Xander H.T. Wehrens

Ca2+/Calmodulin-Dependent Protein Kinase II Phosphorylation Regulates the Cardiac Ryanodine Receptor

Abstract— The cardiac ryanodine receptor (RyR2)/calcium release channel on the sarcoplasmic reticulum is required for muscle excitation-contraction coupling. Using site-directed mutagenesis, we identified the specific Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation site on recombinant RyR2, distinct from the site for protein kinase A (PKA) that mediates the “fight-or-flight” stress response. CaMKII phosphorylation increased RyR2 Ca2+ sensitivity and open probability. CaMKII was activated at increased heart rates, which may contribute to enhanced Ca2+-induced Ca2+ release. Moreover, rate-dependent CaMKII phosphorylation of RyR2 was defective in heart failure. CaMKII-mediated phosphorylation of RyR2 may contribute to the enhanced contractility observed at higher heart rates. The full text of this article is available online at http://circres.ahajournals.org.

Impact of noncardiac comorbidities on morbidity and mortality in a predominantly male population with heart failure and preserved versus reduced ejection fraction

OBJECTIVES The aim of this study was to evaluate the prevalence and prognostic impacts of noncardiac comorbidities in patients with heart failure (HF) with preserved ejection fraction (HFpEF) compared with those with HF with reduced ejection fraction (HFrEF). BACKGROUND There is a paucity of information on the comparative prognostic significance of comorbidities between patients with HFpEF and those with HFrEF. METHODS In a national ambulatory cohort of veterans with HF, the comorbidity burden of 15 noncardiac comorbidities and the impacts of these comorbidities on hospitalization and mortality were compared between patients with HFpEF and those with HFrEF. RESULTS The cohort consisted of 2,843 patients with HFpEF and 6,599 with HFrEF with 2-year follow-up. Compared with patients with HFrEF, those with HFpEF were older and had higher prevalence of chronic obstructive pulmonary disease, diabetes, hypertension, psychiatric disorders, anemia, obesity, peptic ulcer disease, and cancer but a lower prevalence of chronic kidney disease. Patients with HFpEF had lower HF hospitalization, higher non-HF hospitalization, and similar overall hospitalization compared with those with HFrEF (p < 0.001, p < 0.001, and p = 0.19, respectively). An Increasing number of noncardiac comorbidities was associated with a higher risk for all-cause admissions (p < 0.001). Comorbidities had similar impacts on mortality in patients with HFpEF compared with those with HFrEF, except for chronic obstructive pulmonary disease, which was associated with a higher hazard (1.62 [95% confidence interval: 1.36 to 1.92] vs. 1.23 [95% confidence interval: 1.11 to 1.37], respectively, p = 0.01 for interaction) in patients with HFpEF. CONCLUSIONS There is a higher noncardiac comorbidity burden associated with higher non-HF hospitalizations in patients with HFpEF compared with those with HFrEF. However, individually, most comorbidities have similar impacts on mortality in both groups. Aggressive management of comorbidities may have an overall greater prognostic impact in HFpEF compared to HFrEF.

Enhanced Sarcoplasmic Reticulum Ca2+ Leak and Increased Na+-Ca2+ Exchanger Function Underlie Delayed Afterdepolarizations in Patients With Chronic Atrial Fibrillation

Background— Delayed afterdepolarizations (DADs) carried by Na+-Ca2+-exchange current (INCX) in response to sarcoplasmic reticulum (SR) Ca2+ leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined. Methods and Results— Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca2+]i (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca2+]i and SR Ca2+ content (integrated INCX during caffeine-induced Ca2+ transient) were unchanged, whereas diastolic SR Ca2+ leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca2+ leak. Calmodulin expression (60%), Ca2+/calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca2+ leak, the frequency of spontaneous Ca2+ release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca2+ sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca2+]i and INCX density revealed INCX upregulation in cAF. Spontaneous Ca2+ release events accompanied by inward INCX currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca2+]i (diastolic [Ca2+]i–voltage coupling gain) was higher in cAF compared with control. Conclusions— Enhanced SR Ca2+ leak through CaMKII-hyperphosphorylated RyR2, in combination with larger INCX for a given SR Ca2+ release and increased diastolic [Ca2+]i-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

