K201 improves aspects of the contractile performance of human failing myocardium via reduction in Ca2+ leak from the sarcoplasmic reticulum

In heart failure, intracellular Ca2+ leak from cardiac ryanodine receptors (RyR2s) leads to a loss of Ca2+ from the sarcoplasmic reticulum (SR) potentially contributing to decreased function. Experimental data suggest that the 1,4-benzothiazepine K201 (JTV-519) may stabilise RyR2s and thereby reduce detrimental intracellular Ca2+ leak. Whether K201 exerts beneficial effects in human failing myocardium is unknown. Therefore, we have studied the effects of K201 on muscle preparations from failing human hearts. K201 (0.3 μM; extracellular [Ca2+]e 1.25 mM) showed no effects on contractile function and micromolar concentrations resulted in negative inotropic effects (K201 1 μM; developed tension −9.8 ± 2.5% compared to control group; P < 0.05). Interestingly, K201 (0.3 μM) increased the post-rest potentiation (PRP) of failing myocardium after 120 s, indicating an increased SR Ca2+ load. At high [Ca2+]e concentrations (5 mmol/L), K201 increased PRP already at shorter rest intervals (30 s). Strikingly, treatment with K201 (0.3 μM) prevented diastolic dysfunction (diastolic tension at 5 mmol/L [Ca2+]e normalised to 1 mmol/L [Ca2+]e: control 1.26 ± 0.06, K201 1.01 ± 0.03, P < 0.01). In addition at high [Ca2+]e, K201 (0.3 μM) treatment significantly improved systolic function [developed tension +27 ± 8% (K201 vs. control); P < 0.05]. The beneficial effects on diastolic and systolic functions occurred throughout the physiological frequency range of the human heart rate from 1 to 3 Hz. Upon elevated intracellular Ca2+ concentration, systolic and diastolic contractile functions of terminally failing human myocardium are improved by K201

Search for cardiac calcium cycling gene mutations in familial ventricular arrhythmias resembling catecholaminergic polymorphic ventricular tachycardia

<p>Abstract</p> <p>Background</p> <p>Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe inherited cardiac disorder caused by mutations predominantly in the ryanodine receptor (<it>RyR2</it>) gene. We sought to identify mutations in genes affecting cardiac calcium cycling in patients with CPVT and in less typical familial exercise-related ventricular arrhythmias.</p> <p>Methods and Results</p> <p>We recruited 33 consecutive patients with frequent ventricular premature complexes (VPCs) without structural heart disease and often history of syncope or sudden death in family. Sixteen of the patients featured a phenotype typical of CPVT. In 17 patients, VPCs emerged also at rest. Exercise stress test and echocardiography were performed to each patient and 232 family members. Familial background was evident in 42% of cases (n = 14). We sequenced all the coding exons of the <it>RyR2</it>, <it>FKBP1B</it>, <it>ATP2A2 </it>and <it>SLC8A1 </it>genes from the index patients. Single channel recordings of a mutant RyR2 were performed in planar lipid bilayers. Two novel <it>RyR2 </it>missense mutations (R1051P and S616L) and two <it>RyR2 </it>exon 3 deletions were identified, explaining 25% of the CPVT phenotypes. A rare variant (N3308S) with open probabilities similar to the wild type channels <it>in vitro</it>, was evident in a patient with resting VPCs. No disease-causing variants were detectable in the <it>FKBP1B</it>, <it>ATP2A2 </it>or <it>SLC8A1 </it>genes.</p> <p>Conclusion</p> <p>We report two novel CPVT-causing <it>RyR2 </it>mutations and a novel <it>RyR2 </it>variant of uncertain clinical significance in a patient with abundant resting VPCs. Our data also strengthen the previous assumption that exon 3 deletions of <it>RyR2 </it>should screened for in CPVT and related phenotypes.</p

Ca2+-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca2+ Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca2+ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca2+. We show that hyperphosphorylation by exogenous PKA increases open probability (Po) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca2+; lowering [Ca2+] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca2+-sensitive mode of gating. We additionally found that cytosolic incubation with Mg2+/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca2+. We show that the endogenous kinase is both Ca2+-dependent and sensitive to inhibitors of PKC. Moreover, the Ca2+-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca2+-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca2+ regulation

FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death.

