Targeting Pathological Leak of Ryanodine Receptors: Preclinical Progress and the Potential Impact on Treatments for Cardiac Arrhythmias and Heart Failure

Abstract

Introduction:

Type-2 ryanodine receptor (RyR2) located on the sarcoplasmic reticulum initiate systolic Ca²⁺ transients within cardiomyocytes. Proper functioning of RyR2 is therefore crucial to the timing and force generated by cardiomyocytes within a healthy heart. Improper intracellular Ca²⁺ handing secondary to RyR2 dysfunction is associated with a variety of cardiac pathologies including catecholaminergic polymorphic ventricular tachycardia (CPVT), atrial fibrillation (AF), and heart failure (HF). Thus, RyR2 and its associated accessory proteins provide promising drug targets to scientists developing therapeutics for a variety of cardiac pathologies.

Areas Covered:

In this article, we review the role of RyR2 in a variety of cardiac pathologies. We performed a literature search utilizing PubMed and MEDLINE as well as reviewed registries of trials from clinicaltrials.gov from 2010 to 2019 for novel therapeutic approaches that address the cellular mechanisms underlying CPVT, AF, and HF by specifically targeting defective RyR2 channels.

Expert Opinion:

The negative impact of cardiac dysfunction on human health and medical economics are major motivating factors for establishing new and effective therapeutic approaches. Focusing on directly impacting the molecular mechanisms underlying defective Ca²⁺ handling by RyR2 in HF and arrhythmia has great potential to be translated into novel and innovative therapies.

1. Introduction

The release of Ca²⁺ from the sarcoplasm reticulum (SR) through the ryanodine receptor isoform 2 (RyR2) initiates contraction in cardiomyocytes [1]. The regulation of RyR2 therefore impacts the timing and force generated by contracting cardiomyocytes [1]. Dysfunction of RyR2, whether acquired or inherited, leads to dysfunction of cardiomyocytes and subsequently to cardiac disease. Inappropriate activation of RyR2 can lead to dyssynchronous contraction of the myocardium resulting in arrhythmia or even sudden cardiac death [1, 2]. Dysfunction of RyR2 can also deplete SR Ca²⁺ stores, leading to decreased contractile performance, resulting in heart failure (HF). Despite RyR2’s importance in the functioning of healthy cardiomyocytes, there are few therapies for heart disease that directly target RyR2. Here we discuss the important role of RyR2 in heart function, how its dysfunction leads to cardiac disease, and the current state of development of therapeutic approaches to normalize pathological RyR2 function. We argue that RyR2 is a promising drug target due to its role in the cardiac cycle and describe the promising pre-clinical trials that demonstrate its potential therapeutic impact.

2. Cardiac excitation-contraction coupling

The synchronized contraction of cardiac muscle at regular intervals is required to produce life-sustaining cardiac output, the primary function of the heart. Synchronized contraction occurs as a result of properly transmitted electrical signal throughout the cardiac muscle and the translation of that electrical signal into a mechanical force. This translation of electrical activation to mechanical force is known as excitation-contraction (EC) coupling [3]. In EC coupling, Ca²⁺ acts as a key signaling molecule in the transition from action potential propagation (electrical signal) to actin-myosin contraction (mechanical force). Action potential voltage, produced by an influx of Na⁺ into the cell, stimulates Ca²⁺ influx from the extracellular space through L-type Ca²⁺ channels. This influx of Ca²⁺ then stimulates the release of a much large amount of Ca²⁺ from the SR by activating RyR2. Ca²⁺ released from the SR activate actin myofilaments, which contract against myosin fibers, resulting in force generation within cardiomyocytes. Diastolic relaxation occurs when Ca²⁺ is removed from the cytosol either back into the SR through the action of sarco/endoplasmic reticulum Ca²⁺-adenosine triphosphatase type 2a (SERCA2a), which replenishes SR Ca²⁺ stores, or into the extracellular space through the sarcolemmal Na⁺/Ca²⁺-exchanger (NCX). The sequence repeats itself when the next action potential is sensed, continuing the cardiac cycle.

2.1. Role of RyR2 regulation and dysfunction

RyR2’s role in EC coupling is to act as the sentinel channel controlling the release of SR Ca²⁺, which in turn activates actin-myosin contraction. This gate-keeping effect is why RyR2 is so important to healthy cardiomyocyte function and why its dysfunction leads to pathology [4]. RyR2 is a homotetrameric channel comprised of four RyR2 monomers located on the outer SR membrane [5]. Under physiological conditions, RyR2 opening probability is increased by the cytoplasmic L-type Ca²⁺ current. When exposed to increasing local concentrations of intracellular Ca²⁺, RyR2 opens, releasing SR Ca²⁺, which then stimulates contraction [4, 6]. RyR2’s sensitivity to intracellular Ca²⁺ concentration is regulated by its associated accessory proteins, such as FKBP12.6, calmodulin (CaM), and calsequestrin (CASQ2), which induce RyR2 domain rearrangement and conformational changes that alter the open or closed probability of RyR2. The binding of accessory proteins is in turn regulated by the reversible phosphorylation of RyR2 tetrameric monomers, a process controlled by kinases such as protein kinase A (PKA) and calmodulin kinase II (CaMKII), and phosphatases such as protein phosphatase 1 and 2a (PP1, PP2A) [7, 8]. Specifically, phosphorylation of serine at amino acid locations 2808 (S808), 2814 (S2814), and 2030 (S2030) appear to impact the Ca²⁺ release through RyR2 and alter its function in physiological and disease states [9–11].

