The role of leaky ryanodine receptors in heart disease: a novel therapeutic target

Andrew R. MARKS, MD
Department of Physiology and Cellular Biophysics and the Clyde and Helen Wu Center for Molecular Cardiology
College of Physicians and Surgeons of Columbia University, New York
NY 10032, USA

The role of leaky ryanodine receptors in heart disease: a novel therapeutic target

by A. R. Marks, USA

Contraction of the heart and limbs requires activation of muscle by the release of intracellular calcium (Ca2+) from the sarcoplasmic reticulum. Impaired release of Ca2+ can reduce muscle contraction and increased Ca2+ release can be toxic. If intracellular Ca2+ is released at a time when the muscle is supposed to be relaxed, disastrous consequences canoccur, such as impaired relaxation, extrusion of Ca2+ from the cell leading to decreased Ca2+ transients, and weakened contraction, arrhythmias, muscle damage, or even death. Intracellular Ca2+ release is regulated by specialized channels known as ryanodine receptors (RyRs). RyR channels undergo stress-induced posttranslational modifications (chiefly phosphorylation, nitrosylation, and oxidation) that impair binding of the stabilizing subunit calstabin (CALcium release channel STABilizing proteIN; also known as FKBP) to the channels. Calstabin stabilizes the RyR channel closed state and prevents a pathological leak of intracellular Ca2+. Leaky cardiac RyR2s exacerbate heart failure progression and arrhythmias, leaky skeletal muscle RyR1s impair muscle function in muscular dystrophy, and leaky neuronal RyR2s play a key role in post-traumatic stress disorder. In animal models, a novel class of small molecules, known as Rycals, inhibit stress-induced dissociation of calstabin from RyR channels, reduce intracellular Ca2+ leak, and are potential novel therapeutics.

Medicographia. 2014;36:342-348 (see French abstract on page 348)

Excitation-contraction coupling

With each beat of the heart, calcium (Ca2+) is released from the sarcoplasmic reticulum (SR) via the cardiac type 2 ryanodine receptor (RyR2), raising the cytosolic Ca2+ concentration about tenfold (≈1 μM) and activating cardiac muscle contraction (Figure 1).1 The Ca2+ is then pumped back into the SR by the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), lowering the cytosolic Ca2+ concentration to baseline levels (≈100 nM), and causing relaxation. The Ca2+ release and reuptake cycle is initiated by the action potential, an electrical signal that depolarizes the plasma membrane and the specialized invagination of the plasma membrane called the transverse tubule (T tubule). Voltage- gated Ca2+ channels on the T tubule are activated by depolarization and allow a small amount of Ca2+ to run down its concentration gradient from μM external ([Ca2+]ext) to nM cytosolic ([Ca2+]cyt). Ca2+ entering via the plasma membrane voltagegated Ca2+ channels binds to and activates RyR2 channels, which then release Ca2+ stored at high concentration (≈ μM) in the SR. The elevation of [Ca2+]cyt results in the binding of Troponin C, allowing actin-myosin cross-bridging such that the thick and thin filaments of the sarcomere slide past each other, shortening the sarcomere and causing cardiac muscle contraction. The release of Ca2+ from the SR determines the amplitude of the Ca2+ transient, which in turn regulates the force of cardiac muscle contraction. The release and reuptake of intracellular Ca2+, referred to as Ca2+ cycling, drives muscle contraction and relaxation.

Figure 1
Figure 1. Calcium (Ca2+) cycling in cardiac myocytes and regulation
by protein kinase A (PKA).

