Ryanodine receptor 2 inhibition reduces dispersion of cardiac repolarization, improves contractile function, and prevents sudden arrhythmic death in failing hearts.

Sudden cardiac death (SCD) from ventricular tachycardia/fibrillation (VT/VF) is a leading cause of death, but current therapies are limited. Despite extensive research on drugs targeting sarcolemmal ion channels, none have proven sufficiently effective for preventing SCD. Sarcoplasmic ryanodine receptor 2 (RyR2) Ca2+ release channels, the downstream effectors of sarcolemmal ion channels, are underexplored in this context. Recent evidence implicates reactive oxygen species (ROS)-mediated oxidation and hyperactivity of RyR2s in the pathophysiology of SCD. We tested the hypothesis that RyR2 inhibition of failing arrhythmogenic hearts reduces sarcoplasmic Ca2+ leak and repolarization lability, mitigates VT/VF/SCD and improves contractile function. We used a guinea pig model that replicates key clinical aspects of human nonischemic HF, such as a prolonged QT interval, a high prevalence of spontaneous arrhythmic SCD, and profound Ca2+ leak via a hyperactive RyR2. HF animals were randomized to receive dantrolene (DS) or placebo in early or chronic HF. We assessed the incidence of VT/VF and SCD (primary outcome), ECG heart rate and QT variability, echocardiographic left ventricular (LV) structure and function, immunohistochemical LV fibrosis, and sarcoplasmic RyR2 oxidation. DS treatment prevented VT/VF and SCD by decreasing dispersion of repolarization and ventricular arrhythmias. Compared to placebo, DS lowered resting heart rate, preserved chronotropic competency during transient ß-adrenergic challenge, and improved heart rate variability and cardiac function. Inhibition of RyR2 hyperactivity with dantrolene mitigates the vicious cycle of sarcoplasmic Ca2+ leak-induced increases in diastolic Ca2+ and ROS-mediated RyR2 oxidation, thereby reducing repolarization lability and protecting against VT/VF/SCD. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function. These potentially life-saving effects of RyR2 inhibition warrant further investigation, such as clinical studies of repurposing dantrolene as a potential new therapy for heart failure and/or SCD.

Each year, more than 300,000 people experience cardiac arrest or sudden cardiac death. Sudden cardiac death is caused by irregular heartbeats known as ventricular tachycardia or ventricular fibrillation, which prevent the heart from pumping blood. During a regular heart rhythm, the heart muscles contract and relax, regulated by a coordinated rise and fall of calcium ions within heart cells. In the cells of diseased hearts, on the other hand, calcium leaks out of a compartment known as the sarcoplasmic reticulum in an uncontrolled manner. This happens because an ion channel in the membrane of the sarcoplasmic reticulum known as ryanodine receptor 2 becomes hyperactive and releases calcium in an uncontrolled manner. This abnormal calcium release leads to irregular calcium waves, which can make the heart's electrical properties unstable, causing ventricular tachycardia, ventricular fibrillation and sudden cardiac death. Joshi et al. tested whether dantrolene, a molecule that blocks ryanodine receptor 2, can stop calcium leaks from the sarcoplasmic reticulum and prevent lethal arrhythmias and sudden cardiac death in failing hearts. To investigate this, Joshi et al. induced heart failure in guinea pigs that have abnormal heart calcium signalling similar to human heart failure, and then treated the animals with either dantrolene or a placebo. The results indicate that blocking ryanodine receptor 2 hyperactivity with dantrolene prevents lethal arrhythmias and sudden cardiac death by blocking calcium leaks and by preventing the instability of the electrical properties of the heart. Additionally, Joshi et al. found that dantrolene also improved the diseased heart's ability to pump adequate amounts of blood, allowing failing hearts to meet increased cardiovascular demands, and thereby improving the heart's overall function. The proposed studies come from a strong clinical need to improve bad outcomes in people who keep having fatal heart rhythm episodes despite getting the best medical care. Many heart failure patients are plagued by recurrent defibrillator shocks to abort sudden cardiac death from relentless lethal heart rhythms. These shocks are painful, injure the heart, and worsen the quality of life. Unfortunately, management options are extremely limited for these patients. The findings of Joshi et al. indicate that dantrolene may be a potential treatment for people with fatal heart rhythms who are at risk of sudden cardiac death and could have a positive impact on these people's quality of life. However, before this can happen, dantrolene will first have to be thoroughly tested to ensure effectivity and safety in humans. In any case, Joshi et al. have opened a new avenue in the search for medications to treat deadly arrhythmias and sudden cardiac death.

