Chapter 25 RYANODINE RECEPTOR DYSFUNCTION IN

RYANODINE RECEPTOR DYSFUNCTION IN HEART FAILURE AND ARRHYTHMIAS

Stephan E. Lehnart, Xander H.T. Wehrens, and Andrew R. Marks

Dept. of Physiology and Cellular Biophysics, Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia University, New York, NY

INTRODUCTION

Ventricular tachycardias and sudden cardiac death (SCD) are associated

with underlying cardiac disease including ischemic heart disease and heart

failure. Although arrhythmias are a relatively common cause of sudden

cardiac death, the mechanisms that trigger and sustain electrical instability of

the heart are not well understood. Abnormalities in intracellular cycling

have been associated with heart failure (HF), cardiac hypertrophy, and in

several genetic forms of arrhythmias. Aberrant diastolic sarcoplasmic

reticulum (SR) release provides a mechanism for the initiation of

arrhythmias. Abnormal SR release during the relaxation phase of the

heart (diastole), when intracellular concentrations are low, causes

delayed afterdepolarizations (DADs). Afterdepolarizations caused by

abnormal diastolic SR release cause electrical instability of the

membrane potential which may degenerate into arrhythmias. In both the

failing heart and in genetic forms of arrhythmias, defective cardiac

ryanodine receptor (RyR2) release channels have been associated with

abnormal SR release. Patients with missense mutations in the RyR2

gene exhibit exercise-induced arrhythmias in the structurally normal hearts,

which implies a dominant role for aberrant diastolic SR release as a

trigger for cardiac arrhythmias. We will review recent findings that indicate

RyR2-dependent diastolic SR leak as an arrhythmogenic trigger

mechanism and as a new therapeutic target.

HEART FAILURE RESULTS IN DEFECTIVE HANDLING

About 50% of patients with HF die suddenly and a majority of these deaths are related to ventricular tachycardias. Heart failure is characterized by neurohumoral activation as a futile compensatory mechanism to counteract reduced cardiac output, resulting in increased catecholamine concentrations in the plasma and heart.

Chronically increased stimulation of the signaling cascade has been shown to result in maladaptive changes that sustain and worsen HF.

Regardless of the etiology, HF results in defective homeostasis in cardiomyocytes.

Relative to the normal heart, the whole cell transient is reduced in amplitude and the decay of the transients slowed in cardiomyocytes and heart muscles from HF patients and in animal models of HF.

The reduced amplitude of the intracellular transient underlies the reduced force production of failing heart muscle

and is associated with decreased SR concentrations.

SR depletion has been linked to decreased SR uptake in HF.

Reduced SR uptake is related to decreased SR ATPase (SERCA2a) pump function and decreased SR uptake rate due to relative upregulation of, and hypo-phosphorylation of phospholamban (PLB),

which inhibits SERCA2a.

The net result of increased SR leak via RyR2 (see below) and decreased SERCA2a function is reduced SR

concentrations and prolonged decay of the SR release transient, which impairs contractility.

Chronic stimulation of the sympathetic nervous system in HF causes hyperactivity of protein kinase A (PKA) in the terminal or junctional SR (see Chapter 4). PKA hyperphosphorylation of RyR2 release channels results in depletion of the channel-stabilizing subunit calstabin2 (FKBP12.6) from the RyR2 channel complex (see Chapter 15), resulting in increased RyR2 channel activity due to enhanced -dependent activation.

A chronic defect in diastolic RyR2 closure may contribute to SR store depletion.

Our group and others have recently shown that RyR2 is a macromolecular signaling complex in which mAKAP (AKAP6) targets PKA specifically to the RyR2 release channel, thereby establishing compartmentalized PKA phosphorylation-dependent modulation of SR release which is distinct from other handling proteins.

In hearts of HF patients, decreased levels of protein phosphatases PP1 and PP2A were observed in the RyR2 channel complex, contributing to chronic RyR2 PKA hyperphosphorylation.

