Phosphorylation Of Cardiac Ryanodine Receptor Research Articles

Phosphorylation of cardiac ryanodine receptor (RyR2): a structure-function study of phosphomimetic and phospho-null triple mutants

The activity of ryanodine receptor type 2 (RyR2), a key player in cardiac contractility, is modulated by various post-translational modifications. Phosphorylation at multiple sites mediated through a dynamic balance between various site-specific kinases and phosphatases was proposed as an important regulator, but the subject remains contentious. Ser2808 is thought to be phosphorylated by protein kinase A (PKA) and Ca2+/calmodulin dependent protein kinase II (CaMKII), while Ser2814 and Ser2030 are phosphorylated mainly by CaMKII and PKA, respectively.

Autonomous activation of CaMKII exacerbates diastolic calcium leak during beta-adrenergic stimulation in cardiomyocytes of metabolic syndrome rats

Autonomous Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation induces abnormal diastolic Ca2+ leak, which leads to triggered arrhythmias in a wide range of cardiovascular diseases, including diabetic cardiomyopathy. In hyperglycemia, Ca2+ handling alterations can be aggravated under stress conditions via the β-adrenergic signaling pathway, which also involves CaMKII activation. However, little is known about intracellular Ca2+ handling disturbances under β-adrenergic stimulation in cardiomyocytes of the prediabetic metabolic syndrome (MetS) model with obesity, and the participation of CaMKII in these alterations.MetS was induced in male Wistar rats by administering 30 % sucrose in drinking water for 16 weeks. Fluo 3-loaded MetS cardiomyocytes exhibited augmented diastolic Ca2+ leak (in the form of spontaneous Ca2+ waves) under basal conditions and that Ca2+ leakage was exacerbated by isoproterenol (ISO, 100 nM). At the molecular level, [3H]-ryanodine binding and basal phosphorylation of cardiac ryanodine receptor (RyR2) at Ser2814, a CaMKII site, were increased in heart homogenates of MetS rats with no changes in RyR2 expression. These alterations were not further augmented by Isoproterenol. SERCA pump activity was augmented 48 % in MetS hearts before β-adrenergic stimuli, which is associated to augmented PLN phosphorylation at T17, a target of CaMKII. In MetS hearts. CaMKII auto-phosphorylation (T287) was increased by 80 %. The augmented diastolic Ca2+ leak was prevented by CaMKII inhibition with AIP. In conclusion, CaMKII autonomous activation in cardiomyocytes of MetS rats with central obesity significantly contributes to abnormal diastolic Ca2+ leak, increasing the propensity for β-adrenergic receptor-driven lethal arrhythmias.

"Phosphorylation of cardiac ryanodine receptors by protein kinase G: Evaluation of its impact on store overload-induced calcium release

Cardiac Ryanodine receptors (RyR2) are Ca2+ channels located within the sarcoplasmic reticulum (SR) membrane of cardiac myocytes and play a key role in excitation-contraction coupling. RyR2 can also open spontaneously in a SR store dependent manner, a process known as Store Overload-Induced Ca2+ Release (SOICR). We and others previously demonstrated PKG-dependent phosphorylation of RyR2 at Ser2808 in cardiac myocytes but its functional impact on SOICR remains unknown. Our objective is to study the effects promoted by PKG-dependent phosphorylation of the RyR2, with respect to SOICR frequency and amplitude.

Ryanodine Receptor Phosphorylation and Heart Failure

By the time the heart reaches the pathological state clinically recognized as heart failure (HF), it has undergone profound and often irreversible alterations in structure and function at the molecular, cellular, and organ level. Although the etiologies of HF are diverse (hypertension, myocardial infarction, atherosclerosis, valvular insufficiency, mutations in genes encoding sarcomeric proteins, etc), some alterations are commonly found in most forms of HF, and they may account for the maladaptive structural remodeling and systolic dysfunction that characterize this syndrome. At the cellular level, there are well-documented changes in ionic channel density and function (electric remodeling), increased ROS production, mitochondrial dysfunction, imbalanced energy intake and consumption, genetic reprogramming, altered excitation-contraction coupling, and, in general, dysregulation of a multitude of other processes and pathways that are essential for proper cardiac function.1 Combined, this myriad of alterations leads to loss in contractility and ejection fraction, ventricular wall remodeling, increased vascular resistance, and dysregulated fluid homeostasis. In this issue of Circulation Research , Respress et al2 report that preventing phosphorylation of cardiac ryanodine receptors (RyR2) at a single residue, S2814, is sufficient to avert many of these alterations and improve cardiac function in HF. The results presented follow a string of papers that touch on the delicate and controversial subject of ryanodine receptor phosphorylation and HF. They offer a new twist to a contentious story and attempt to reconcile many apparently contradicting results, but key issues remain. Article, see p 1474 ### Calcium Leak in HF Although, as mentioned above, the etiologies of HF are diverse, it appears that suppressing the dysfunction of a select group of biological and molecular signaling pathways may substantially improve or even reverse the cardiac deterioration observed in HF. For example, correcting the characteristically depressed sarcoplasmic reticulum (SR) calcium content of failing cardiomyocytes is a target of HF gene therapy.3 …