Sudden Death in Familial Polymorphic Ventricular Tachycardia Associated With Calcium Release Channel (Ryanodine Receptor) Leak

Background— Familial polymorphic ventricular tachycardia (FPVT) is characterized by exercise-induced arrhythmias and sudden cardiac death due to missense mutations in the cardiac ryanodine receptor (RyR2), an intracellular Ca2+ release channel required for excitation-contraction coupling in the heart. Methods and Results— Three RyR2 missense mutations, P2328S, Q4201R, and V4653F, which occur in Finnish families, result in similar mortality rates of ≈33% by age 35 years and a threshold heart rate of 130 bpm, above which exercise induces ventricular arrhythmias. Exercise activates the sympathetic nervous system, increasing cardiac performance as part of the fight-or-flight stress response. We simulated the effects of exercise on mutant RyR2 channels using protein kinase A (PKA) phosphorylation. All 3 RyR2 mutations exhibited decreased binding of calstabin2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. After PKA phosphorylation, FPVT-mutant RyR2 channels showed a significant gain-of-function defect consistent with leaky Ca2+ release channels and a significant rightward shift in the half-maximal inhibitory Mg2+ concentration (IC50). Treatment with the experimental drug JTV519 enhanced binding of calstabin2 to RyR2 and normalized channel function. Conclusions— Sympathetic activation during exercise induces ventricular arrhythmias above a threshold heart rate in RyR2 mutation carriers. Simulating the downstream effects of the sympathetic activation by PKA phosphorylation of RyR2 channels containing these FPVT missense mutations produced a consistent gain-of-function defect. RyR2 function and calstabin2 depletion were rescued by JTV519, suggesting stabilization of the RyR2 channel complex may represent a molecular target for the treatment and prevention of exercise-induced arrhythmias and sudden death in these patients.

β-Blockers Restore Calcium Release Channel Function and Improve Cardiac Muscle Performance in Human Heart Failure

Background—Chronic &bgr;-adrenergic receptor (&bgr;-AR) blockade improves cardiac contractility and prolongs survival in patients with heart failure; however, the mechanisms underlying these favorable responses are poorly understood. Stress-induced activation of the sympathetic nervous system results in protein kinase A (PKA)-mediated phosphorylation of the calcium (Ca2+) release channel/cardiac ryanodine receptor (RyR2), required for cardiac excitation-contraction (EC) coupling, activating the RyR2 channel, and increasing cardiac contractility. The hyperadrenergic state of heart failure results in leaky RyR2 channels attributable to PKA hyperphosphorylation and depletion of the stabilizing FK506 binding protein, FKBP12.6. We tested the hypothesis that improved cardiac muscle function attributable to &bgr;-AR blockade is associated with restoration of normal RyR2 channel function in patients with heart failure. Methods and Results—We assessed the effects of &bgr;-AR blockade on left ventricular volume using isolated perfused hearts and &bgr;-agonist responsiveness using muscle strips from patients undergoing transplantation. Twenty-four human hearts were examined, 10 from patients with heart failure treated with &bgr;-AR blockers (carvedilol, metoprolol, or atenolol), 9 from patients with heart failure without &bgr;-AR blocker treatment, and 5 normal hearts. RyR2 PKA phosphorylation was determined by back-phosphorylation, FKBP12.6 in the RyR2 macromolecular complex was determined by coimmunoprecipitation, and channel function was assayed using planar lipid bilayers. &bgr;-AR blockers reduced left ventricular volume (reverse remodeling) and restored &bgr;-agonist response in cardiac muscle from patients with heart failure. Improved cardiac muscle function was associated with restoration of normal FKBP12.6 levels in the RyR2 macromolecular complex and RyR2 channel function. Conclusions—Improved cardiac muscle function during &bgr;-AR blockade is associated with improved cardiac Ca2+ release channel function in patients with heart failure.