Ryanodine receptor 2 inhibition reduces dispersion of cardiac repolarization, improves contractile function and prevents sudden arrhythmic death in failing hearts.

Introduction: Sudden cardiac death (SCD) from ventricular tachycardia/fibrillation (VT/VF) are a leading cause of death, but current therapies are limited. Despite extensive research on drugs targeting sarcolemmal ion channels, none have proven sufficiently effective for preventing SCD. Sarcoplasmic ryanodine receptor 2 (RyR2) Ca 2+ release channels, the downstream effectors of sarcolemmal ion channels, are underexplored in this context. Recent evidence implicates reactive oxygen species (ROS)- mediated oxidation and hyperactivity of RyR2s in the pathophysiology of SCD. Objective: To test the hypothesis that RyR2 inhibition of failing arrhythmogenic hearts reduces sarcoplasmic Ca 2+ leak and repolarization lability, mitigates VT/VF/SCD and improves contractile function. Methods: We used a guinea pig model that replicates key clinical aspects of human nonischemic HF, such as a prolonged QT interval, a high prevalence of spontaneous arrhythmic SCD, and profound Ca 2+ leak via a hyperactive RyR2. HF animals were randomized to receive dantrolene (DS) or placebo in early or chronic HF. We assessed the incidence of VT/VF and SCD (primary outcome), ECG heart rate and QT variability, echocardiographic left ventricular (LV) structure and function, immunohistochemical LV fibrosis, and sarcoplasmic RyR2 oxidation. Results: DS treatment prevented VT/VF and SCD by decreasing dispersion of repolarization and ventricular arrhythmias. Compared to placebo, DS lowered resting heart rate, preserved chronotropic competency during transient ß-adrenergic challenge, and improved heart rate variability and cardiac function. Conclusion: Inhibition of RyR2 hyperactivity with dantrolene mitigates the vicious cycle of sarcoplasmic Ca 2+ leak-induced increases in diastolic Ca 2+ and ROS-mediated RyR2 oxidation, thereby increasing repolarization lability and protecting against VT/VF/SCD. Moreover, the consequent increase in sarcoplasmic Ca 2+ load improves contractile function. These potentially life-saving effects of RyR2 inhibition warrant further investigation, such as clinical studies of repurposing dantrolene as a potential new therapy for heart failure and/or SCD.

Cardiac sympathetic denervation prevents sudden cardiac arrest and improves cardiac function by enhancing mitochondrial-antioxidant capacity.

RATIONALE: Sudden cardiac arrest (SCA) and heart failure (HF) are leading causes of death. The underlying mechanisms are incompletely understood, limiting the design of new therapies. Whereas most autonomic modulation therapies have not shown clear benefit in HF patients, growing evidence indicates cardiac sympathetic denervation (CSD) exerts cardioprotective effects. The underlying molecular and cellular mechanisms remain unexplored. OBJECTIVE: Based on the hypothesis that mitochondrial reactive oxygen species (mROS) drive the pathogenesis of HF and SCA, we investigated whether CSD prevents SCA and HF by improving mitochondrial antioxidant capacity and redox balance, to correct impaired Ca2+ handling and repolarization reserve. METHODS AND RESULTS: We interrogated CSD-specific responses in pressure-overload HF models with spontaneous SCA using in vivo echocardiographic and electrocardiographic studies and in vitro biochemical and functional studies including ratiometric measures of mROS, Ca2+ and sarcomere dynamics in left ventricular myocytes. Pressure-overloaded HF reduced mitochondrial antioxidant capacity and increased mROS, which impaired ß-adrenergic signaling and caused SR Ca2+ leak, reducing SR Ca2+ and increasing diastolic Ca2+, impaired myofilament contraction and further increased the sympathetic stress response. CSD improved contractile function and mitigated mROS-mediated diastolic Ca2+ overload, dispersion of repolarization, triggered activity and SCA by upregulating mitochondrial antioxidant and NADPH-producing enzymes. CONCLUSIONS: Our findings support a fundamental role of sympathetic stress-induced downregulation of mROS scavenging enzymes and RyR-leak mediated diastolic Ca2+ overload in HF and SCA pathogenesis that are mitigated by CSD. This first report on the molecular and cellular mechanisms of CSD supports its evaluation in additional high-risk patient groups.