Excitation-contraction coupling in the heart is initiated by depolarization of the
transverse tubule (T-tubule) that activates voltage-gated L-type Ca2+ channels
(LTCC) in the plasma membrane. Ca2+ influx via LTCC triggers Ca2+ release from
the sarcoplasmic reticulum (SR) via type 2 cardiac ryanodine receptors (RyR2).
Catecholamine activation of β-adrenergic receptors (β-ARs) stimulates adenylyl
cyclase (AC), and the generation of cyclic adenosine monophosphate (cAMP),
which in turn activates PKA. PKA phosphorylates several targets, augmenting
the Ca2+ transient by increasing the activities of LTCC, RyR2, and sarcoplasmic/
endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a). Relaxation occurs after the
Ca2+ is pumped out of the cytoplasm and into the SR by SERCA2a, which is
regulated by phospholamban (PLB). In addition, Ca2+ is extruded from the cell
by the sarcolemmal Na+/Ca2+ exchanger (NCX). Chronic activity of the sympathetic
nervous system leads to hyperphosphorylation (indicated with the red “P”)
of the β-AR, activation of β-AR kinase (βARK), and desensitization of β-ARs.
In heart failure (HF), RyR2 is hyperphosphorylated by PKA, leading to an increased
open probability at low [Ca2+], consistent with leak of Ca2+ during diastole.
The long-term effect of increased Ca2+ release and diastolic Ca2+ leak is depletion
of SR Ca2+ stores. SERCA2a expression and activity is decreased in HF,
which is linked to PLB hypophosphorylation. Arrows indicate increased or decreased
expression or activity in HF.
Abbreviations: Gs, heterotrimeric G protein.
Adapted from reference 1: Marx SO et al. J Mol Cell Cardiol. 2013;58:225-231.
© 2013 Elsevier Ltd.

Ryanodine receptors are highly specialized calcium release channels

RyR channels are the largest known ion channels possessing enormous cytoplasmic domains that contain binding sites for regulatory subunits that integrate extracellular signals to modulate the release of intracellular Ca2+. Along with the closely related inositol 1,4,5-trisphosphate receptors, they are the only channels that release Ca2+ from intracellular stores. RyR1 is required for skeletal muscle contraction and RyR2 for cardiac muscle contraction. The cytoplasmic domains of the RyR channels are enormous macromolecular complexes (>3 million daltons) that include kinases, phosphatases, phosphodiesterases, and their targeting proteins, as well as regulatory subunits such as calstabin (CALcium release channel STABilizing proteIN, also known as FK506 binding protein [FKBP]) (Figure 2, page 344).2

RyR2 are homotetrameric intracellular Ca2+ release channels on the SR with at least two functional domains: the carboxyterminus containing the transmembrane segments forming the Ca2+ conducting pore and the large amino-terminus (termed the “foot” structure), which contains modulatory binding sites. Several modulatory elements are bound to it, including FKBP (aka calstabin), a member of the immunophilin familyof cis-trans peptidyl-prolyl isomerases (FKBP12 in skeletal muscle3 and FKBP12.6 in cardiac muscle)4,5; protein kinase A (PKA) and its anchoring protein, muscle A kinase anchoring protein (mAKAP); protein phosphatase 1 (PP1); and protein phosphatase 2A (PP2A).6 FKBP12 (calstabin1) and FKBP12.6 (calstabin2), endogenous modulators of RyR1 and RyR2 respectively, have been shown to stabilize the channel complex, resulting in channels that demonstrate full conductance.4,7 The recruitment of the kinase(s) and phosphatases into the complex is mediated through specific leucine/isoleucine zippers present on both the targeting/anchoring proteins and RyRs.8

Role of calstabins in modulating RyR2 function

We originally reported that calstabin1 is a subunit of RyR13 that is required to stabilize the closed state of the channel and prevent a pathological intracellular Ca2+ leak through the channel.7 We subsequently discovered that diastolic SR Ca2+ leak plays a prominent role in heart failure (HF) progression.6 At the time, this was highly controversial because the dogma was that there was SR Ca2+ overload in HF and we reported the opposite—that leaky RyR2 channels leading to SRCa2+ depletion accounted for the diminished Ca2+ transient observed in HF and the weakened heart muscle contraction. Subsequently, others have confirmed our work showing that the diastolic SR Ca2+ leak through the RyR channels is indeed pathological and contributes to heart disease, and that SR Ca2+ is depleted in failing hearts.9 Moreover, calstabin24,10 plays an important physiological role in stabilizing the closed state of RyR2 and is required to prevent the diastolic SR Ca2+ leak.6

Figure 2
Figure 2. Remodeling of RyR2 macromolecular complex in failing hearts and impact of treatment.