Defective Cardiac Ryanodine Receptor Regulation During Atrial Fibrillation

Background—Ca2+ leak from the sarcoplasmic reticulum (SR) may play an important role in triggering and/or maintaining atrial arrhythmias, including atrial fibrillation (AF). Protein kinase A (PKA) hyperphosphorylation of the cardiac ryanodine receptor (RyR2) resulting in dissociation of the channel-stabilizing subunit calstabin2 (FK506-binding protein or FKBP12.6) causes SR Ca2+ leak in failing hearts and can trigger fatal ventricular arrhythmias. Little is known about the role of RyR2 dysfunction in AF, however. Methods and Results—Left and right atrial tissue was obtained from dogs with AF induced by rapid right atrial pacing (n=6 for left atrial, n=4 for right atrial) and sham instrumented controls (n=6 for left atrial, n=4 for right atrial). Right atrial tissue was also collected from humans with AF (n=10) and sinus rhythm (n=10) and normal cardiac function. PKA phosphorylation of immunoprecipitated RyR2 was determined by back-phosphorylation and by immunoblotting with a phosphospecific antibody. The amount of calstabin2 bound to RyR2 was determined by coimmunoprecipitation. RyR2 channel currents were measured in planar lipid bilayers. Atrial tissue from both the AF dogs and humans with chronic AF showed a significant increase in PKA phosphorylation of RyR2, with a corresponding decrease in calstabin2 binding to the channel. Channels isolated from dogs with AF exhibited increased open probability under conditions simulating diastole compared with channels from control hearts, suggesting that these AF channels could predispose to a diastolic SR Ca2+ leak. Conclusions—SR Ca2+ leak due to RyR2 PKA hyperphosphorylation may play a role in initiation and/or maintenance of AF.

Circadian rhythms govern cardiac repolarization and arrhythmogenesis.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada’s syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.

Cellular and Molecular Mechanisms of Atrial Arrhythmogenesis in Patients With Paroxysmal Atrial Fibrillation

Background— Electrical, structural, and Ca2+-handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca2+-release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca2+-release events in paroxysmal AF (pAF) are unknown. Method and Results— Right-atrial appendages from control sinus rhythm patients or patients with pAF (last episode a median of 10–20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca2+ current, and Na+/Ca2+-exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca2+ leak and incidence of delayed after-depolarizations in pAF. Ca2+-transient amplitude and sarcoplasmic reticulum Ca2+ load (caffeine-induced Ca2+-transient amplitude, integrated Na+/Ca2+-exchange current) were larger in pAF. Ca2+-transient decay was faster in pAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting increased SERCA2a function. In agreement, phosphorylation (inactivation) of the SERCA2a-inhibitor protein phospholamban was increased in pAF. Ryanodine receptor fractional phosphorylation was unaltered in pAF, whereas ryanodine receptor expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both ryanodine receptor dysregulation and enhanced SERCA2a activity promote increased sarcoplasmic reticulum Ca2+ leak and sarcoplasmic reticulum Ca2+-release events, causing delayed after-depolarizations/triggered activity in pAF. Conclusions— Increased diastolic sarcoplasmic reticulum Ca2+ leak and related delayed after-depolarizations/triggered activity promote cellular arrhythmogenesis in pAF patients. Biochemical, functional, and modeling studies point to a combination of increased sarcoplasmic reticulum Ca2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.

Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias

Catecholaminergic polymorphic ventricular tachycardia is a form of exercise-induced sudden cardiac death that has been linked to mutations in the cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutations significantly decrease the binding affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak triggers ventricular tachycardia (VT) and sudden cardiac death. In calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak, monophasic action potential alternans, and bidirectional VT. Calstabin-deficient cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents in diastole consistent with delayed after-depolarizations. The 1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2 for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential alternans and triggered arrhythmias. Our data suggest that calstabin-2 deficiency is as a critical mediator of triggers that initiate cardiac arrhythmias.

Ryanodine Receptor Phosphorylation by Calcium/Calmodulin-Dependent Protein Kinase II Promotes Life-Threatening Ventricular Arrhythmias in Mice With Heart Failure

Background— Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca2+ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca2+ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca2+/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results— Mice in which the S2814 Ca2+/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca2+ release events, resulting in reduced sarcoplasmic reticulum Ca2+ load on confocal microscopy. These Ca2+ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca2+/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. Conclusions— Our results suggest that Ca2+/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca2+ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.