Artificial Intelligence, Wearables and Remote Monitoring for Heart Failure: Current and Future Applications.

Substantial milestones have been attained in the field of heart failure (HF) diagnostics and therapeutics in the past several years that have translated into decreased mortality but a paradoxical increase in HF-related hospitalizations. With increasing data digitalization and access, remote monitoring via wearables and implantables have the potential to transform ambulatory care workflow, with a particular focus on reducing HF hospitalizations. Additionally, artificial intelligence and machine learning (AI/ML) have been increasingly employed at multiple stages of healthcare due to their power in assimilating and integrating multidimensional multimodal data and the creation of accurate prediction models. With the ever-increasing troves of data, the implementation of AI/ML algorithms could help improve workflow and outcomes of HF patients, especially time series data collected via remote monitoring. In this review, we sought to describe the basics of AI/ML algorithms with a focus on time series forecasting and the current state of AI/ML within the context of wearable technology in HF, followed by a discussion of the present limitations, including data integration, privacy, and challenges specific to AI/ML application within healthcare.

Contemporary Applications of Machine Learning for Device Therapy in Heart Failure.

Despite a better understanding of the underlying pathogenesis of heart failure (HF), pharmacotherapy, surgical, and percutaneous interventions do not prevent disease progression in all patients, and a significant proportion of patients end up requiring advanced therapies. Machine learning (ML) is gaining wider acceptance in cardiovascular medicine because of its ability to incorporate large, complex, and multidimensional data and to potentially facilitate the creation of predictive models not constrained by many of the limitations of traditional statistical approaches. With the coexistence of "big data" and novel advanced analytic techniques using ML, there is ever-increasing research into applying ML in the context of HF with the goal of improving patient outcomes. Through this review, the authors describe the basics of ML and summarize the existing published reports regarding contemporary applications of ML in device therapy for HF while highlighting the limitations to widespread implementation and its future promises.

Impaired Right Ventricular Calcium Cycling Is an Early Risk Factor in R14del-Phospholamban Arrhythmias.

The inherited mutation (R14del) in the calcium regulatory protein phospholamban (PLN) is linked to malignant ventricular arrhythmia with poor prognosis starting at adolescence. However, the underlying early mechanisms that may serve as prognostic factors remain elusive. This study generated humanized mice in which the endogenous gene was replaced with either human wild type or R14del-PLN and addressed the early molecular and cellular pathogenic mechanisms. R14del-PLN mice exhibited stress-induced impairment of atrioventricular conduction, and prolongation of both ventricular activation and repolarization times in association with ventricular tachyarrhythmia, originating from the right ventricle (RV). Most of these distinct electrocardiographic features were remarkably similar to those in R14del-PLN patients. Studies in isolated cardiomyocytes revealed RV-specific calcium defects, including prolonged action potential duration, depressed calcium kinetics and contractile parameters, and elevated diastolic Ca-levels. Ca-sparks were also higher although SR Ca-load was reduced. Accordingly, stress conditions induced after contractions, and inclusion of the CaMKII inhibitor KN93 reversed this proarrhythmic parameter. Compensatory responses included altered expression of key genes associated with Ca-cycling. These data suggest that R14del-PLN cardiomyopathy originates with RV-specific impairment of Ca-cycling and point to the urgent need to improve risk stratification in asymptomatic carriers to prevent fatal arrhythmias and delay cardiomyopathy onset.

