Sarcoplasmic reticulum (SR) Ca

Key point• 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.Abstract While β-adrenergic receptor (β-AR) stimulation leads to positive inotropic effects, it can also induce arrhythmogenic Ca 2+ waves. β-AR stimulation increases mitochondrial oxygen consumption and, thereby, the production of reactive oxygen species (ROS). We therefore investigated the role of ROS in the generation of Ca 2+ waves during β-AR stimulation in rabbit ventricular myocytes. Isoproterenol (ISO) increased Ca 2+ transient amplitude during systole, sarcoplasmic reticulum (SR) Ca 2+ load and the occurrence of Ca 2+ waves during diastole. These effects, however, developed at different time points during ISO application. While SR Ca 2+ release and load reached a maximum level after 3 min, Ca 2+ waves occurred at the highest frequency only after 6 min of ISO application. Measurement of intra-SR-free Ca 2+ concentration ([Ca 2+ ] SR ) showed an initial increase of SR Ca 2+ load followed by a gradual decline over time during ISO application. This decline of [Ca 2+ ] SR was not due to decreased SR Ca 2+ uptake, but instead was the result of increased SR Ca 2+ leak mainly in the form of Ca 2+ waves. ISO application led to significant RyR phosphorylation at the protein kinase A (PKA)-specific site, which remained relatively stable throughout β-AR activation. Moreover, β-AR stimulation significantly increased ROS production after 4-6 min of ISO application. The ROS scavenger Tiron and the superoxide dismutase mimetic MnTBPA abolished the ISO-mediated ROS production. The mitochondria-specific antioxidant Mito-Tempo and an inhibitor of the electron transport chain, rotenone, also effectively prevented the ISO-mediated ROS production. Scavenging ROS during ISO application decreased the occurrence of Ca 2+ waves and partially prevented augmentation of SR Ca 2+ leak, but did not affect

Zebrafish serves as a promising transgenic animal model that can be used to study cardiac Ca2+ regulation. However, mechanisms of sarcoplasmic reticulum (SR) Ca2+ handling in zebrafish heart have not been systematically explored. We found that in zebrafish ventricular myocytes the action potential (AP)-induced Ca2+ transient is mainly (80%) mediated by Ca2+ influx via L-type Ca2+ channels (LTCC) and only 20% by Ca2+ released from the SR. This small contribution of the SR to the Ca2+ transient was not the result of depleted SR Ca2+ load. We found that the ryanodine receptor (RyR) expression level in zebrafish myocytes was 78% lower compared to rabbit myocytes. In permeabilized myocytes, increasing cytosolic [Ca2+] from 100 to 350 nM did not trigger SR Ca2+ release. However, an application of a low dose of caffeine activated Ca2+ sparks. These results show that the zebrafish cardiac RyR is not sensitive to the mechanism of Ca2+-induced Ca2+ release. Activation of protein kinase A by forskolin increased phosphorylation of the RyR in zebrafish myocardium. In half of the studied cells, an increased Ca2+ transient by forskolin was entirely mediated by augmentation of LTCC current. In the remaining myocytes, the forskolin action was associated with an increase of both LTCC and SR Ca2+ release. These results indicate that the mechanism of excitation-contraction coupling in zebrafish myocytes differs from the mammalian one mainly because of the small contribution of SR Ca2+ release to the Ca2+ transient. This difference is due to a low sensitivity of RyRs to cytosolic [Ca2+].

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.

The sarcoplasmic reticulum calcium pump SERCA plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.