Elisa Bovo

Ca2+ spark-dependent and -independent sarcoplasmic reticulum Ca2+ leak in normal and failing rabbit ventricular myocytes

Sarcoplasmic reticulum (SR) Ca2+ leak is an important component of cardiac Ca2+ signalling. Together with the SR Ca2+‐ATPase (SERCA)‐mediated Ca2+ uptake, diastolic Ca2+ leak determines SR Ca2+ load and, therefore, the amplitude of Ca2+ transients that initiate contraction. Spontaneous Ca2+ sparks are thought to play a major role in SR Ca2+ leak. In this study, we determined the quantitative contribution of sparks to SR Ca2+ leak and tested the hypothesis that non‐spark mediated Ca2+ release also contributes to SR Ca2+ leak. We simultaneously measured spark properties and intra‐SR free Ca2+ ([Ca2+]SR) after complete inhibition of SERCA with thapsigargin in permeabilized rabbit ventricular myocytes. When [Ca2+]SR declined to 279 ± 10 μm, spark activity ceased completely; however SR Ca2+ leak continued, albeit at a slower rate. Analysis of sparks and [Ca2+]SR revealed, that SR Ca2+ leak increased as a function of [Ca2+]SR, with a particularly steep increase at higher [Ca2+]SR (>600 μm) where sparks become a major pathway of SR Ca2+ leak. At low [Ca2+]SR (<300 μm), however, Ca2+ leak occurred mostly as non‐spark‐mediated leak. Sensitization of ryanodine receptors (RyRs) with low doses of caffeine increased spark frequency and SR Ca2+ leak. Complete inhibition of RyR abolished sparks and significantly decreased SR Ca2+ leak, but did not prevent it entirely, suggesting the existence of RyR‐independent Ca2+ leak. Finally, we found that RyR‐mediated Ca2+ leak was enhanced in myocytes from failing rabbit hearts. These results show that RyRs are the main, but not sole contributor to SR Ca2+ leak. RyR‐mediated leak occurs in part as Ca2+ sparks, but there is clearly RyR‐mediated but Ca2+ sparks independent leak.

Reactive oxygen species contribute to the development of arrhythmogenic Ca2+ waves during β-adrenergic receptor stimulation in rabbit cardiomyocytes

•  β‐Adrenergic receptor (β‐AR) stimulation is the most important positive inotropic effect on the heart, but it can also induce cardiac arrhythmias. •  In rabbit ventricular myocytes, short‐term β‐AR stimulation induced a positive inotropic effect that was associated with increased ryanodine receptor phosphorylation. •  However, prolonged β‐AR stimulation increased the occurrence of calcium waves during diastole. This effect was associated with an increase in the reactive oxygen species production and oxidation of thiol groups on ryanodine receptors. •  These results suggest that phosphorylation combined with oxidation of ryanodine receptors during β‐AR stimulation increases the receptor activity to a critical level leading to the generation of arrhythmogenic calcium waves. •  Thus, attenuating reactive oxygen species production during β‐AR stimulation may be a promising therapeutic strategy to prevent the occurrence of arrhythmias, while at the same time preserving cardiac positive inotropy.

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.

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca2+ Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca(2+) release during systole. However, it remains obscure how the dyadic organization affects SR Ca(2+) handling during diastole. By measuring intraluminal SR Ca(2+) ([Ca(2+)]SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca(2+) is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca(2+) that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca(2+)-ATPase (SERCA). Detubulation decreased [Ca(2+)]SR decline during rest, thus making the leaked SR Ca(2+) more accessible for SERCA. These results suggest that Ca(2+) extrusion systems are localized in T-tubules. Inhibition of Na(+)-Ca(2+) exchanger (NCX) slowed [Ca(2+)]SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca(2+)]SR decline. Despite a significant SR Ca(2+) leak, Ca(2+) sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca(2+) spark activity independent of SR Ca(2+) load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca(2+) that leaks from the SR. This can be explained if the majority of SR Ca(2+) leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca(2+)] by NCX play a critical role in suppressing Ca(2+) sparks during rest.

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.

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.

R-CEPIA1er as a new tool to directly measure sarcoplasmic reticulum [Ca] in ventricular myocytes.

In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR; [Ca]SR) partially determines the amplitude of cytosolic Ca transient that, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best approach available to directly measure [Ca]SR was to use low-affinity Ca indicators (e.g., Fluo-5N). However, this approach presents several limitations, including nonspecific cellular localization, dye extrusion, and species limitation. Recently a new genetically encoded family of Ca indicators has been generated, named Ca-measuring organelle-entrapped protein indicators (CEPIA). Here, we tested the red fluorescence SR-targeted Ca sensor (R-CEPIA1er) as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat ventricular myocytes with an adenovirus expressing the R-CEPIA1er gene displayed prominent localization in the SR and nuclear envelope. Calibration of R-CEPIA1er in myocytes resulted in a Kd of 609 μM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR [Ca]SR dynamics measured with R-CEPIA1er were compared with [Ca]SR measured with Fluo5-N. We found that both the time course of the [Ca]SR depletion and fractional SR Ca release induced by an action potential were similar between these two Ca sensors. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, measurement of [Ca]SR with R-CEPIA1er can be combined with cytosolic [Ca] measurements (with Fluo-4) to obtain more detailed information regarding Ca handling in cardiac myocytes. In conclusion, R-CEPIA1er is a promising tool that can be used to measure [Ca]SR dynamics in myocytes from different animal species.

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.