Mitochondrial ROS Drive Sudden Cardiac Death and Chronic Proteome Remodeling in Heart Failure.

RATIONALE: Despite increasing prevalence and incidence of heart failure (HF), therapeutic options remain limited. In early stages of HF, sudden cardiac death (SCD) from ventricular arrhythmias claims many lives. Reactive oxygen species (ROS) have been implicated in both arrhythmias and contractile dysfunction. However, little is known about how ROS in specific subcellular compartments contribute to HF or SCD pathophysiology. The role of ROS in chronic proteome remodeling has not been explored. OBJECTIVE: We will test the hypothesis that elevated mitochondrial ROS (mROS) is a principal source of oxidative stress in HF and in vivo reduction of mROS mitigates SCD. METHODS AND RESULTS: Using a unique guinea pig model of nonischemic HF that recapitulates important features of human HF, including prolonged QT interval and high incidence of spontaneous arrhythmic SCD, compartment-specific ROS sensors revealed increased mROS in resting and contracting left ventricular myocytes in failing hearts. Importantly, the mitochondrially targeted antioxidant (MitoTEMPO) normalized global cellular ROS. Further, in vivo MitoTEMPO treatment of HF animals prevented and reversed HF, eliminated SCD by decreasing dispersion of repolarization and ventricular arrhythmias, suppressed chronic HF-induced remodeling of the expression proteome, and prevented specific phosphoproteome alterations. Pathway analysis of mROS-sensitive networks indicated that increased mROS in HF disrupts the normal coupling between cytosolic signals and nuclear gene programs driving mitochondrial function, antioxidant enzymes, Ca2+ handling, and action potential repolarization, suggesting new targets for therapeutic intervention. CONCLUSIONS: mROS drive both acute emergent events, such as electrical instability responsible for SCD, and those that mediate chronic HF remodeling, characterized by suppression or altered phosphorylation of metabolic, antioxidant, and ion transport protein networks. In vivo reduction of mROS prevents and reverses electrical instability, SCD, and HF. Our findings support the feasibility of targeting the mitochondria as a potential new therapy for HF and SCD while identifying new mROS-sensitive protein modifications.

Anterior pericardial access to facilitate electrophysiology study and catheter ablation of ventricular arrhythmias: A single tertiary center experience.

INTRODUCTION: Epicardial ablation is becoming an important part of management in patients with ventricular tachycardia (VT). Posterior epicardial access via the Sosa or needle-in-needle (NIN) approach for epicardial VT ablation is considered to be the method of choice for most electrophysiologists. Anterior epicardial access as an alternative technique has recently been proposed, but there are limited data about its safety, efficacy, and the rate of immediate complications. In this study, we report our experience with anterior epicardial access between 2009 and 2016. METHODS: Between 2009 and June 2016, 100 consecutive patients underwent epicardial VT ablation using an anterior approach. The success rate, epicardial bleeding, and other complications related to the epicardial access in these patients were compared to the previously reported rate of complications in patients whom epicardial access was performed using the NIN or Sosa techniques. RESULTS: Anterior epicardial access was obtained successfully in 100% of patients in the first attempt. The success rate of the anterior approach was comparable with the reported success rate of the NIN technique (100% vs. 100%, P value not significant) but better than the Sosa technique (100% vs. 94%, P = 0.012). None of the patients in the anterior approach series suffered from significant pericardial bleeding (defined as greater than 80 mL of blood loss), RV puncture/damage, or need for an emergent cardiac surgery. CONCLUSION: An anterior epicardial approach is feasible and appears to have an acceptable safety profile in comparison with other epicardial approaches.