FKBP12.6, for example, when bound to RyR2, stabilizes the tetrameric macromolecular complex, helping to maintain RyR2 in a closed state during the myocyte resting phase [9, 12, 13]. However, when PKA phosphorylates RyR2, it disrupts FKBP12.6 binding, leading to an increased open probability [9]. When persistently activated, PKA phosphorylation can lead to SR Ca²⁺ leak, resulting in pre-mature contractions and arrhythmias, or depleting SR Ca²⁺ stores, a potential mechanism of contractile dysfunction in HF [12, 14–16].

In addition, RyR2 activity is modulated by calmodulin (CaM), a Ca²⁺ sensitive regulator. At low Ca²⁺ concentrations, CaM binds to RyR2, inhibiting the channel. However, at high Ca²⁺ concentrations, CaM instead binds to Ca²⁺ reversing its inhibition on the RyR2 channel [17].

CaM also modulates the activity of Ca²⁺/CaM-dependent protein kinase II (CaMKII), a kinase which phosphorylates RyR2 in response to stresses on the cardiovascular system. When activated by beta-adrenergic stimulation, as in exercise or under physiologic stress, CaMKII phosphorylates RyR2 and leads to more frequent and more forceful contraction [10, 18].

Binding of a variety of accessory proteins can modulate RyR2 channel activity. Moreover, the level of RyR2 phosphorylation at various residues also allows for refinement of the action of RyR2 in the cardiac cycle, allowing the timing and force generated by contraction to be altered in response to external stimuli or intracellular conditions [19]. Mutations in RyR2 or any of its associated proteins therefore may leads to dysregulation of RyR2-mediated Ca²⁺ release, leading to cardiac pathology.

In additional to modulation of accessory proteins, other posttranslational modifications can impact the function of RyR2, especially in the diseased state. The role of oxidation, in particular, has been of interest, as cardiac disease can lead to increased oxidative states and RyR2 is particularly sensitive to oxidative stress [20, 21]. This sensitivity is due to the abundance of cysteines present throughout RyR2. When RyR2 is exposed to reactive oxygen species and becomes oxidized, Ca²⁺ leak increases, a process that is reversed when oxidation is reduced [20, 22]. Working in a cardioprotective manner, S-nitrosylation is another posttranslational modification of RyR2 that is active in the setting of oxidative stress [23]. S-nitrosylation reduces RyR2 activity, helping to maintain SR Ca²⁺ load and preventing the luminal Ca²⁺ levels from exceeding the thresholds for store induced Ca²⁺ release [20, 21, 23]. The interplay between tissue oxygenation, formation of reactive oxygen species, and nitrosylation and how the resulting posttranslational modifications impact cardiac function is an interested future avenue for therapeutic development, especially in the realm of heart failure. However, it is important to note that the specific oxidation active sites on RyR2 have yet to be characterized, making targeted drug development based on this mechanism of action difficult [21].

2.2. Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited, stress-induced arrhythmia disorder with a high incidence of sudden cardiac death. Caused in up to 60% of patients by mutations in RyR2, CPVT leads to diastolic Ca²⁺ leak in the presence of beta-adrenergic stimulation. This leak stimulates activation of the Na⁺/Ca²⁺-exchanger, resulting in delayed afterdepolarizations (DADs) and triggered activity, which can lead to potentially fatal ventricular tachyarrhythmias [2]. Mutated RyR2 results in diastolic leak in CPVT only when exposed beta-adrenergic stimulation and not at rest [12]. It has been shown that beta-adrenergic stimulation reduces the affinity of FKBP12.6, a channel-stabilizing accessory protein, to CPVT-mutant RyR2 channels [12]. In addition, it has been shown that activation of CaMKII by beta-adrenergic stimulation or exercise can exacerbate SR Ca²⁺ leak through mutant RyR2 channels [24, 25]. This mechanism is likely to play a major role since ventricular tachycardia is only observed at faster heart rates in CPVT patients [26]. Recent work has also shown that CaM plays an important role in CPVT related RyR2 dysfunction associated with select RyR2 mutations, as CaM binding affinity is decreased, and spontaneous Ca²⁺ leak is increased in these models [27, 28].

Another proposed theory is that mutations in RyR2 result in weakened binding between the N-terminal and central domains of the protein, resulting in conformational changes, destabilizing the closed state of the channel, eventually leading to Ca²⁺ leak, a process known as domain unzipping [29]. A third proposed mechanism for DADs and triggered activity in CPVT is that Ca²⁺ accumulation in the SR stimulates spontaneous SR Ca²⁺ release, a process known as store-overload induced-Ca²⁺ release [30]. As Ca²⁺ handling is altered in CPVT, Ca²⁺ sensing by RyR2 may be impacted on both the cytosolic and luminal side of the channel, with work supporting the role of cardiac calsequestrin (Casq2) in luminal Ca²⁺ sensing and handling [31, 32].

While a rare cause of CPVT, mutations in CaM and Casq2 have also been associated with CPVT phenotype, both with diastolic Ca²⁺ leak leading to DADs and triggered activity resulting in lethal ventricular arrhythmias in the presence of beta-adrenergic stimulation [32, 33].