Thus, PKA hyperphosphorylation of

and calstabin2 depletion result in “leaky” RyR2 channels

and

increased diastolic SR leak

, which have been demonstrated in HF patients and multiple animal models of HF (Fig. 25-1 A).

Figure 25-1. Model of the RyR2 release channel in heart failure and CPVT. A. In

failing hearts, PKA hyperphosphorylation of in RyR2 due to chronic activation of the

sympathetic nervous system and reduced amounts of protein phosphatases 1 and 2A (PP1,

PP2A) in the RyR2 macromolecular complex, depletes calstabin2 (FKBP12.6) from the RyR2

complex. Increased diastolic RyR2 open probability results in depletion of SR stores,

contributing to reduced systolic RyR2 release. In addition, leak during diastole can

initiate arrhythmias. B. In CPVT, inherited RyR2 missense mutations reduce the calstabin2

binding affinity for RyR2. During exercise, PKA phosphorylation of RyR2 further depletes

calstabin2 from the channel complex leading to increased -dependent activation and

aberrant diastolic SR release that can activate DADs and trigger fatal ventricular

arrhythmias.

Chapter 25 The diastolic SR leak via PKA hyperphosphorylated RyR2 in cardiomyocytes from failing hearts, combined with decreased SR uptake due to decreased SERCA2a activity, results in partial SR depletion such that action potentials leading to activation of induced

release (CICR) release less resulting in decreased contractility.

Chronic sympathetic stimulation that characterizes heart failure causes PKA- hyperphosphorylation of RyR2, thereby further increasing SR leak in HF.

Additional changes occurring in HF contribute to defective intracellular homeostasis. Increased exchanger (NCX) expression and function have been demonstrated in HF.

Since NCX and SERCA2a compete for extrusion, more is extruded from the cytosol to the extracellular space, contributing to SR store depletion in HF.

In the context of a prolonged intracellular transient,

extrusion via NCX compensates for decreased SR uptake at the cost of increased membrane depolarization.

On the other hand, reverse mode NCX influx during the late phase of the action potential has been demonstrated to contribute to increased intracellular in HF cardiomyocytes.

To summarize, HF results in altered function of several key handling proteins involved in excitation- contraction coupling resulting in partial SR store depletion that is associated with RyR2-dependent diastolic SR leak.

ARRHYTHMIC MECHANISMS IN HEART FAILURE

Alterations of intracellular cycling have been described consistently in failing hearts, and represent a candidate mechanism for the initiation of DADs and triggered arrhythmias. Alterations in transmembrane ion transport exacerbate the electrical instability of the cardiomyocyte membrane. Among several alterations in HF, expression and function of the is decreased, resulting in a reduction of outward repolarizing current, increased intracellular concentrations, increased reverse-mode NCX current and intracellular concentrations.

Functional downregulation of currents delays repolarization and shifts the resting membrane potential to more positive levels.

Abnormalities in membrane repolarization and action potential prolongation contribute to arrhythmogenesis and sudden death in HF patients.

Diastolic SR leak can activate a transient inward current which causes membrane depolarizations resulting in DADs and triggered arrhythmias.

It is thought that results from forward mode NCX net

influx

or from a current

In the failing

heart, a prominent increase in NCX function contributes significantly more

depolarizing current, and increases the propensity for arrhythmias triggered by DADs

and early afterdepolarizations (EADs).

Acutely, the propensity for triggered arrhythmias may be further increased by activation of signaling due to upregulation of SR uptake and leak in the context of reduced repolarizing currents in HF.

Indeed, mapping studies in animal models and in patients with heart failure have identified repetitive focal activity as a mechanism underlying ventricular tachycardia consistent with DADs due to SR leak in cardiomyocytes.

INHERITED CARDIAC ARRHYTHMIA SYNDROMES DISTURBING INTRACELLULAR CYCLING

Inherited mutations in genes that are involved in intracellular handling increase the propensity for SCD and ventricular tachycardias (VT).

Autosomal-dominant inherited missense mutations in the RyR2 gene have been linked to exercise-induced arrhythmias,

known as catecholaminergic polymorphic ventricular tachycardia (CPVT).