Central role for GSK3ß in the pathogenesis of arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is characterized by redistribution of junctional proteins, arrhythmias, and progressive myocardial injury. We previously reported that SB216763 (SB2), annotated as a GSK3ß inhibitor, reverses disease phenotypes in a zebrafish model of ACM. Here, we show that SB2 prevents myocyte injury and cardiac dysfunction in vivo in two murine models of ACM at baseline and in response to exercise. SB2-treated mice with desmosome mutations showed improvements in ventricular ectopy and myocardial fibrosis/inflammation as compared with vehicle-treated (Veh-treated) mice. GSK3ß inhibition improved left ventricle function and survival in sedentary and exercised Dsg2mut/mut mice compared with Veh-treated Dsg2mut/mut mice and normalized intercalated disc (ID) protein distribution in both mutant mice. GSK3ß showed diffuse cytoplasmic localization in control myocytes but ID redistribution in ACM mice. Identical GSK3ß redistribution is present in ACM patient myocardium but not in normal hearts or other cardiomyopathies. SB2 reduced total GSK3ß protein levels but not phosphorylated Ser 9-GSK3ß in ACM mice. Constitutively active GSK3ß worsens ACM in mutant mice, while GSK3ß shRNA silencing in ACM cardiomyocytes prevents abnormal ID protein distribution. These results highlight a central role for GSKß in the complex phenotype of ACM and provide further evidence that pharmacologic GSKß inhibition improves cardiomyopathies due to desmosome mutations.

Entropy of cardiac repolarization predicts ventricular arrhythmias and mortality in patients receiving an implantable cardioverter-defibrillator for primary prevention of sudden death.

AIMS: The need for a readily available, inexpensive, non-invasive method for improved risk stratification of heart failure (HF) patients is paramount. Prior studies have proposed that distinct fluctuation patterns underlying the variability of physiological signals have unique prognostic value. We tested this hypothesis in an extensively phenotyped cohort of HF patients using EntropyXQT, a novel non-linear measure of cardiac repolarization dynamics. METHODS AND RESULTS: In a prospective, multicentre, observational study of 852 patients in sinus rhythm undergoing clinically indicated primary prevention implantable cardioverter-defibrillator (ICD) implantation (2003-10), exposures included demographics, history, physical examination, medications, laboratory results, serum biomarkers, ejection fraction, conventional electrocardiographic (ECG) analyses of heart rate and QT variability, and EntropyXQT. The primary outcome was first 'appropriate' ICD shock for ventricular arrhythmias. The secondary outcome was composite events (appropriate ICD shock and all-cause mortality). After exclusions, the cohort (n = 816) had a mean age of 60 ± 13 years, 28% women, 36% African Americans, 56% ischaemic cardiomyopathy, and 29 ± 16% Seattle HF risk score (SHFS) 5-year predicted mortality. Over 45 ± 24 months, there were 134 appropriate shocks and 166 deaths. After adjusting for 30 exposures, the hazard ratios (comparing the 5th to 1st quintile of EntropyXQT) for primary and secondary outcomes were 3.29 (95% CI 1.74-6.21) and 2.28 (1.53-3.41), respectively. Addition of EntropyXQT to a model comprised of the exposures or SHFS significantly increased net reclassification and the ROC curve area. CONCLUSIONS: EntropyXQT measured during ICD implantation strongly and independently predicts appropriate shock and all-cause mortality over follow-up. EntropyXQT complements conventional risk predictors and has the potential for broad clinical application.

Cardiac resynchronization therapy restores sympathovagal balance in the failing heart by differential remodeling of cholinergic signaling.