(A) The cardiac type 2 ryanodine receptor (RyR2) macromolecular complex is comprised of four RyR2 subunits (1-4 indicate the four monomers). Each RyR2 subunit binds one molecule of calstabin2 (also known as FKBP12.6) and muscle A kinase anchoring protein (mAKAP), which is the targeting protein for the protein kinase A (PKA) catalytic and regulatory subunits and phosphodiesterase 4D3 (PDE4D3); protein phosphatase 2A (PP2A) and its targeting protein PR130; and protein phosphatase 1 (PP1) and its targeting protein sphinophilin (accessory molecules are shown for one of the four RyR2 subunits, except calstabin2, which is shown for all four RyR2 subunits). The β-adrenergic signaling pathway can activate PKA through the second messenger cyclic adenosine monophosphate (cAMP). (B) In heart failure, PKA hyperphosphorylation of RyR2 at Ser2809, due to reduced PDE4D3, PP1, and PP2A levels in the RyR2 macromolecular complex, depletes calstabin2 from the RyR2 channel. This stress-induced remodeling of RyR2, causing a diastolic sarcoplasmic reticulum (SR) calcium (Ca2+) leak due to abnormal RyR2 channel openings, may be prevented by treatment with β-blockers (BBs), which interfere with the upstream β-adrenergic receptors (β-AR) signaling pathway, or with Rycals, which selectively increase the binding affinity of calstabin2 to PKA-phosphorylated, nitrosylated, and oxidized RyR2.
Adapted from reference 2: Marks AR.
J Clin Invest. 2013;123:46-52.© 2013, American Society for Clinical Investigation.

PKA hyperphosphorylation of RyR2 causes depletion of calstabin2 from the RyR2 complex.6 We found that oxidation and nitrosylation of RyR2 also cause depletion of calstabin2 from the RyR2 macromolecular complex.11,12 Moreover, the combination of oxidation, nitrosylation, and Ser2808 phosphorylation depleted nearly all of the calstabin2 from the channel complex.11,12 These observations have been supported by data from RyR2-S2808A mice, which harbor RyR channels that cannot be PKA phosphorylated and are therefore protected against PKA phosphorylation–induced depletion of calstabin2, and from phosphomimetic RyR2-S2808D mice that exhibit decreased levels of calstabin2 in the RyR2 complex.11-14

Regulation of ryanodine receptors: role of phosphorylation

Activation of β-adrenergic receptors (β-ARs) leads to elevated cyclic adenosine monophosphate (cAMP) and activation of PKA (Figure 1).1 β-AR activation has multiple targets, including voltage-dependent L-type Ca2+ channels (Cav1.2), RyR2, and phospholamban, the regulator of SERCA (Figure 1).1

PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulation of RyR2 channel function plays an important role in modulating cardiac contractility and arrhythmogenesis. We have shown that there is a single functional PKA phosphorylation site, a separate single functional CaMKII phosphorylation site on RyR2, and a single functional PKA phosphorylation site on RyR115 yielding four sites for each type of phosphorylation on each homotetrameric channel. Confusion has been created in the field because Ser2809 on canine and human RyR2 (Ser2808 in murine RyR2) was originally identified as both a PKA and CaMKII phosphorylation site.16,17 This was based on phosphopeptide mapping,16 which identified a phosphorylated peptide that contained two sites, one for PKA and one for CaMKII, but this was not appreciated until years later when site-directed mutagenesis studies and knock-in mice showed that Ser2808 is exclusively phosphorylated by PKA,18 and Ser2815 (Ser2814 in murine RyR2) is phosphorylated by CaMKII.15,19,20 Using site-directed mutagenesis to ablate the phosphorylation sites by substituting an alanine residue for the target serine residues, we showed that the channels can no longer be phosphorylated (RyR1-S2844A ablates PKA phosphorylation of RyR1, RyR2-S2808A ablates PKA phosphorylation of RyR2, and RyR2-S2814A ablates CaMKII phosphorylation of RyR2).15 Knock-in mice for each of these mutations confirmed that the respective channels have only a single PKA and CaMKII phosphorylation site.18,19 Mice engineered with a RyR2-S2808A mutation have blunted inotropic and chronotropic responses to catecholamines.11,12,18 Mice engineered with a RyR2-S2814A mutation have RyR2 channels that cannot be phosphorylated by CaMKII, and exhibit a blunted positive force frequency relationship.19 Mice engineered with a phosphomimetic mutation (substituting an aspartic acid residue for serine) of the RyR2 PKA phosphorylation site showed an age-dependent cardiomyopathy and arrhythmias.12 Based on these extensive studies there is no longer any rational basis for referring to the sites for PKA and CaMKII phosphorylation of RyR2 as “controversial.”