Arrhythmias, a common cause of sudden cardiac death, can occur in structurally normal hearts, although the mechanism is not known. In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum releases the calcium required for muscle contraction. The FK506 binding protein (FKBP12.6) stabilizes RyR2, preventing aberrant activation of the channel during the resting phase of the cardiac cycle. We show that during exercise, RyR2 phosphorylation by cAMP-dependent protein kinase A (PKA) partially dissociates FKBP12.6 from the channel, increasing intracellular Ca(2+) release and cardiac contractility. FKBP12.6(-/-) mice consistently exhibited exercise-induced cardiac ventricular arrhythmias that cause sudden cardiac death. Mutations in RyR2 linked to exercise-induced arrhythmias (in patients with catecholaminergic polymorphic ventricular tachycardia [CPVT]) reduced the affinity of FKBP12.6 for RyR2 and increased single-channel activity under conditions that simulate exercise. These data suggest that "leaky" RyR2 channels can trigger fatal cardiac arrhythmias, providing a possible explanation for CPVT

Abnormalities of calcium metabolism and myocardial contractility depression in the failing heart

Heart failure (HF) is characterized by molecular and cellular defects which jointly contribute to decreased cardiac pump function. During the development of the initial cardiac damage which leads to HF, adaptive responses activate physiological countermeasures to overcome depressed cardiac function and to maintain blood supply to vital organs in demand of nutrients. However, during the chronic course of most HF syndromes, these compensatory mechanisms are sustained beyond months and contribute to progressive maladaptive remodeling of the heart which is associated with a worse outcome. Of pathophysiological significance are mechanisms which directly control cardiac contractile function including ion- and receptor-mediated intracellular signaling pathways. Importantly, signaling cascades of stress adaptation such as intracellular calcium (Ca2+) and 3′-5′-cyclic adenosine monophosphate (cAMP) become dysregulated in HF directly contributing to adverse cardiac remodeling and depression of systolic and diastolic function. Here, we provide an update about Ca2+ and cAMP dependent signaling changes in HF, how these changes affect cardiac function, and novel therapeutic strategies which directly address the signaling defects

K201 (JTV-519) alters the spatiotemporal properties of diastolic Ca2+ release and the associated diastolic contraction during β-adrenergic stimulation in rat ventricular cardiomyocytes

K201 has previously been shown to reduce diastolic contractions in vivo during β-adrenergic stimulation and elevated extracellular calcium concentration ([Ca2+]o). The present study characterised the effect of K201 on electrically stimulated and spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release and contractile events in isolated rat cardiomyocytes during β-adrenergic stimulation and elevated [Ca2+]o. Parallel experiments using confocal microscopy examined spontaneous diastolic Ca2+ release events at an enhanced spatiotemporal resolution. 1.0 μmol/L K201 in the presence of 150 nmol/L isoproterenol (ISO) and 4.75 mmol/L [Ca2+]o significantly decreased the amplitude of diastolic contractions to ~16% of control levels. The stimulated free Ca2+ transient amplitude was significantly reduced, but stimulated cell shortening was not significantly altered. When intracellular buffering was taken into account, K201 led to an increase in action potential-induced SR Ca2+ release. Myofilament sensitivity to Ca2+ was not changed by K201. Confocal microscopy revealed diastolic events composed of multiple Ca2+ waves (2–3) originating at various points along the cardiomyocyte length during each diastolic period. 1.0 μmol/L K201 significantly reduced the (a) frequency of diastolic events and (b) initiation points/diastolic interval in the remaining diastolic events to 61% and 71% of control levels respectively. 1.0 μmol/L K201 can reduce the probability of spontaneous diastolic Ca2+ release and their associated contractions which may limit the propensity for the contractile dysfunction observed in vivo

The effects of over-expression of the FK506-binding protein FKBP12.6 on K+ currents in adult rabbit ventricular myocytes