RATIONALE: Cardiac resynchronization therapy (CRT) is the only heart failure (HF) therapy documented to improve left ventricular function and reduce mortality. The underlying mechanisms are incompletely understood. Although ß-adrenergic signaling has been studied extensively, the effect of CRT on cholinergic signaling is unexplored. OBJECTIVE: We hypothesized that remodeling of cholinergic signaling plays an important role in the aberrant calcium signaling and depressed contractile and ß-adrenergic responsiveness in dyssynchronous HF that are restored by CRT. METHODS AND RESULTS: Canine tachypaced dyssynchronous HF and CRT models were generated to interrogate responses specific to dyssynchronous versus resynchronized ventricular contraction during hemodynamic decompensation. Echocardiographic, electrocardiographic, and invasive hemodynamic data were collected from normal controls, dyssynchronous HF and CRT models. Left ventricular tissue was used for biochemical analyses and functional measurements (calcium transient, sarcomere shortening) from isolated myocytes (n=42-104 myocytes per model; 6-9 hearts per model). Human left ventricular myocardium was obtained for biochemical analyses from explanted failing (n=18) and nonfailing (n=7) hearts. The M2 subtype of muscarinic acetylcholine receptors was upregulated in human and canine HF compared with nonfailing controls. CRT attenuated the increased M2 subtype of muscarinic acetylcholine receptor expression and Gαi coupling and enhanced M3 subtype of muscarinic acetylcholine receptor expression in association with enhanced calcium cycling, sarcomere shortening, and ß-adrenergic responsiveness. Despite model-dependent remodeling, cholinergic stimulation completely abolished isoproterenol-induced triggered activity in both dyssynchronous HF and CRT myocytes. CONCLUSIONS: Remodeling of cholinergic signaling is a critical pathological component of human and canine HF. Differential remodeling of cholinergic signaling represents a novel mechanism for enhancing sympathovagal balance with CRT and may identify new targets for treatment of systolic HF.

Inhibiting mitochondrial Na+/Ca2+ exchange prevents sudden death in a Guinea pig model of heart failure.

RATIONALE: In cardiomyocytes from failing hearts, insufficient mitochondrial Ca(2+) accumulation secondary to cytoplasmic Na(+) overload decreases NAD(P)H/NAD(P)(+) redox potential and increases oxidative stress when workload increases. These effects are abolished by enhancing mitochondrial Ca(2+) with acute treatment with CGP-37157 (CGP), an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger. OBJECTIVE: Our aim was to determine whether chronic CGP treatment mitigates contractile dysfunction and arrhythmias in an animal model of heart failure (HF) and sudden cardiac death (SCD). METHODS AND RESULTS: Here, we describe a novel guinea pig HF/SCD model using aortic constriction combined with daily ß-adrenergic receptor stimulation (ACi) and show that chronic CGP treatment (ACi plus CGP) attenuates cardiac hypertrophic remodeling, pulmonary edema, and interstitial fibrosis and prevents cardiac dysfunction and SCD. In the ACi group 4 weeks after pressure overload, fractional shortening and the rate of left ventricular pressure development decreased by 36% and 32%, respectively, compared with sham-operated controls; in contrast, cardiac function was completely preserved in the ACi plus CGP group. CGP treatment also significantly reduced the incidence of premature ventricular beats and prevented fatal episodes of ventricular fibrillation, but did not prevent QT prolongation. Without CGP treatment, mortality was 61% in the ACi group <4 weeks of aortic constriction, whereas the death rate in the ACi plus CGP group was not different from sham-operated animals. CONCLUSIONS: The findings demonstrate the critical role played by altered mitochondrial Ca(2+) dynamics in the development of HF and HF-associated SCD; moreover, they reveal a novel strategy for treating SCD and cardiac decompensation in HF.

Dynamic analysis of cardiac rhythms for discriminating atrial fibrillation from lethal ventricular arrhythmias.

BACKGROUND: Implantable cardioverter-defibrillators (ICDs), the first line of therapy for preventing sudden cardiac death in high-risk patients, deliver appropriate shocks for termination of ventricular tachycardia (VT)/ventricular fibrillation. A common shortcoming of ICDs is imperfect rhythm discrimination, resulting in the delivery of inappropriate shocks for atrial fibrillation (AF). An underexplored area for rhythm discrimination is the difference in dynamic properties between AF and VT/ventricular fibrillation. We hypothesized that the higher entropy of rapid cardiac rhythms preceding ICD shocks distinguishes AF from VT/ventricular fibrillation. METHODS AND RESULTS: In a multicenter, prospective, observational study of patients with primary prevention ICDs, 119 patients received shocks from ICDs with stored, retrievable intracardiac electrograms. Blinded adjudication revealed shocks were delivered for VT/ventricular fibrillation (62%), AF (23%), and supraventricular tachycardia (15%). Entropy estimation of only 9 ventricular intervals before ICD shocks accurately distinguished AF (receiver operating characteristic curve area, 0.98; 95% confidence intervals, 0.93-1.0) and outperformed contemporary ICD rhythm discrimination algorithms. CONCLUSIONS: This new strategy for AF discrimination based on entropy estimation expands on simpler concepts of variability, performs well at fast heart rates, and has potential for broad clinical application.

