Ernst Niggli

In this narrative review an example of a cardiac arrhythmia with a mechanistically appealing molecular pathomechanism is highlighted. This example will be used to delineate how recent basic science findings, which are summarized, can be used to obtain a deeper understanding of pathological behavior from the molecular to the cellular level. The condition in question is the highly lethal catecholaminergic polymorphic ventricular tachycardia (CPVT) resulting from a point mutation of the cardiac ryanodine receptor. For this deep dive the RyR2^R420Q phenotype will be discussed in detail. Interestingly, these findings and the conclusions which could be drawn from them were very much unexpected, but could be relevant for pharmacological treatments. To make the transition from the molecular and cellular findings to the patient will require the translation across several layers of complexity. Ultimately, such detailed understanding will lead to improved therapies tailored to each individual case and the specific RyR2 mutation carried by a particular family, in the framework of precision medicine.

During exercise or stress, the sympathetic system stimulates cardiac contractility via β‐adrenergic receptor (β‐AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation–contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca ²⁺ /calmodulin kinase type‐2‐specific site. To examine the contribution of these phosphosites to Ca ²⁺ signalling, we generated double knock‐in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2‐S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca ²⁺ imaging and electrophysiology experiments. Isoprenaline was used to stimulate β‐ARs. Measurements of Ca ²⁺ waves and latencies in myocytes revealed an increased propensity for spontaneous Ca ²⁺ releases in DKI myocytes, both in control conditions and during β‐AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca ²⁺ inside the sarcoplasmic reticulum, suggesting higher Ca ²⁺ sensitivity of the RyRs. The refractoriness of Ca ²⁺ spark triggering depends on the Ca ²⁺ sensitivity of the RyR2. We found that RyR2‐S2808A/S2814A channels were more Ca ²⁺ sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2‐Ser2808 and RyR2‐S2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca ²⁺ releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for β‐AR‐mediated sensitization of RyRs. image Key points: Phosphorylation of cardiac sarcoplasmic reticulum Ca ²⁺ ‐release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function. Ablation of both the RyR2‐Ser2808 and RyR2‐Ser2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca ²⁺ releases, as previously suggested for hyperphosphorylated RyRs. The intra‐sarcoplasmic reticulum Ca ²⁺ threshold for spontaneous Ca ²⁺ wave generation is lower in RyR2‐double‐knock‐in cells. The RyR2 from double‐knock‐in cells exhibits increased Ca ²⁺ sensitivity. Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel. Phosphorylation of Ser2030 might be sufficient for a β‐adrenergic response.

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible n-butyl ester protecting group to deliver amphipathic cargoes to the cytosol. We show that the size of the caging chromophore conditions the ability of intracellular probe delivery and esterase charge unmasking.

Key points: In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery. Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias. We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two‐photon photolysis of a new ultra‐effective caged calcium compound. The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation. Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types. In cardiac myocytes Ca²⁺‐induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca²⁺ release occurs via subcellular Ca²⁺ signalling events, Ca²⁺ sparks. Local recovery of Ca²⁺ release depends on both SR refilling and restoration of Ca²⁺ sensitivity of the RyRs. We used two‐photon (2P) photolysis of the ultra‐effective caged Ca²⁺ compound BIST‐2EGTA and laser‐scanning confocal Ca²⁺ imaging to probe refractoriness of local Ca²⁺ release in control conditions and in the presence of cAMP or low‐dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST‐2EGTA and rhod‐2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca²⁺ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self‐learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyR^R420Q mutation were used for further validation and revealed a higher Ca²⁺ sensitivity of CICR. This new 2P approach provides composite information of Ca²⁺ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca²⁺ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca ²⁺ release channel/RyR2 (type 2 ryanodine receptor) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2 ^R420Q . Objective: To determine the arrhythmogenic mechanisms of this mutation. Methods and Results: Ventricular tachycardias under stress conditions were observed in both patients with catecholaminergic polymorphic ventricular tachycardia and knock-in mice. During action potential recording (by patch-clamp in knock-in mouse cardiomyocytes and by microelectrodes in mutant human induced pluripotent stem cell-derived cardiomyocytes), we observed an increased occurrence of delayed afterdepolarizations under isoproterenol stimulation, associated with increased Ca ²⁺ waves during confocal Ca ²⁺ recording in both mouse and human RyR2 ^R420Q cardiomyocytes. In addition, Ca ²⁺ -induced Ca ²⁺ -release, as well as a rough indicator of fractional Ca ²⁺ release, were higher and Ca ²⁺ sparks longer in the RyR2 ^R420Q -expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum measured by gated stimulated emission depletion super-resolution and electron microscopy, respectively. The increase in junctional sarcoplasmic reticulum width might be due to the impairment of RyR2 ^R420Q binding to JPH2 (junctophilin-2), as there were less junctophilin-2 coimmunoprecipitated with RyR2 ^R420Q . At the single current level, the RyR2 ^R420Q channel dwells longer in the open state at low intracellular Ca ²⁺ ([Ca ²⁺ ] _i ), but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N terminus and the core solenoid, a RyR2 interdomain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death. Conclusions: The RyR2 ^R420Q catecholaminergic polymorphic ventricular tachycardia mutation modifies the interdomain interaction of the channel and weakens its association with JPH2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction.

This editorial refers to ‘Compartmentalized β₁-adrenergic signalling synchronizes excitation–contraction coupling without modulating individual Ca²⁺ sparks in healthy and hypertrophied cardiomyocytes’, by H.-Q. Yang et al., pp. 2069–2080.

Key points: Increased protein phosphatase 1 (PP‐1) activity has been found in end stage human heart failure. Although PP‐1 has been extensively studied, a detailed understanding of its role in the excitation–contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP‐1 activity on local Ca²⁺ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP‐1 protein. We report that acute de‐phosphorylation may increase the sensitivity of RyR2 channels to Ca²⁺ in situ, and that the RyR2‐serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP‐1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca²⁺ leak in the diseased heart. Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered to be important regulatory and disease related post‐translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase‐1 (PP‐1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca²⁺ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP‐1 activity, we investigated how the PP‐1 catalytic subunit (PP‐1c) alters Ca²⁺ sparks in permeabilized cardiomyocytes and we also applied a PP‐1‐disrupting peptide (PDP3) to specifically activate endogenous PP‐1, including the one anchored on the RyR2 macromolecular complex. We compared wild‐type and transgenic mice in which the usually highly phosphorylated site RyR2‐S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A^+/+). In wild‐type myocytes, PP‐1 increased Ca²⁺ spark frequency by two‐fold, followed by depletion of the sarcoplasmic reticulum Ca²⁺ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca²⁺ load. RyR2 Ca²⁺ sensitivity, which was assessed by Ca²⁺ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A^+/+ cardiomyocytes did not respond to both PP‐1c and PDP3 treatment. Our results suggest an increased Ca²⁺ sensitivity of RyR2 upon de‐phosphorylation by PP‐1. Furthermore, we have confirmed the S2808 site as a target for PP‐1 and as a potential link between RyR2s modulation and the cellular response.

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca²⁺ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A^+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca²⁺ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca²⁺ loading and L-type Ca²⁺ current (I_Ca) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca²⁺ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca²⁺ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A^+/+ myocytes. Using Ca²⁺ spark recovery analysis, we found that mutant RyR Ca²⁺ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca²⁺ waves and increases latency to the first spontaneous Ca²⁺ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs. Graphical Abstract