In addition to dysfunction found in ventricular CMs as discussed above, additional work has demonstrated that CPVT Purkinje cells may exhibit a particularly high rate of spontaneous SR Ca²⁺ release events, suggesting that focal arrhythmias might in part originate from the conduction system [34]. Other recent studies have found that CPVT-related RyR2 mutations are associated with sinoatrial node dysfunction, establishing a potential role for pacemaker cells as well [35]. Studies in various mouse models and induced pluripotent stem cells (iPSC)-derived cardiomyocytes generated from CPVT patients all show that abnormal SR Ca²⁺ release events lead to delayed after-depolarizations and triggered activity [1], suggesting that targeting the RyR2 channel using pharmacological or possibly gene therapy approaches might represent a viable treatment option. For a more in-depth review of the current understanding of RyR2 and its role in CPVT, the authors would recommend the following reviews [1, 30].

2.3. Atrial fibrillation

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia. AF can lead to serious clinical complications including HF, myocardial infarction, stroke, cognitive impairment, and sudden cardiac death. Genetic mutations, extra-cardiac factors (i.e., obesity, hypertension, inflammation), as well as remodeling of the cardiac tissue can contribute to the formation of AF in patients [1, 36, 37]. AF’s natural history is a progression from intermittent arrhythmic activity in early disease, known as paroxysmal AF (pAF), to persistent arrhythmic activity, known as chronic AF (cAF).

The mechanisms underlying pAF include a combination of increased SR Ca²⁺ load and increased SR Ca²⁺ leak [1, 38]. SR Ca²⁺ leak in the context of pAF is caused by a combination of increased RyR2 protein expression, secondary to disruptions in micro-RNA regulation, as well as increased RyR2 activity independent of changes in PKA or CaMKII phosphorylation, secondary to changes in expression levels of RyR2-accessory proteins [39–41]. Increased SR Ca²⁺ load is also promoted by increased SERCA2a activity, a result of inappropriate regulation of SERCA2a inhibitory proteins [38].

In patients with more advanced stage disease (cAF), hyperactivity of the RyR2 channel is primarily caused by enhanced phosphorylation of RyR2 by kinases such as PKA and CaMKII [14, 39, 42]. In additional, NCX is upregulated in cAF, leading to increased triggered activity as a result of RyR2 SR Ca²⁺ leak, a significant contributor to the disorganized atrial contraction that is pathognomonic of the disease [42]. There has been a paper suggesting a role for oxidation of RyR2 in AF, although its mechanism in progression from pAF to cAF wasn’t elucidated [43].

2.4. Heart Failure

HF occurs when the myocardium is no longer capable of pumping adequate blood volume throughout the body to maintain proper tissue oxygenation. HF is a progressive disease, which involves remodeling of the whole myocardium, including dilation the chambers, development of interstitial fibrosis, and alterations to the cellular microstructure [44–46]. Given the importance of EC coupling to the proper functioning of a healthy cardiomyocyte, it is not surprising to see the associated dysfunction in EC coupling, and specifically Ca²⁺ handling in HF [45, 46].

In HF, dysfunction in Ca²⁺ handling leads to decreased SR Ca²⁺ stores, leading to decreased Ca²⁺ transient amplitude and increased Ca²⁺ transient duration during systole. These altered Ca²⁺ transients result in decreased contractility with a subsequent decrease in cardiac output [45, 46]. The depleted SR Ca²⁺ stores are the result of a combination of decreased SERCA2a Ca²⁺ re-uptake, increased NCX activity, and diastolic RyR2 leak [45, 46]. Decreased SERCA2a Ca²⁺ re-uptake is due to a decrease in SERCA2a expression and activity in heart failure [47, 48]. This results in decreased SR Ca²⁺ loading, a condition further exacerbated by increased NCX activity [49]. The role of RyR2-dependent diastolic Ca²⁺ leak in heart failure is well established. First, it further depletes SR Ca²⁺ stores, leading to decreased and prolonged Ca²⁺ transients [45, 46]. Second, diastolic Ca²⁺ leak, along with increased NCX activity can lead to DADs and triggered activity, leading to increased arrhythmic activity within the failing myocardium [12, 42]. RyR2 leak in heart failure can be caused by enhanced PKA and CaMKII-dependent phosphorylation, reduced protein phosphophatase levels, and altered levels of other RyR2-associated regulatory proteins (e.g., CaM, junctophilin-2, striated muscle-specific protein kinase) [9, 50–52]. For example, it was been found that chronically elevated levels of CaMKII can promote SR Ca²⁺ leak via RyR2 and the development of HF [53, 54]. CaMKII phosphorylates RyR2 at serine 2814 (S2814) [10]. Persistent CaMKII hyper- phosphorylation of RyR2 at S2814 leads to the development of HF, while inhibition of S2814 phosphorylation protects against the development of HF in mouse models [55, 56].

The relative importance of phosphorylation at specific sites on RyR2 and the role of altered RyR2 phosphorylation in HF remain controversial. For example, hyperphosphorylation of RyR2 at serine 2808 (S808) by PKA in human and animal models of HF was associated with FKBP12.6-RyR2 dissociation, resulting in Ca²⁺ leak and decreased cardiac function in the context of beta-adrenergic stimulation [9, 15, 57]. However, other studies found that replacing the serine located at 2808 with an alanine, thus preventing phosphorylation at this site, did not prevent heart failure progression nor beta adrenergic response in a mouse model [58, 59]. Other studies point to a possible important role for the phosphorylation of S2030, but not S2808, by PKA in HF in the context of beta-adrenergic stimulation [11]. These findings are summarized well in the following reviews [19, 46, 60].