Mutation carriers characteristically develop arrhythmias during exercise or emotional stress but not during rest (Fig. 25-2 A). Overall mortality rates are 30-50% at age 35 (Fig. 25-2 B),

and only partial protection from syncope and SCD can be achieved by treatment with treatment.

RyR2 missense mutations have also been linked to arrhythmogenic right ventricular dysplasia/cardiomyopathy type 2 (ARVD/C2).

Importantly, arrhythmias in CPVT mutation carriers occur in the structurally normal heart, whereas ARDV/C2 is associated with dysplasia of the right ventricle.

Studies of single RyR2 channels containing missense mutations found in CPVT patients demonstrated a gain-of-function defect following PKA phosphorylation, consistent with ‘leaky’ RyR2 channels (diastolic SR leak) during stress/exercise.

Consistent with these findings, intracellular

leak was observed after stimulation in atrial tumor cells expressing the same CPVT mutant RyR2 channels.

The observation that RyR2 missense mutations only result in leaky release channels under conditions that mimic activation of the signaling cascade

is in agreement with the finding in affected mutation carriers that arrhythmias only occur during stress or exercise.

An important finding in CPVT mutant RyR2 channels is a decreased binding affinity of the channel-stabilizing subunit calstabin2, contributing to enhanced dissociation of calstabin2 from the RyR2 channel complex during PKA phosphorylation, which enhances diastolic SR leak.

However, it is important

to understand that calstabin2 is bound to CPVT mutant RyR2 channels under

resting conditions, in contrast to calstabin2-depletion due to PKA

Chapter 25 hyperphosphorylation of RyR2 in failing hearts, which sustains diastolic SR

leak even at rest (Fig. 25-1).

Figure 25-2. Ryanodine receptor dysfunction causes ventricular arrhythmias in CPVT patients and calstabin2-deficient mice. A. Example of a ventricular tachycardia degenerating into ventricular fibrillation in a CPVT patient carrying a RyR2 missense mutation. Reproduced with permission from Priori et al.

B. Kaplan-Meyer curve showing high mortality rates in carriers of the RyR2-Q4201R missense mutation linked to CPVT.

Reproduced with permission from Lehnart et al.

C. Electrocardiogram of a

haploinsufficient mouse, showing polymorphic ventricular tachycardia recorded by telemetry during exercise testing. Reproduced with permission from Wehrens et al.

Missense or nonsense mutations in the calsequestrin 2 gene (CSQ2) have

been liked to an autosomal-recessive form of CPVT.

Calsequestrin 2 is

the most abundant binding protein within the SR store of cardiomyocytes, and functions as a high capacity low affinity buffer presumably regulating RyR2 function. It was proposed that CSQ2 missense mutations decrease intra-SR binding, which would increase the concentration of free SR consistent with SR overload and diastolic SR leak as a mechanism causing DADs and arrhythmias.

For details about RyR2 regulation by luminal SR and calsequestrin, please refer to Chapter 7. An autosomal-dominant form of the long-QT syndrome (LQT4), associated with cardiac arrhythmias and sudden cardiac death, was linked to inactivating point mutations in the ankyrin-B gene (AnkB).

Interestingly, arrhythmias and SCD typically occurred in AnkB mutation carriers after physical exertion and emotional stress.

It is thought that AnkB mutations cause defective membrane targeting of handling proteins, including the inositol 1,4,5-trisphosphate receptor NCX and the resulting in SR overload and diastolic SR leak as a trigger of DADs and arrhythmias.

To summarize, a variety of mutations that affect intracellular handling directly or indirectly cause arrhythmias by alterations in intracellular cycling all of which are consistent with diastolic SR serving as the trigger for fatal cardiac arrhythmias. Functional defects in intracellular handling become exaggerated during stimulation, activating inward depolarizing currents and DADs that are the likely trigger mechanism for cardiac arrhythmias.