The stress of heart failure

A key characteristic of HF is the chronic activation of the sympathetic nervous system (SNS) resulting from a maladaptive physiological response to cardiac dysfunction designed to maintain or improve cardiac function. Acute activation of the SNS maintains cardiac function, but at a high cost. β-Adrenergic agonists or phosphodiesterase inhibitors, which are used to treat acute decompensated HF, increase contractility by increasing cAMP levels and activating PKA, which in turn activates key regulators of excitation-contraction coupling including RyR2, thereby increasing Ca2+ release. However, these treatments also increase mortality, in part because they are proarrhythmic and increase energy consumption.On the other hand, blocking neurohormonal pathways is the current mainstay of HF therapy. The agents used to block neurohormonal pathways (angiotensin-converting enzyme [ACE] inhibitors and β-adrenergic receptor blockers [β-blockers]) improve cardiac function and survival, but they do so at a high cost. First, their use is limited due to side effects (side effects of βblockers include depression, lethargy, impotence, blunting of the physiological response to hypoglycemia [a major problem in diabetic patients taking insulin], and impaired exercise capacity). Moreover, initiation of β-blocker and ACE inhibitor therapy, particularly in the outpatient setting, poses challenges for both the physician and patient. It is necessary to carefully titrate β-blockers and ACE inhibitors, both of which have hemodynamic effects that are particularly problematic in HF patients, gradually increasing the dose while monitoring the heart rate and blood pressure. These side effects and physiological response to β-blockers and ACE inhibitors likely explain why only a relatively small percentage of HF patients receive the recommended doses of β-blockers and ACE inhibitors, and why most HF patients are not physiologically β-blocked. Thus, it is clear that the neurohormonal response (eg, activation of SNS and renin-angiotensin signaling) to cardiac dysfunction is maladaptive, because enhancing this response (eg, with β-AR agonists) causes increased mortality and blocking the SNS response improves survival.

Another factor confounding the use of β-blockers in HF is that their mechanism of action in HF is uncertain. Is it merely slowing the heart rate that explains the benefit of β-blockers in HF? Slowing the heart rate is likely a component of the beneficial effect of β-blockers in HF, as ivabradine, a blocker of the pacemaker current, decreased hospital visits and adverse cardiac events in the SHIFT (Systolic Heart failure treatment with the IF inhibitor ivabradine Trial) study.21 We have shown that β-blockers indirectly “fix” the leak in RyR2; this is achieved in both animal models of HF22 and in patients with HF23 by blocking the β-AR, resulting in reduced PKA phosphorylation and oxidation of RyR2, and preventing depletion of calstabin2 from the RyR2 macromolecular complex. Thus, it appears that Rycals and β-blockers share a common target— the cardiac RyR2 channel leak. In support of this unexpected finding is the fact that mice expressing the phosphomimetic RyR2-S2808D channel with post–myocardial infarction (MI) HF are resistant to β-blockers, while they respond to Rycals the same as wild-type mice.12