As mentioned previously, posttranslational modifications in the form of oxidation, which promotes Ca²⁺ leak, and S-nitoslyation, which is cardioprotective during oxidative stress, also play an important role in the progression of HF [20–23]. As HF, by definition, is a state of oxidative stress as the cardiovascular system struggles to supply the body’s tissues with proper oxygenation, the impact of these modifications on cardiac performance cannot be overlooked. The lack of identifiable, specific, active oxidation sites on RyR2 remains a barrier to progress towards developing therapeutic approaches addressing this mechanism within HF at this time [21].

3. Therapies under development that target RyR2

Given the central role of RyR2 dysfunction in the pathogenesis of various cardiac arrhythmia disorders and heart failure, various groups have set out to develop drug compounds that normalize RyR2 channel activity. Here we will focus on several FDA approved drugs that modulate RyR2 activity, as well as small molecules in preclinical development and in clinical trials. A literature search was conducted using the PubMed and MEDLINE databases. The search terms RyR2, arrhythmia, AF, CPVT, HF, and therapy were used and articles from 2010 to 2019 were reviewed. Once identified, a specific search for each compound was conducted in the PubMed and MEDLINE databases as well as a specific search of the clinicaltrials.gov registry. Any previous significant work on each compound was included in this review, even if the work was conducted prior to our specified timeline. If a compound had not been published on within our 10-year timeline it was excluded from this review. We will not discuss the natural alkaloid ryanodine and various toxins but instead refer the reader to previous reviews, as these compounds have provided inspiration for but are not considered viable therapeutic options at this time [1, 61].

3.1. Flecainide

Flecainide, a class IC antiarrhythmic drug, has been approved for medical use in the United States since 1985. Nevertheless, 35 years later there remains controversy on its precise mechanisms of action [1]. Flecainide is best known as a Na⁺ channel blocker, but more recent studies have shown that flecainide’s Na⁺ channel blockade alone cannot explain its efficacy in CPVT [1, 62]. In fact, there is evidence that flecainide works, in part, by directly blocking the open state of RyR2, decreasing or eliminating aberrant RyR2 activity, and reducing spontaneous Ca²⁺ leak in both mouse models of CPVT and patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) [62–64]. However, RyR2 blockade alone does not explain flecainide’s full mechanism of action either [65]. Reduction of Na⁺ current due to Na⁺ channel inhibition resulting in decreasing elevated intracellular Ca²⁺ levels via the NCX is also thought to decrease inappropriate Ca²⁺ waves [65]. Furthermore, it has been shown that flecainide increases the threshold for action potential onset through its Na⁺ channel inhibition mechanism, which could also play a role in its antiarrhythmic activity [63]. Thus, a triple mode of action has been proposed for flecainide with all three proposed mechanisms contributing to decrease spontaneous Ca²⁺ release from RyR2 [66, 67] (Table 1).

Table 1.

Current therapies targeting RyR2 in clinical practice, clinical trails, and preclinical trials for the treatment of cardiac arrythmias and heart failure.

Drug Name	Class	Current Proposed Mechanism	Potency (IC50)	Development stage	Clinical considerations and side effects	References
Flecainide	Class IC antiarrhythmic	Triple action mechanism: Sodium channel blocker RyR2 open state blocker Increases action potential onset threshold	16μM	Used clinically to treat AF 53 total current and past clinical trials AF and CPVT mouse models AF and CPVT human iPSC-CMs	Second line treatment for CPVT Black box warning in CHD Contraindicated in HF	[64, 66–70, 72]
Propafenone	Class IC antiarrhythmic	Sodium channel blocker RyR2 open state block	20μM	Used clinically to treat AF and other arrhythmia disorders 40 total current and past clinical trials AF and CPVT mouse models	Off-label use in paroxysmal AF Blackbox warning for history of MI Contraindicated in structural heart disease, HF, AV block	[72, 75, 109, 110]
R-propafenone	Class IC antiarrhythmic enantiomer	Specific RyR2 open state blocker	2μM	Active clinical trial for AF Casq2−/− mouse model of CPVT Casq2−/− rabbit ventricles	No clinical indication	[72–76]
K201 (JTV519)	Benzothiazepine derivatives	Stabilization of RyR2-FKBP12.6 in closed state Conformational change in RyR2 decreasing open probability Potentially mutation-dependent effects Inhibits annexin V and K+ channels, SERCA2a, SR Ca2+ ATPase (off-target effects)	5.7μM	3 previous clinical trials in AF terminated or completed and unreported FKBP12.6+/− mouse CPVT Canine HF model Explanted human tissue HF Oubain-induced Ca2+ leak mouse CMs Non-failing human CMs	No clinical indication	[29, 78–83, 85–87, 94]
S107	Benzothiazepine derivatives	Specific stabilization of RyR2-FKBP12.6 in closed state	n/a	RyR2-mutant CPVT mouse RyR2-mutant AF mouse RyR2-mutant CPVT human iPSC-CMs	No clinical indication	[88–91]
EL9	Tetracaine derivative	Derived from tetracaine (Na+ channel blocker with state-independent RyR2 inhibition) More selective, more potent for RyR2 than tetracaine	13nM	RyR2-mutant CPVT mouse	No clinical indication	[94]
EL20	Tetracaine derivative	RyR2 stabilization in the absence of CaM	35nM	RyR2-mutant CPVT mouse	No clinical indication	[95]
Dantrolene	Hydantoin derivative	CaM-dependent RyR2 stabilization	0.3 μM	Clinical trial in CPVT currently recruiting Canine HF Rabbit HF CMs Mouse HF RyR2-mutant CPVT mouse models RyR2-mutant iPSC-CMs from CPVT patients	Clinically used for the treatment of malignant hyperthermia	[96–102]
VK-II-86	Non-beta blocking carvedilol analogue	Reduces open time and open probability of RyR2 channels No beta-blocking effect	n/a	RyR2-mutant CPVT mouse Rat cardiomyocytes	No clinical indication	[103–105]
ent-1 verticilide	Unnatural verticilide enantiomer	RyR2 blockade that saturates at 20% Precise mechanism unknown	0.1μM	Casq2−/− mouse CPVT	No clinical indication	[106]