Phosphodiesterase 4D Regulates Baseline Sarcoplasmic Reticulum Ca 2+ Release and Cardiac Contractility, Independently of L-Type Ca 2+ Current

Baseline contractility of mouse hearts is modulated in a phosphatidylinositol 3-kinase-γ-dependent manner by type 4 phosphodiesterases (PDE4), which regulate cAMP levels within microdomains containing the sarcoplasmic reticulum (SR) calcium ATPase type 2a (SERCA2a). The goal of this study was to determine whether PDE4D regulates basal cardiac contractility. At 10 to 12 weeks of age, baseline cardiac contractility in PDE4D-deficient (PDE4D(-/-)) mice was elevated mice in vivo and in Langendorff perfused hearts, whereas isolated PDE4D(-/-) cardiomyocytes showed increased whole-cell Ca2+ transient amplitudes and SR Ca2+content but unchanged L-type calcium current, compared with littermate controls (WT). The protein kinase A inhibitor R(p)-adenosine-3',5' cyclic monophosphorothioate (R(p)-cAMP) lowered whole-cell Ca2+ transient amplitudes and SR Ca2+ content in PDE4D(-/-) cardiomyocytes to WT levels. The PDE4 inhibitor rolipram had no effect on cardiac contractility, whole-cell Ca2+ transients, or SR Ca2+ content in PDE4D(-/-) preparations but increased these parameters in WT myocardium to levels indistinguishable from those in PDE4D(-/-). The functional changes in PDE4D(-/-) myocardium were associated with increased PLN phosphorylation but not cardiac ryanodine receptor phosphorylation. Rolipram increased PLN phosphorylation in WT cardiomyocytes to levels indistinguishable from those in PDE4D(-/-) cardiomyocytes. In murine and failing human hearts, PDE4D coimmunoprecipitated with SERCA2a but not with cardiac ryanodine receptor. PDE4D regulates basal cAMP levels in SR microdomains containing SERCA2a-PLN, but not L-type Ca2+ channels or ryanodine receptor. Because whole-cell Ca2+ transient amplitudes are reduced in failing human myocardium, these observations may have therapeutic implications for patients with heart failure.

Cardiac Ryanodine Receptor Phosphorylation at Ser2808 is Involved in Intra-SR Calcium Sensing

During stress, cardiac function is enhanced via β-adrenergic stimulation. Phosphorylation of cardiac ryanodine receptors (RyR2) has been proposed to lead to increased Ca2+ release from the sarcoplasmic reticulum (SR), but experimental findings are controversial. Using cardiomyocytes lacking the PKA phosphorylation site Ser2808 on RyR2 we previously observed that this site contributes to improved spatio-temporal synchronization of Ca2+ release during EC-coupling under β-adrenergic stimulation, presumably by a Ca2+ sensing mechanism inside the SR. Here, we further tested the hypothesis that Ser2808 phosphorylation modulates RyR2 function via its dependence on [Ca2+]SR. Cardiomyocytes from control and S2808A mice were loaded with fluo-3-AM and Ca2+ was imaged with confocal line-scans. Ca2+ waves were induced by increasing external [Ca2+] to 10mM and analyzed in control and during β-stimulation with isoproterenol (1µM). In control conditions Ca2+ transients during a wave showed a comparable amplitude and time course. However, wave speed was reduced by 25% in S2808A cells, possibly related to a lack of background phosphorylation at this site. While ISO increased wave speed by 15% in control cells (from 98 to 113µm/s), it remained unaffected in S2808A cells (from 73 to 75µm/s). This situation could be reproduced in control cells preincubated with the PKA inhibitor H89 or the CaMKII inhibitor KN93, indicating a complex regulation of CICR that is pharmacologically difficult to dissect in intact cells. Taken together our findings in S2808A myocytes reveal a lack of Ca2+ release synchronization (via an intra-SR Ca2+ sensing mechanism) and an ablation of Ca2+ wave speed modulation (presumably by the same mechanism) by β-adrenergic stimulation. Thus, Ser2808 phosphorylation may change the sensitivity of the RyR2 for intra-SR Ca2+. Hyperphosphorylation of Ser2808, as in failing hearts, may thereby render RyR2 hypersensitive and contribute to increased Ca2+ leak.