This study examines the effects of the intracellular protein FKBP12.6 on action potential and associated K+ currents in isolated adult rabbit ventricular cardiomyocytes. FKBP12.6 was over-expressed by ~6 times using a recombinant adenovirus coding for human FKBP12.6. This over-expression caused prolongation of action potential duration (APD) by ~30%. The amplitude of the transient outward current (Ito) was unchanged, but rate of inactivation at potentials positive to +40 mV was increased. FKBP12.6 over-expression decreased the amplitude of the inward rectifier current (IK1) by ~25% in the voltage range −70 to −30 mV, an effect prevented by FK506 or lowering intracellular [Ca2+] below 1 nM. Over-expression of an FKBP12.6 mutant, which cannot bind calcineurin, prolonged APD and affected Ito and IK1 in a similar manner to wild-type protein. These data suggest that FKBP12.6 can modulate APD via changes in IK1 independently of calcineurin binding, suggesting that FKBP12.6 may affect APD by direct interaction with IK1

A negative screen for mutations in calstabin 1 and 2 genes in patients with dilated cardiomyopathy

<p>Abstract</p> <p>Background</p> <p>Calstabins 1 and 2 bind to Ryanodine receptors regulating muscle excitation-contraction coupling. Mutations in Ryanodine receptors affecting their interaction with calstabins lead to different cardiac pathologies. Animal studies suggest the involvement of calstabins with dilated cardiomyopathy.</p> <p>Results</p> <p>We tested the hypothesis that calstabins mutations may cause dilated cardiomyopathy in humans screening 186 patients with idiopathic dilated cardiomyopathy for genetic alterations in calstabins 1 and 2 genes (<it>FKBP12 </it>and <it>FKBP12.6)</it>. No missense variant was found. Five no-coding variations were found but not related to the disease.</p> <p>Conclusions</p> <p>These data corroborate other studies suggesting that mutations in <it>FKBP12 </it>and <it>FKBP12.6 </it>genes are not commonly related to cardiac diseases.</p

FKBP12 Activates the Cardiac Ryanodine Receptor Ca2+-Release Channel and Is Antagonised by FKBP12.6

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca2+-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca2+-induced Ca2+-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca2+, whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca2+-wave frequency and decreased the SR Ca2+-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12

A bootstrap approach for assessing the uncertainty of outcome probabilities when using a scoring system

Background: Scoring systems are a very attractive family of clinical predictive models, because the patient score can be calculated without using any data processing system. Their weakness lies in the difficulty of associating a reliable prognostic probability with each score. In this study a bootstrap approach for estimating confidence intervals of outcome probabilities is described and applied to design and optimize the performance of a scoring system for morbidity in intensive care units after heart surgery. Methods: The bias-corrected and accelerated bootstrap method was used to estimate the 95% confidence intervals of outcome probabilities associated with a scoring system. These confidence intervals were calculated for each score and each step of the scoring-system design by means of one thousand bootstrapped samples. 1090 consecutive adult patients who underwent coronary artery bypass graft were assigned at random to two groups of equal size, so as to define random training and testing sets with equal percentage morbidities. A collection of 78 preoperative, intraoperative and postoperative variables were considered as likely morbidity predictors. Results: Several competing scoring systems were compared on the basis of discrimination, generalization and uncertainty associated with the prognostic probabilities. The results showed that confidence intervals corresponding to different scores often overlapped, making it convenient to unite and thus reduce the score classes. After uniting two adjacent classes, a model with six score groups not only gave a satisfactory trade-off between discrimination and generalization, but also enabled patients to be allocated to classes, most of which were characterized by well separated confidence intervals of prognostic probabilities. Conclusions: Scoring systems are often designed solely on the basis of discrimination and generalization characteristics, to the detriment of prediction of a trustworthy outcome probability. The present example demonstrates that using a bootstrap method for the estimation of outcome-probability confidence intervals provides useful additional information about score-class statistics, guiding physicians towards the most convenient model for predicting morbidity outcomes in their clinical context