AF: atrial fibrillation; CaM: calmodulin; Casq2: calsequestrin 2; CHD: congenital heart disease; CM: cardiomyocyte; CPVT: catecholaminergic polymorphic ventricular tachycardia; HF: heart failure; MI: myocardial infarction.

Flecainide is used clinically in the treatment of AF and supraventricular tachycardia [64, 68–70]. It has also been shown to be an effective treatment for CPVT in clinical studies [64, 69, 71]. On the other hand, flecainide has several drawbacks, including a black box warning due to pro-arrhythmic behavior leading to possible lethal arrhythmias in patients with structural heart disease [68, 70]. It is also contraindicated in HF due to its negative ionotropic effects [68].

3.2. Propafenone and propafenone derivatives

Propafenone is a clinically available class IC antiarrhythmic drug, with a mechanism of action similar to flecainide. Like flecainide, propafenone is a Na⁺ channel blocker with additional activity on the open state of RyR2 [72]. Propafenone - similar to flecainide - is contraindicated in patients with AV block, Brugada syndrome, a history of cardiac ischemia, and HF [72].

Propafenone is a compound that is a racemic mixture of both S-propafenone and R-propafenone enantiomers [73]. In studies evaluating the two enantiomers separately, R-propafenone is more specific for RyR2 open state blockage, with an IC₅₀ = 2μM [72, 73]. Further exploration of R-propafenone revealed that it has an increased potency in preventing pro-arrhythmic behavior in a Casq2^−/− CPVT mouse model [74]. This increased specificity to RyR2 has also been shown to prevent atrial arrhythmias in the same Casq2^−/− mouse model, in contrast to other Na⁺ channel blockers that were unable to prevent AF in this model [75]. This was further supported by work that demonstrated that R-propafenone had increased efficacy to prevent Ca²⁺ sparks compared to lidocaine in explanted rabbit hearts [76]. There is currently an active clinical trial in the recruitment stage to investigate whether R-propafenone is more effective than S-propafenone in preventing AF (clinicaltrials.gov, Trial ID: ).

3.3. Benzothiazepine derivatives

Diltiazem, a voltage-gated Ca²⁺ channel blocker used in the treatment of arrhythmias and hypertension, is a 1,5 benzothiazepine derivative. The clinical efficacy of diltiazem lead to the exploration of the potential therapeutic properties of additional benzothiazepine derivatives for the treatment of heart diseases, leading to the discovery of K201, aka JTV519, a 1,4 benzothiazepine derivative [77].

Initial studies revealed that K201 stabilizes the RyR2-FKBP12.6 interaction [78–80]. Because biochemical studies revealed that RyR2 channels containing a missense mutation linked to CPVT become hyperactive as a result of reduced FKBP12.6 binding [12], Wehrens et al.[78] tested whether JTV519 could inhibit exercise-induced ventricular arrhythmias in a FKBP12.6+/− mouse model of CPVT. The results of these studies revealed that K201 enhances FKBP12.6 binding to RyR2 thus blunting spontaneous diastolic re-openings of mutant RyR2. Studies conducted at that time also confirmed K201’s ability to normalize RyR2 Ca²⁺ handling in a canine HF model [80].

Later studies, however, demonstrated that K201 remains effective even after FKBP12.6 dissociation from RyR2 [81]. Therefore, its activity seems to be partially independent of FKBP12.6. Follow up work found that K201 induces a conformational change in RyR2 which decreases its open probability, [82] which it does independent of CaMKII’s action on RyR2 [83].

While investigating its mechanism of action, it was found that the efficacy of K201 on RyR2 stabilization might be dependent on the location of the CPVT mutation within the RyR2 channel protein [61]. K201 has been effective in stabilization of RyR2 with mutations in the central and N-terminal domains [29], but has required significantly higher concentrations or has been unable to correct RyR2 Ca²⁺ handling when CPVT mutations were located in the C-terminal domain [29, 81]. These observations were made using small RyR2 peptides, so it remains to be established whether there is mutation-dependent efficacy of K201 in vivo in mice carrying CPVT-mutations in RyR2. Nevertheless, the potential mutation-specific efficacy of K201 is possibly due to the fact that the binding site of K201 is in the central domain of the RyR2 protein [84].