Phosphorylation of Ryanodine Receptor At Serine-2809 Modulates Sarcoplasmic Reticulum Ca Release in Rabbit Ventricular Myocytes

The role of protein kinase A (PKA)-dependent phosphorylation of cardiac ryanodine receptor (RyR) is highly controversial. Here we studied a functional link between RyR phosphorylation at serine-2809 (PKA-specific site) and sarcoplasmic reticulum (SR) Ca release and leak in cardiomyocytes. We simultaneously measured intra-SR free Ca ([Ca]SR) with Fluo-5N and cytosolic Ca with Rhod-2 in permeabilized rabbit ventricular myocytes. RyR phosphorylation at site serine-2809 was measured with a phospho-specific antibody (Badrilla, UK). We found that cAMP (10 μM) increased Ca spark frequency by ∼2.6 times. This effect was associated with an increase in SR Ca load from 0.84 to 1.24 mM. PKA inhibitory peptide (10 μM) abolished cAMP-mediated increase of SR Ca load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR-mediated Ca leak. The lack of cAMP effect on RyR function can be explained by almost maximal phosphorylation of RyR at serine-2809 after membrane permeabilization and also argues against the functional importance of another PKA-specific site (serine-2031) for SR Ca release. This high phosphorylation level of RyR could be due to a shift of the balance between protein kinase and phosphatase activity after permeabilization. Preventing this increase in phosphorylation with staurosporine (1 μM) decreased RyR-mediated SR Ca leak. Surprisingly, further dephosphorylation of RyR at serine-2809 with protein phosphatase 1 (PP1; 2 U/ml) markedly increased Ca leak. However, it is important to note that PP1 and staurosporine possibly affected other phosphorylation sites of RyR as well. In conclusion, our results provide direct evidence that RyR phosphorylation at serine-2809 modulates channel function and SR Ca release in rabbit ventricular myocytes.

Cardiac ryanodine receptor phosphorylation by CaM Kinase II: keeping the balance right

Phosphorylation of the cardiac ryanodine receptor (RyR2) is a key mechanism regulating sarcoplasmic reticulum (SR) Ca2+ release. Differences in opinion have arisen over the importance assigned to specific phosphorylation sites on RyR2, over the kinase (s) suggested to directly phosphorylate RyR2 and surrounding the possibility that altered phosphorylation of RyR2 is associated with contractile dysfunction observed in heart failure. Ca2+/calmodulin dependent protein kinase II (CaMKII) can phosphorylate RyR2 and modulate its activity. This phosphorylation positively modulates cardiac inotropic function but in extreme situations such as heart failure, elevated CaMKII activity can adversely increase Ca2+ release from the SR and lead to arrhythmogenesis. Although other kinases can phosphorylate RyR2, most notably cAMP-dependent protein kinase (PKA), evidence for a key role of CaMKII in mediating RyR2-dependent Ca2+ release is emerging. Future challenges include (i) fully identifying mechanisms of CaMKII interaction with the RyR2 complex and (ii) given the ubiquitous expression of CaMKII, developing selective strategies to modulate RyR2-targeted CaMKII activity and allow improved understanding of its role in normal and diseased heart.

Maximum Phosphorylation of the Cardiac Ryanodine Receptor at Serine-2809 by Protein Kinase A Produces Unique Modifications to Channel Gating and Conductance Not Observed at Lower Levels of Phosphorylation

It is suggested that protein kinase A (PKA)-dependent phosphorylation of cardiac ryanodine receptors (RyR2) is linked to the development of heart failure and the generation of fatal cardiac arrhythmias. It is also suggested that RyR2 is phosphorylated to 75% of maximum levels in heart failure resulting in leaky, unregulated channels gating in subconductance states. We now demonstrate that this is unlikely, as RyR2 isolated from nonfailing cardiac muscle is phosphorylated to 75% of maximum at serine-2809, and in this situation, RyR2 displays low open probability (P(o)) (0.059+/-0.010 [SEM]; n=30) and normal regulation of gating by Ca(2+) and other ligands. However, when serine-2809 is PKA phosphorylated to maximum levels on RyR2, unique changes in channel behavior are observed. The channel displays enhanced single-channel conductance, very long open states causing large increases in P(o), and no evidence of subconductance states. Dephosphorylation of channels by protein phosphatase 1 (from 75% to near 0% at serine-2809) also enhances RyR2 channel activity through abbreviation of closed lifetimes. We propose that channels phosphorylated to 75% of maximum at serine-2809 occupy a natural low point in the RyR2 activity landscape. This optimizes channel control, which can be accomplished either by enhanced or decreased phosphorylation, making the channel particularly sensitive to the kinase:phosphatase balance. Pathological situations such as heart failure might upset this balance and thereby permit prolonged stoichiometric phosphorylation of serine-2809, which would be required for dysregulation of SR Ca(2+) release.

