The yellow brick road to understanding the RyR2 signalosome

Fine-tuned intracellular Ca²⁺ cycling in cardiomyocytes is crucial 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 contributory factor in heart failure and arrhythmogenesis (see review, Terentyev & Hamilton, 2016). RyR2 is readily responsive to post-translational 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 regarding which phosphorylation site is relevant functionally, which kinase acts on which site, and how these kinases are associated with the channel. Adding to the complexity is the activity of serine–threonine phosphatases, known to be increased in disease states such as heart failure. Dephosphorylation of RyR2 has also been shown to increase RyR2 channel activity (Terentyev & Hamilton, 2016).

Although there are 42 different potential phosphorylation sites for human RyR2, three have fuelled significant research for >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 review, Woll & Van Petegem, 2022). Traditionally, Serine-2808 is considered a primary protein kinase A (PKA) target, whereas Serine-2814 is 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. Although structural data have significantly advanced our knowledge regarding RyR2 phosphorylation, much is to be learned.

Highlighting intricacies of RyR2 phospho-signalling is recent work from the Moore laboratory linking the phosphorylation state of RyR2 with its tetramer arrangements in clusters (Asghari et al., 2024). Moore and colleagues 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 in conditions mimicking stress, rearrangements of RyR2s within clusters are driven primarily by Serine-2030 phosphorylation. However, it remained unclear whether rearrangements of RyR2 tetramers in cardiomyocytes with phospho-ablated Serine-2808 and Serine-2814 seen in baseline conditions can have any functional consequences leading to increased SR Ca²⁺ leak.

In this issue of The Journal of Physiology, Niggli, Valdivia and colleagues help to dissect the role of Serine-2030 by generating double knock-in (RyR2-DKI) mice with Serine-2808 and Serine-2814 phospho-ablated (Janicek et al., 2024). 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 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 that 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⁺ leak (Janicek et al., 2024). Moreover, given that 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 the functional relevance of Serine-2030 in the β-adrenergic response. The differences in spontaneous Ca²⁺ wave frequency of RyR2-DKIs and wild-type cardiomyocytes are much smaller under β-adrenergic stimulation. However, permeabilized RyR2-DKI cardiomyocytes showed increased spark frequency when treated with cyclic AMP vs. control cardiomyocytes. This confirms that phosphorylation of Ser-2030, the only phosphorylatable site left of three, increases RyR2 activity, suggesting that this site is 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 remodelling in conditions of permanently increased SR Ca²⁺ leak? In the present manuscript, the authors report decreased expression of phospholamban in RyR2-DKI cardiomyocytes (Janicek et al., 2024). This can be crucial for maintaining SR Ca²⁺ content, preserving Ca²⁺ transient amplitude and contractility. However, such a persistent increase in SR Ca²⁺ leak/uptake is energetically costly and can elicit adverse consequences, such as mitochondrial damage or endoplasmic reticulum stress, especially in conditions with increased metabolic demand.

Notably, RyR2-DKI mice exhibit mild arrhythmia patterns of primarily sustained bigeminy when injected with β-adrenergic agonist (Janicek et al., 2024). 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 remodelling or cardiomyopathy (Bhuiyan et al. 2007; Tiso et al. 2001), whereas RyR2-DKI hearts showed no signs of macroscopic structural remodelling 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 in 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 depends crucially on careful untangling of intricate channel regulatory mechanisms, which will require significant effort from the research community in the future.