In addition to its action on RyR2 channels, K201 also inhibits annexin V and potassium channels [85, 86]. Additionally, K201 inhibits SERCA2a and sarcolemmal ion-channels in a concentration dependent manner [82, 87]. This non-specificity might contribute to its efficacy in the absence of FKBP12.6 but is concerning for off-target effects and possible pro-arrhythmia behavior [1, 61].

In additional to its efficacy in preventing arrhythmias in certain CPVT models, K201 improved contractile performance in muscle preparations obtained from the explanted hearts of human HF patients [82]. In terms of treating the potential arrhythmias that can result from RyR2-dependent SR Ca²⁺ leak, K201 decreased SR Ca²⁺ leak and pro-arrhythmic Ca²⁺ sparks both in the context of increased phosphorylation, as seen in HF and AF [78], and in the absence of increased phosphorylation, relevant to other pro-arrhythmic conditions, in both mouse and human cells [83]. Three clinical trials were registered with clinicaltrials.gov using K201 to treat AF between 2009–2011, with two terminated and one completed without reported results.

Another 1,4-benzothiaxepine derivative, S107, was developed as a K201 derivative and screened for the specific mechanism of stabilizing RyR2 and FKBP12.6 when RyR2 is in the closed state [88]. S107 was found to be a more specific RyR2 blocker, effectively blocking RyR2 at low nanomolar concentrations, but having no effect on over 400 ion channels, kinases, G-protein receptors, and other drug targets at a concentration of 10μM [89].

S107 has been shown to decrease SR Ca²⁺ leak and prevent arrhythmias in a mouse model of CPVT [89]. It has been similarly effective in decreasing RyR2 diastolic leak and pacing-induced AF in RyR2 mutant mouse models [90]. Finally, it has been tested in iPSC-derived cardiomyocytes from a CPVT patient and shown to decrease pro-arrhythmic DADs from 88% to 25% [91]. To date, there are no clinical trials of the efficacy of S107 in human patients.

3.3. Tetracaine derivatives

Tetracycline is an ester-type local anesthetic that not only blocks Na⁺ channels but also inhibits RyR2. Unlike flecainide, however, tetracaine is a state-independent blocker of RyR2, resulting in prolonged channel closing [64]. This leads to SR Ca²⁺ build up, which leads to paradoxical Ca²⁺ spark activity as Ca²⁺ build up eventually self-activates Ca²⁺ release through Ca²⁺-dependent Ca²⁺ channels [92, 93].

EL9 was developed using an unbiased pharmaceutical approach to create new tetracycline derivatives with higher affinity for RyR2 and fewer off-target side effects. In testing conducted in RyR2-mutant CPVT mouse models, EL9 was able to suppress ventricular tachycardia in mice and to reduce the frequency of pro-arrhythmic Ca²⁺ sparks in cardiomyocytes isolated from these mouse hearts [94]. The IC₅₀ of EL9 was about 400x lower than that of K201 [94]. A related compound, known as EL20, 2-(diethylamino)ethyl 4-(butylamino)-2-methoxybenzoate, had similar effects on a CPVT mouse model [95]. EL20 reduced VT frequency in CPVT mice while also decreasing Ca²⁺ spark formation in a dose-dependent manner in CPVT RyR2 mutated mouse cardiomyocytes [95]. It did this while maintaining normal systolic Ca2⁺ handling parameters. EL20 had an IC₅₀ of 35nM, similar to that of EL9, 13nM, making both compounds attractive therapeutic candidates given their potency [94, 95].

Since these next generation tetracaine derivatives showed great promise in mouse models of CPVT, additional structure-activity relationship studies are ongoing to identify promising candidates for further preclinical development and eventually clinical trials. This class of compounds may also present a new therapeutic option for other arrhythmia disorders, such as atrial fibrillation and ventricular tachycardias in failing hearts. However, their efficacy in in vitro and in vivo models of AF and HF still need to be established.

3.4. Hydantoin derivatives

Dantrolene is a hydantoin derivative that is currently used clinically for the treatment of malignant hyperthermia (MH), a condition caused by pathological SR Ca²⁺ leak through ryanodine receptor isoform 1 (RyR1) in skeletal muscle.[96] Dantrolene’s mechanism of action involves CaM-dependent stabilization of the interaction between the N-terminus and central domains, which is defective in case of RyR1 channels with MH-associated mutations [96, 97]. Dantrolene was also found to be effective in stabilizing CPVT-mutant RyR2 channels in a similar fashion [98]. Dantrolene decreased SR Ca²⁺ leak and inhibited Ca²⁺ sparks and DADs in cardiomyocytes isolated from failing dog hearts [98]. Similar results were obtained in ventricular myocytes isolated from rabbits with HF, in which dantrolene decreased Ca²⁺ sparks and increased SR Ca²⁺ stores, a sign of preserved intracellular inotropy [99]. Dantrolene was also found to significantly decrease epinephrine induced VT and inhibited Ca²⁺ sparks and transients in a pressure overload-induced HF mouse model [100]. Dantrolene decreased the frequency of premature ventricular contractions at rest and eliminated exercise-induced VT while reducing epinephrine-induced VT in a mouse model of CPVT [96]. Dantrolene also normalized Ca²⁺ spark activity back to control-levels and eliminated DADs in iPSC-CMs from CPVT patients with RyR2 mutations [101].