Cardiac ryanodine receptor phosphorylation: target sites and functional consequences

A study by Xiao and co-workers in this issue of the Biochemical Journal demonstrates PKA (protein kinase A)-dependent phosphorylation of Ser-2030 on the cardiac ryanodine receptor (RyR2) that is activated by beta-adrenergic agonists. They show that RyR2 phosphorylation at this site is not appreciably altered in heart failure samples, but retains PKA-dependence of phosphorylation. They contrast this with RyR2 phosphorylation at Ser-2808, a site previously reported to be the key and only PKA target site on RyR2. Here Ser-2808 phosphorylation was found to be relatively insensitive to either PKA activation or inhibition. These results add important new information to a highly controversial field. This issue is important because it is increasingly clear that altered regulation of the gating of the RyR2 sarcoplasmic reticulum Ca2+-release channel (e.g. by phosphorylation) is critically important in mediating altered diastolic sarcoplasmic reticulum Ca2+ release. This may contribute to both reduced cardiac function and arrhythmogenesis in humans carrying mutations in the RyR2 gene and with acquired heart failure of varied aetiology. This study brings some new answers, but also raises additional new questions that will require further investigation.

Stoichiometric phosphorylation of cardiac ryanodine receptor on serine 2809 by calmodulin-dependent kinase II and protein kinase A.

The ryanodine receptor of cardiac muscle performs a central role in excitation-contraction coupling. Phosphorylation of the channel on serine 2809 (in rabbit or the corresponding serine 2808 in man) alters function in vitro, although the impact of this in vivo has not been established. We have produced a pair of antisera to the serine 2809 phosphorylation site to aid description of the incidence and consequence of phosphorylation of this receptor. One of these antisera is specific for the serine 2809 phosphorylated form of the cardiac ryanodine receptor; the other antiserum is specific for the serine 2809 dephosphorylated receptor. These antibodies have been used to demonstrate that both protein kinase A and calmodulin-dependent kinase II are capable of phosphorylating serine 2809 in vitro. Both kinases phosphorylate serine 2809 to full stoichiometry, but this is accompanied by the incorporation of more (radioactive) phosphate into the receptor by calmodulin-dependent kinase II than by protein kinase A. These data suggest that calmodulin-dependent kinase II phosphorylates at least four sites in addition to serine 2809 in vitro.

Gating kinetics and ligand sensitivity modified by phosphorylation of cardiac ryanodine receptors.

The effects of protein-kinase- (PKA-) dependent phosphorylation on the stationary gating kinetics of single ryanodine receptor (RyR) channels was defined. The single-channel activity from canine cardiac RyR was reconstituted into planar lipid bilayers. Exogenously applied PKA increased the single-channel open probability ( P(o)) of both native and purified cardiac RyR channels, after preincubation with ATP and Mg2+. The action of PKA on the RyR channel occurred only in the presence of ATP and adenosine 5'- O-(3-thiotriphosphate) (ATPgammaS), but not in the presence of 5'-adenylimidodiphosphate (AMP-PCP). Thus, the action of PKA requires the presence of a hydrolyzable ATP analog. PKA-induced channel activation was blocked by specific PKA inhibitors. All these results confirmed that the RyR channel can be phosphorylated by exogenous protein kinase. The gating kinetics of single RyR channels before PKA treatment were significantly altered by ATP and Mg2+ as physiological ligands. In contrast, after PKA treatment, neither ATP nor Mg2+ significantly alters the gating kinetics of these channels. PKA-dependent phosphorylation thus decreases the ATP and Mg2+ apparent sensitivity in most of the gating parameters of single RyR channels. The phosphorylated RyR channels open and close more frequently, stay open for longer, and stay closed for shorter periods. The dwell-time histograms obtained demonstrate that the phosphorylated and the dephosphorylated channels have strikingly different open and closed kinetics at physiological cytoplasmic concentrations of Mg and ATP.

Phosphorylation of the Cardiac Ryanodine Receptor by cAMP-Dependent Protein Kinase1

The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by the catalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (Mr 400,000) in cardiac microsomes was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiac ryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (Mr 400,000-450,000) in canine skeletal microsomes isolated from extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers were not significantly phosphorylated. In agreement with these findings, the ryanodine receptor purified from cardiac microsomes was also phosphorylated by cAMP-dependent protein kinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purified preparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Upon phosphorylation of the cardiac ryanodine receptor, the levels of [3H]ryanodine binding at saturating concentrations of this ligand increased by up to 30% in the presence of Ca2+ concentrations above 1 microM in both cardiac microsomes and the purified cardiac ryanodine receptor preparation. In contrast, the Ca2+ concentration dependence of [3H]ryanodine binding did not change significantly. These results suggest that phosphorylation of the ryanodine receptor by cAMP-dependent protein kinase may be an important regulatory mechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.