Cardiac contractility is determined by the amplitude and kinetics of Ca2+ cycling. We reported that diastolic SR Ca2+ leak via RyR2 channels contributes to HF progression6 and fatal ventricular arrhythmias.24-27 Prior to the diastolic SR Ca2+ leak model of HF, the dogma was that HF was associated with SR Ca2+ overload. Subsequent to our discovery of the diastolic SR Ca2+ leak in HF,6 the presence of both the SR Ca2+ depletion and the diastolic SR Ca2+ leak were confirmed in HF by most of the leading experts in the field.9,13,28-30

The defective SR Ca2+ handling is characterized by leaky RyR2 channels due to stress-induced dissociation of the stabilizing RyR2 subunit calstabin2 (principally manifested as PKA phosphorylation, oxidation, and nitrosylation of the channel), resulting in a diastolic SR Ca2+ leak, reduced SR Ca2+ content, and decreased Ca2+ transient.6,12,24,31-33 Compounding this problem is impaired SR Ca2+ uptake due to reduced activity of SERCA2a as a consequence of reduced SERCA2a expression, increased inhibition of the pump by phospholamban,34 and enhanced Na+/Ca2+ exchanger (NCX) activity (Figure 1).1,35 Thus, these dysfunctional processes conspire to deplete the SR of Ca2+ and lead to impaired cardiac contractility.36 Not surprisingly, therefore, both the RyR2 leak and the impaired uptake have been targeted with novel therapeutics, which are now undergoing clinical testing in HF patients.

Multiple animal models have been used to support a role for PKA hyperphosphorylation of RyR2 in HF progression.37 Genetically altered mice harboring RyR2 that cannot be PKA phosphorylated (RyR2-S2808A), were protected against calstabin2 depletion from the RyR2 complex and HF progres sion 4 weeks post-MI.18 Phosphodiesterase 4D3 (PDE4D3)– deficient mice develop an age-dependent cardiomyopathy and arrhythmias, RyR2 PKA hyperphosphorylation, and calstabin2 depletion. Crossing the PDE4D3-deficient mice and RyR2-S2808A mice was protective.33

Figure 3
Figure 3. Parallels between leaky RyR2 in heart
failure and catecholaminergic polymorphic ventricular
tachycardia (CPVT).

(A,B) In heart failure, protein kinase A (PKA; purple)
hyperphosphorylation of Ser2809 due to reduced protein
phosphatase 1 (PP1; green) and protein phosphatase 22A
(PP22A; grey) in the cardiac type 2 ryanodine receptor (RyR2)
macromolecular complex depletes calstabin2 (FKBP12.6;
yellow) from RyR2 (orange). This results in a diastolic sarcoplasmic
reticulum (SR) calcium (Ca2+) leak, depletion of
SR Ca2+ stores, and functional uncoupling of RyR2, which
can trigger arrhythmias in patients with heart failure. (C,D)
In CPVT, an inherited RyR2 mutation decreases the binding
affinity of calstabin2 to RyR2. After exercise, two to four
calstabin2 are released from the channel complex, leading
to increased Ca2+-dependent activation and diastolic SR
Ca2+ release that can trigger delayed afterdepolarizations
and fatal ventricular arrhythmias.
Adapted from reference 40: Wehrens XH, Marks AR.
Trends Biochem Sci. 2003;28:671-678. © 2003 Elsevier Ltd.

Arrhythmias due to abnormal RyR2 function

Increased RyR2 activity has been shown to cause arrhythmias, particularly associated with increased catecholaminergic stimulation. This is best exemplified by catecholaminergic polymorphic ventricular tachycardia (CPVT), a rare inherited form of exercise-induced sudden cardiac death that occurs in individualswith structurally normal hearts andnormal electrocardiograms. Mutations in RyR2 have been linked to CPVT.38,39 RyR2 withCPVT mutations have reduced affinity for calstabin2, which results in leaky channels during exercise (Figure 3).25,40

Calstabin2-deficient and haploinsufficient mice exhibit CPVT,27 but do not develop HF. Presumably the reason for the lack of cardiac dysfunction is that in contrast to the post-MI model, where calstabin2 depletion from the RyR2 complex has been shown to promote HF progression, the calstabin2-deficient mice have otherwise normal cardiac function and are able to compensate for the loss of calstabin in the absence of a compromised ventricle (eg, no MI). Calstabin2-deficient mice exhibit delayed after depolarizations and exercise-induced ventricular tachycardia,27 and RyR2 from calstabin2-deficient mice exhibit slightly increased open probability at baseline that increases substantially when the mice are exercised.