Dantrolene has recently advanced into proof-of-principle studies in human subjects, in which its anti-arrhythmic potential in CPVT patients is being investigated [102]. In this study, 6 subjects underwent exercise-stress testing one day before, the day of, and one day after the intravenous administration of 1.5mg/kg of dantrolene. Dantrolene decreased the number of premature ventricular contractions in a mutation-dependent fashion [102]. Patients with mutations located in the N-terminal or central regions experienced a reduction in PVC burden of 88–97%, while those with mutations just outside the central region of the RyR2 protein experienced decreased PVC burden of 33–77%. Meanwhile those mutations near or in the transmembrane region only experienced a 1–2% decreased in PVCs. Patient-derived iPSC-CMs exhibited a decreased Ca²⁺ spark frequency following exposure to dantrolene, again in a mutation-dependent fashion, with those with mutations in the N-terminal or central regions having Ca²⁺ spark reductions greater than 50% and those with mutations outside of those regions having reductions less than 50% [102]. The first clinical study of dantrolene in CPVT patients demonstrated its potential anti-arrhythmic potential in patients with mutations corresponding to dantrolene’s region stabilization within RyR2 [102]. Although these results are encouraging in a personalized medicine context, its effectiveness may be limited to a subset of CVPT-associated mutations within RyR2. This specificity also limits its applicability as a potential generalized treatment for CPVT, and could potentially limit its usefulness in AF and other arrhythmia disorders as these disorders are usually more multi-factorial and not solely the result of dysfunction within a specific region of RyR2. Dantrolene’s efficacy in variety of HF models does provide optimism regarding its possible generalized use, however, and we would welcome additional studies in a variety of disease models demonstrating its therapeutic versatility. A new clinical trial exploring the efficacy of dantrolene in CPVT patients is currently in the recruitment phase (clinicaltrials.gov, Trial ID: ).

3.5. Non-beta blocking carvedilol analogue

Carvedilol is a beta-blocker used clinically to prevent ventricular tachyarrhythmias in patients with HF [103]. In fact, it was found to be the only beta blocker capable of suppressing store-overload induced Ca²⁺ release, a pro-arrhythmic process mediated by defective RyR2 [103]. This is the result of carvedilol reducing the open time and open probability of RyR2 channels [103]. In an attempt to separate the beta-blocking and RyR2-modulating effects of carvedilol, VK-II-86 was developed [103, 104]. VK-II-86 suppressed store overload-induced Ca²⁺ release in both WT and RyR2-mutant mice while not effecting the heart rate [103]. This compound also suppressed ventricular tachyarrhythmias and pro-arrhythmic behavior in a CPVT mouse model, but was less effective than when combined with a drug with beta-blocker activity [103].

VK-II-86 has also been investigated as a potential treatment of the side effects associated with chemotherapy treatment. One of the major downsides to a recently developed class of chemotherapy agents known as class I kinase inhibitors is that they promote RyR2 activation, leading to store-overload induced Ca²⁺ release [104]. This process can lead to arrhythmias and HF in patients undergoing cancer treatment with these agents. In an in vitro cell culture model, VK-II-86 was shown to be effective in decreasing store-overload induced Ca²⁺ release in cells treated with the class I kinase inhibitors, CX-4545 (silmitasertib) and sunitinib [104]. Additionally, VK-II-86 was shown to prevent ouabain-induced cardiotoxicity, decreasing Ca²⁺ spark frequency and apoptosis in rat cardiomyocytes exposed to the inotrope ouabain [105]. The efficacy of VK-II-86 in non-drug-induced pathology would need to be further characterized to expand its potential therapeutic utility beyond preventing side effects of cardiotoxic therapies.

3.6. Unnatural verticilide enantiomer

In an attempt to develop a new therapeutic molecule for inhibiting RyR2 in a more specific manner, ent-1 verticilide was derived from fungal cycloligomeric depsipeptide, a known RyR antagonist [106]. The resulting compound had an IC₅₀ of 0.1 μM and saturated at 20% binding, a feature that could confer safety in therapeutic use by preventing complete blockage of RyR2 at higher doses [106]. ent-1 verticilide decreased Ca²⁺ spark frequency in cardiomyocytes and decreased VT occurrence in vivo in a Casq2^−/− CPVT mouse model [106]. Additionally, CPVT mice and CPVT mouse cardiomyocytes treated with ent-1 verticilide had decreased incidence of VT and decreased DADs compared to those treated with dantrolene, flecainide, and tetracaine [106]. Given these promising results, further refinement of ent-1 verticilide or a similar compound has the potential to develop into a potential therapy for SR Ca²⁺ leak-induced arrhythmia, HF, or CPVT. Additional work is needed to determine ent-1 verticilide’s viability beyond animal models of CPVT.

