The yellow brick road to understanding the RyR2 signalosome

Fine-tuned intracellular Ca²⁺ cycling in cardiomyocytes is critical for a graded response to the ever-changing metabolic demands of the body. Phosphorylation of the cardiac ryanodine receptor (RyR2), the major sarcoplasmic reticulum (SR) Ca²⁺ release channel, is a crucial regulator of its function. Changes in RyR2 phosphorylation patterns were implicated as an important contributing factor in heart failure and arrhythmogenesis (see review¹). RyR2 is readily responsive to posttranslational modifications by multiple kinases and phosphatases at many potential phosphorylation sites within the channel tetramer. This vast phosphorylation signalosome creates potential for huge channel diversity but is difficult to untangle experimentally. The general consensus is that phosphorylation of RyR2 by protein serine-threonine kinases increases Ca²⁺ sensitivity and channel activity. However, debates remain as to which phosphorylation site is functionally relevant, which kinase acts on which site, and how do these kinases associate with the channel. Adding to the complexity is activity of serine-threonine phosphatases, known to be increased in disease states such as heart failure. Dephosphorylation of RyR2 has been shown to increase RyR2 channel activity as well¹.

Although there are 42 different potential phosphorylation sites for human RyR2, three have fueled significant research for over 20 years. These are Serine-2808, Serine-2030 and Serine-2814. Serine-2808 and Serine-2814 are both found in the same ‘hotspot’ domain of RyR2, at the top of the cytosolic channel face (see review²). Traditionally, Serine-2808 is considered a primary protein kinase A (PKA) target, while Serine-2814 a Ca²⁺/calmodulin kinase II (CaMKII) target. Intriguingly, PKA-sensitive Serine-2030 is in a completely different, peripheral channel region. From a structural standpoint, it is unclear how phosphorylation at a site so far away from the pore can affect channel function. It also remains unclear where protein phosphatases directly interact with the channel. While structural data has significantly advanced our knowledge regarding RyR2 phosphorylation, much is to be learned.

Highlighting intricacies of RyR2 phospho-signaling is recent work of the Moore laboratory linking the phosphorylation state of RyR2 with its tetramer arrangements in clusters³. Asghari et al. demonstrated an increased abundance of isolated tetramers or orphaned RyR2 channels from mice rendered unphosphorylatable at Serine-2808, Serine-2814, or Serine-2030. Under β-adrenergic stimulation, normal channel clustering is largely restored in Ser-2808 and Ser-2814 ablated mouse cardiomyocytes, but not for Serine-2030 ablated. This suggests that under conditions mimicking stress, RyR2s rearrangements within clusters are primarily driven by Serine-2030 phosphorylation. However, it remained unclear whether rearrangements of RyR2 tetramers in cardiomyocytes with phospho-ablated Serine-2808 and Serine-2814 seen under baseline conditions can have any functional consequences leading to increased SR Ca²⁺ leak.

In this issue of The Journal of Physiology, Niggli and colleagues help to dissect the role of Serine-2030 by generating double knock-in mice (RyR2-DKI) with Serine-2808 and Serine-2814 phospho-ablated⁴. This rendered only Serine-2030 available for phosphorylation. An important finding of this work is that even in the absence of β-adrenergic stimulation, cardiomyocytes from RyR2-DKI mice exhibited more pro-arrhythmic spontaneous Ca²⁺ waves (SCWs) consistent with hyperactivation of RyR2 clusters. In control cardiomyocytes, acute dephosphorylation of RyR2 by application of protein phosphatase 1 (PP1) significantly increased Ca²⁺ spark frequency. Conversely, PP1 was completely ineffective in RyR2-DKI myocytes, suggesting these two sites confer most, if not all, RyR2 sensitivity to phosphatases. These data agree with previous findings that not only phosphorylation, but also dephosphorylation increases RyR2-mediated Ca²⁺ leak¹. Moreover, as RyR2-DKI mice exhibit increased susceptibility for adrenergically induced arrhythmias, it demonstrates the importance of dephosphorylation of these two sites in arrhythmogenesis.

In addition, this work carefully illuminates functional relevance of Serine-2030 in the β-adrenergic response. The differences in SCW frequency RyR2-DKIs and WTs are much smaller under β-adrenergic stimulation. However, permeabilized RyR2-DKI cardiomyocytes showed increased spark frequency when treated with cyclic AMP vs controls. This confirms that phosphorylation of Ser-2030, the only phosphorylatable site left of three, increases RyR2 activity, suggesting this site as the major site that confers PKA sensitivity. Moreover, this experiment demonstrates that Serine-2030 phosphorylation can further increase activity of already hyperactive mutant RyR2s.

Several important questions remain. How much of an alteration in intracellular Ca²⁺ handling in RyR2-DKI mice is determined by a loss of RyR2 phosphorylation at Serine-2808 and Serine-2814 vs secondary remodeling in conditions of permanently increased SR Ca²⁺ leak? In the present manuscript, authors report decreased expression of phospholamban in RyR2-DKI cardiomyocytes⁴. This can be critical for maintaining SR Ca²⁺ content preserving Ca²⁺ transient amplitude and contractility. However, such persistent increase in SR Ca²⁺ leak/uptake is energetically costly thus can elicit adverse consequences, such as mitochondrial damage or ER stress, especially under conditions with increased metabolic demand.

Notably, RyR2-DKI mice exhibit mild arrhythmia patterns of primarily sustained bigeminy when injected with β-adrenergic agonist⁴. In comparison, models with RyR2 mutations linked to the arrhythmia syndrome catecholaminergic polymorphic tachycardia often demonstrate more severe sustained bidirectional/polymorphic ventricular tachycardia under catecholaminergic challenge. Furthermore, several human RyR2 mutations were shown to cause significant tissue remodeling or cardiomyopathy^5,6, while RyR2-DKI hearts showed no signs of macroscopic structural remodeling and preserved function. This highlights that RyR2 hyperactivity is not a basic, all-or-none event. On the contrary, it encompasses a spectrum of functional changes, which can lead to very different outcomes under different genetic and/or environmental circumstances.

RyR2 is an attractive therapeutic target, given its central role in cardiac arrhythmogenesis and failure. However, successful development of safe effective RyR2-based treatment strategies critically depends on careful untangling of intricate channel regulatory mechanisms, which will require significant effort from the research community in the future.