induced by Redox-Mediated Intersubunit Cross-Linking

We have recently discovered a novel post-translational modification of type-2 ryanodine receptor (cardiac RyR2) induced by oxidative stress: intersubunit cross-linking (XL). Because RyR gating is associated with large-scale intersubunit dynamics, we hypothesized that XL is functionally the most important redox modification of RyR2. We have developed a simple cell model system to define the functional importance of RyR2 XL. We found that co-expressing recombinant human RyR2 and the SR Ca-ATPase (SERCA2a) in HEK293 cells generates Ca oscillations with similar kinetics to cardiac Ca waves. The frequency of these “cardiac-like” Ca waves was significantly increased in the presence of oxidants, and this effect was associated with RyR2 XL. By analyzing intra-luminal [Ca] dynamics, we concluded that activation of Ca waves by oxidants was due to increased RyR2-mediated Ca leak (but not Ca uptake). Pretreating HEK293 cells co-expressing RyR2/SERCA2a with nitric oxide (NO) donors (30 μM GSNO or SNAP) effectively prevented RyR2 XL and the augmentation of Ca waves induced by oxidants. NO donors by themselves did not affect Ca waves in control conditions. It has been shown in the homologous type-1 RyR (skeletal RyR1) that cysteine 3635 is involved in intersubunit XL as well as in S-nitrosylation. Accordingly, we tested whether mutation of the homologous cysteine in cardiac RyR2 (Cys-3602) abolishes XL. We found that mutation of this cysteine to alanine did not prevent intersubunit XL or the augmentation of Ca waves during oxidative stress. Thus, despite high homology between RyR1 and RyR2, it appears that these channels exhibit important structural and functional differences in redox regulation. Importantly, these results illustrate that S-nitrosylation of RyR2 can be an effective approach to prevent RyR2 XL and, thus, SR Ca mishandling during oxidative stress.

Regulation of sarcoplasmic reticulum Ca2+leak by cytosolic Ca2+in rabbit ventricular myocytes: Cytosolic Ca2+regulates SR Ca2+leak

Non‐technical summary  Contraction and relaxation of the heart strongly depend on calcium (Ca2+) stored in the sarcoplasmic reticulum (SR). Ca2+ stored within the SR is determined by the balance between Ca2+ uptake and Ca2+ leak that occurs mainly via Ca2+ release channels, called ryanodine receptors (RyRs). Alterations in the RyR activity can lead to enhanced SR Ca2+ leak and arrhythmias. Ca2+ tightly regulates the RyR activity from both sides of the SR (cytosolic and luminal). In this work, we studied the effects of cytosolic Ca2+ on SR Ca2+ leak in isolated ventricular myocytes. Elevation of cytosolic Ca2+ increased SR Ca2+ leak by a direct activation of RyRs. In intact myocytes, at the end of contraction and at the beginning of the relaxation phase, SR Ca2+ leak remains relatively constant due to the coordinated regulation of RyRs by cytosolic and luminal Ca2+. Thus, this dual regulation of the RyR contributes to the control of the SR Ca2+ content, preventing excessive loss of Ca2+ that could lead to pathological conditions such as cardiac arrhythmia.