4. Conclusion

Ca²⁺ plays a critical role in the function of the cardiovascular system as the lynchpin molecule in EC coupling within the cardiomyocyte. Central to the proper cycling of Ca²⁺ within the cytoplasm of the cell is the RyR2 channel, whose opening and subsequent release of SR Ca²⁺ heralds the beginning of Ca²⁺-stimulated cardiomyocyte contraction. Dysfunction in RyR2 or its associated proteins, either by genetic variants or acquired through myocardial injury, can result in a variety of pathology including AF, CPVT, and HF. Therefore, RyR2 remains an important potential druggable target for therapeutic treatment of cardiac disease. Current clinically available options for RyR2 modulation are non-specific, with primary mechanisms of action on other channels. The off-target effects are limiting in clinical situations as they may lead to pro-arrhythmic events or worsening of cardiac contractility. Newly developed treatments, summarized in Table 1, look to isolate the activity of the drug to RyR2 channels and its directly associated proteins in an effort to limit these off-target effects. Many of these new therapies have shown promise, some of which have undergone or are currently undergoing clinical trials in patients, but new hurdles have appeared in the development pipeline. Controversy over the mechanism of action of several of these therapies has made it difficult to understand why certain therapies have been effective in normalizing some mutant RyR2 channels but not others. Extreme specificity for certain regions of RyR2 have led other therapies to be mutation-dependent, potentially limiting their clinical impact. New insights into the structure of the RyR2 channel at the molecular level may aid the development of small molecule compounds that can eventually be used to treat patients suffering from arrhythmias and heart failure [107, 108].

5. Expert Opinion

The importance of RyR2 to the healthy function of cardiomyocytes has been well established in this review. It is our opinion, therefore, that RyR2 altering compounds are a promising, underutilized potential therapeutic option for the treatment of a variety of cardiac pathology, including AF, CPVT, and HF. The clinical success of compounds acting on RyR2 such as flecainide demonstrate the potential of such an approach. Current clinically available options, however, are not specifically targeted to RyR2, and their primary mechanism of action is through action on other channels. We believe that this negatively impacts the side effect profiles of these compounds and leads to the contraindications associated with these therapeutics. We propose that further development of compounds that exclusively modulate RyR2 would be beneficial. An ideal agent would correct aberrant SR Ca²⁺ release via the RyR2 channel while limiting adverse side effects such as pro-arrhythmic behavior or reduced contractility.

An important factor in determining which compounds warrant further investigation is the potency and selectivity of the compound. The lack of selectivity appears to lead to the negative side effect profile of flecainide and seems to have negatively impacted the efficacy of K201 as it moved towards clinical trials. We propose that newer compounds with higher specificity and potency, such as EL20, EL9, and R-propefenone, will achieve similar efficacy in counteracting the Ca²⁺ leak and triggered activity of AF and CPVT with lower side effect profiles than currently available therapeutics. However, it is also important to note that highly potent and sensitive compounds have revealed a new potential limitation, that of mutation-location dependence. As demonstrated in clinical trials in dantrolene, certain compounds are only effective in treating disorders caused by mutations within specific domains of the RyR2 protein. Further exploration into the mechanisms that result in one compound being location specific while another provides broader coverage should be encouraged, as these insights could better inform future drug development.

Further investigation into promising existing compounds and future investigation into novel compounds should pay special attention to exploring the following pitfalls to success of RyR2 active therapeutics. First, given the location dependence of previous work, care should be taken to explore the compounds efficacy across multiple mutation models if such models are used to demonstrate efficacy during the development pathway. Second, given RyR2’s importance in EC coupling, any RyR2 active compound moving towards clinical trials should explore the compound’s effect on contractility both in the short and long term. Given the life-long nature of the cardiac pathology in question, most patients will be dependent on these compounds for the rest of their lives. Therefore, it will be important to ensure that the RyR2 altering properties do not lead to pathologic remodeling or decreased contractility with long-term use. Third, consideration should be given to conducting pre-clinical trials in a variety of animal and human models of disease, given cross-species variability, as is standard practice for all drug development.

With properly robust investigation into the potential of RyR2 compounds, we are confident that the field of cardiovascular therapeutics can be advanced to better treat AF, CPVT and HF by addressing the underlying pathologic mechanism of disease, the malfunctioning RyR2.

Figure 1.

A. Schematic overview of key ion channels and transporters involved in excitation-contraction coupling in cardiac myocytes. Calcium (Ca²⁺) enters via voltage-activated L-type Ca²⁺ channels (LTCC), which in turn triggers the release of Ca²⁺ from the sarcoplasmic reticulum (SR) via type-2 ryanodine receptors (RyR2). During diastole, Ca²⁺ is pumped back into the SR via sarco/endoplasmic reticulum ATPase (SERCA2a), which is modulated by phospholamban (PLN), or extruded from the cell via the Na⁺/Ca²⁺-exchanger (NCX). B. In normal, healthy hearts, RyR2 channels release Ca²⁺ during systole, but remain closed during diastole. In disease hearts, however, inherited mutations of acquired defects in RyR2 prevent the channel from remaining fully closed during diastole, leading to pathological SR Ca²⁺ leak.

Article Highlights.

• The type-2 ryanodine receptor plays a key role in excitation-contraction coupling in cardiomyocytes

• Inherited mutations or acquired defects in RyR2 and its regulatory subunits promote diastolic Ca²⁺ leak from the sarcoplasmic reticulum

• Pathogenic SR Ca²⁺ leak can promote 1) delayed afterdepolarizations resulting in arrhythmias and 2) depletion of the SR Ca²⁺ content leading to impaired contractility associated with heart failure

• Several FDA approved anti-arrhythmic drugs including flecainide, propafenone and dantrolene suppress pathogenic, diastolic RyR2 openings associated with heart disease but they also inhibit other ion channels

• Various new RyR2 modulating drugs are in preclinical development, and normalize RyR2 channel activity via distinct molecular mechanisms