MOUSE MODELS OF TRIGGERED ARRHYTHMIAS

Genetic mouse models have been employed to explore defective intracellular cycling as a trigger mechanism of arrhythmias. Since CPVT mutant RyR2 channels display decreased calstabin2 binding affinities,

we hypothesized that depletion of calstabin2 from the RyR2 channel complex facilitates RyR2 leak which may cause DADs and initiate arrhythmias.

Indeed, knockout mice consistently develop DADs, ventricular tachycardias, and SCD following exercise and

stimulation (Fig. 25-2 C).

These observations are supported by our findings that missense mutations in RyR2 linked to CPVT reduce the affinity of calstabin2 for RyR2 and increase single-channel activity under conditions that simulate exercise during diastole.

Previous studies have found that interventions that reduce calstabin2

binding to RyR2 increase the channel open probability causing SR leak

and altered EC coupling.

Consistent with these findings, RyR2

channels from knockout mice show increased open probabilities

under exercised conditions during diastole.

Whereas diastolic SR leak may become effectively buffered under resting conditions,

the pronounced gain-of-function defect seen during exercise and sympathetic activation causes pronounced diastolic SR leak, which triggers DADs and arrhythmias.

PHARMACOLOGIC TARGETING OF DEFECTIVE INTRACELLULAR RELEASE

There are only a few antiarrhythmic drugs that reduce the incidence of SCD, however, with a low efficacy providing only partial protection. The beneficial effects of in heart failure are, at least in part, related to the reduction of RyR2 PKA hyperphosphorylation and normalization of RyR2 open probability.

It has become clear that drugs that block or channel function may increase mortality from arrhythmias.

Therefore, an alternative rationale would be to develop drugs that stabilize, rather than block channel function. Since calstabin2 stabilizes the RyR2 closed state, we hypothesized that rebinding of calstabin2 to the channel complex may reduce SR leak and prevent cardiac arrhythmias.

We showed that a calstabin2 (FKBP12.6)-D37S mutant subunit binds to

PKA hyperphosphorylated RyR2 with a higher affinity compared to wild-

type calstabin2, which has markedly reduced affinity for PKA

phosphorylated RyR2 as compared to unphosphorylated RyR2.

Calstabin2-D37S reduced the open probability of the PKA

hyperphosphorylated RyR2 channel and rescued the gain-of-function defect

that causes diastolic SR leak.

Adenoviral overexpression of wild-type

calstabin2 in cardiomyocytes may also reduce SR leak, implying that

supra-physiologic intracellular calstabin2 levels may promote calstabin2

binding to PKA phosphorylated RyR2.

Recently, we demonstrated that the

1,4-benzothiazepine derivative JTV519 can prevent arrhythmias and sudden

cardiac death in haploinsufficient but not deficient

mice, by increasing the binding of calstabin2 to the RyR2 channel

complex.

Moreover, JTV519 normalizes the gain-of-function defect in

CPVT-mutant RyR2 channels.

These results strongly support the concept

that JTV519 inhibits diastolic SR leak and cardiac arrhythmias by

increasing the binding of calstabin2 to RyR2 (see also Chapter 26).

CONCLUDING REMARKS

Altered RyR2 function plays an important role in heart failure and in genetic forms of arrhythmias. RyR2 dysfunction causes diastolic SR leak, which can deplete SR stores in failing hearts, and induce delayed afterdepolarizations that trigger arrhythmias. New pharmacological interventions that increase calstabin2 binding to PKA-phosphorylated RyR2 have been shown to increase contractility in a dog model of heart failure. and prevent cardiac arrhythmias in a mouse model of arrhythmias and sudden cardiac death. These studies have identified prevention of diastolic SR leak as a new therapeutic strategy for heart failure and certain forms of sudden cardiac death.

ACKNOWLEDGMENTS

Supported by grants to A.R.M. from the NIH and a postdoctoral grant from the Deutsche Forschungsgemeinschaft (DFG) to S.E.L. A.R.M. is the Doris Duke Charitable Foundation Distinguished Clinical Scientist;

X.H.T.W. is a recipient of the Glorney-Raisbeck fellowship of the New

York Academy of Medicine.