Early studies on canine atrial myocytes reported altered Ca2+ handling in cells isolated from the atria with atrial fibrillation.41 RyR2 from canine atria with atrial fibrillation are PKA phosphorylated, depleted of calstabin2, and have increased open probability at diastolic [Ca2+].42 Calstabin2-deficient mice have an increased incidence of atrial fibrillation.43 Atrial fibrillation could be induced by intraesophageal burst pacing protocol in 3 CPVT mouse models, RyR2-R2474S(+/–), RyR2-N2386I (+/–), RyR2-L433P(+/–), but not in wild type mice.44 Consistent with these in vivo results, there was a significant diastolic SR Ca2+ leak in atrial myocytes isolated from these CPVT mouse models.

Fixing leaky RyR2 channels: a novel therapeutic approach for heart and muscle diseases

The identification of the diastolic SR Ca2+ leak via RyR2 as a mechanism underlying HF progression and cardiac arrhythmias has led to novel therapeutic approaches. Matsuzaki and colleagues reported that JTV-519 (K201), a 1,4-benzothiazepene, improved cardiac function in a canine model of pacing- induced HF.45 Testing the drug in calstabin2-deficient mice showed that the ability of JTV-519 to prevent HF progression and fatal cardiac arrhythmias requires stabilization of the closed state of RyR2 by calstabin2.25,46 Moreover, JTV-519 had no effect on the gating properties of normal RyR channels and no effect in healthy dogs and mice.32 We generated many derivatives of the 1,4-benzothiazepine JTV-519 and have developed a novel class of Ca2+ release channel stabilizers known as Rycals. An orally available Rycal, S107, improves skeletal muscle force generation and exercise capacity, reduces arrhythmias and improves muscle function in mice with Duchenne muscular dystrophy by reducing pathologic SR Ca2+ leak in cardiac and skeletal muscle.12,32,47-50 Rycals are protective against post-MI HF progression,11,12 and suppressed ventricular tachycardia/ventricular fibrillation and sudden cardiac death in murine models of human CPVT. S107 also raises the seizure threshold in mice with leaky neuronal RyR2 channels and improves exercise capacity in mouse models of sarcopenia (age-related loss of muscle function).24,26,32,33,48 Leaky RyR2 channels in hippocampal neurons play a key role in stress-induced cognitive dysfunction. Treatment with S107 prevented stress-induced cognitive dysfunction in a murine model, suggesting a novel mechanism and therapeutic approach to post–traumatic stress disorder.41


It is evident that the RyR channel plays an important role in cardiac physiology and pathophysiology. Modulating its activity is important for flight-or-fight responses. Long-term activation of the channel, however, is detrimental, causing progression of HF and arrhythmogenesis. Limiting the diastolic leak using either genetic manipulation of RyR (for instance, alanine substitution of PKA phosphorylation site) or Rycals yields clinical benefit in terms of cardiac function post-MI and incidence of arrhythmias (ventricular tachycardia and atrial fibrillation) in mice.

Conflict of interest: Dr Marks is a consultant for and owns shares in ARMGO Pharma Inc, a startup company developing RyR targeted therapeutics. Acknowledgments: Much of the work referred to in this review has been supported by the National Heart Lung and Blood Institute (NHLBI), the Fondation Leducq, the Doris Duke Charitable Foundation, and the Ellison Foundation.

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Keywords: calstabin; excitation-contraction coupling; heart failure; intracellular calcium release channel; macromolecular complex; oxidation; phosphorylation; Rycal