Should statin therapy be allocated on the basis of global risk or on the basis of randomized trial evidence?

Current clinical guidelines recommend the use of a global risk assessment tool, such as those pioneered by the Framingham Heart Study, to determine eligibility for statin therapy in patients with absolute risk levels greater than a certain threshold. In support of this approach, several randomized trials have reported that patients with high absolute risk clearly benefit from statin therapy. Therefore, the guideline recommendations would seem intuitive and effective, albeit on the core assumption that the mortality and morbidity benefits associated with statin therapy would be greatest in those with high predicted absolute risk. However, if this assumption is incorrect, using predicted absolute risk to guide statin therapy could easily result in underuse in some groups and overuse in others. Herein, the authors question the utility of global risk assessment strategies based on the Framingham risk score for guiding statin therapy in light of current data that have become available from more recent and robust prospective randomized clinical trials since the publication of the National Cholesterol Education Program Adult Treatment Panel III guidelines. Moreover, the Adult Treatment Panel III guidelines do not support treatment of some patients who may benefit from statin therapy. In conclusion, the authors propose an alternative approach for incorporating more recent randomized trial data into future statin allocation algorithms and treatment guidelines.

The kinetics of competitive antagonism of nicotinic acetylcholine receptors at physiological temperature.

Detailed information about the ligand-binding site of nicotinic acetylcholine receptors has emerged from structural and mutagenesis experiments. However, these approaches provide only static images of ligand-receptor interactions. Kinetic measurements of changes in protein function are needed to develop a more dynamic picture. Previously, we measured association and dissociation rate constants for competitive inhibition of current through embryonic muscle acetylcholine receptor channels at 25 degrees C. Little is known about competitive antagonism at physiological temperatures. Here, we performed measurements at 37 degrees C and used thermodynamics to estimate the energetics of antagonism. We used rapid solution exchange protocols to determine equilibrium and kinetics of inhibition of acetylcholine-activated currents in outside-out patches by (+)-tubocurarine, pancuronium and cisatracurium. Kinetic rates as high as 600 s(-1) were resolved by this technique. Binding was primarily enthalpy driven. The 12 degrees C increase in temperature decreased equilibrium antagonist binding by 1.7- to 1.9-fold. In contrast, association and dissociation rate constants increased 1.9- to 6.0-fold. Activation energies for dissociation were 90 +/- 6, 106 +/- 8 and 116 +/- 10 kJ mol(-1) for cisatracurium, (+)-tubocurarine and pancuronium, respectively. The corresponding apparent activation energies for association were 38 +/- 6, 85 +/- 6 and 107 +/- 13 kJ mol(-1). The higher activation energy for association of (+)-tubocurarine and pancuronium compared with cisatracurium is notable. This may arise from either a more superficial binding site for the large antagonist cisatracurium compared to the other ligands, or from a change in receptor conformation upon binding of (+)-tubocurarine and pancuronium but not cisatracurium. Differences in ligand desolvation and ligand conformation are not likely to be important.

Roles of amino acids and subunits in determining the inhibition of nicotinic acetylcholine receptors by competitive antagonists.

BACKGROUND: Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the alpha-delta and alpha-epsilon subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the alpha-epsilon interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. METHODS: HEK293 cells were transfected with wild-type or mutant (alphaY198F, epsilonD59A, epsilonD59N, epsilonD173A, epsilonD173N, deltaD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. RESULTS: Effects of mutations were antagonist specific. alphaY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to epsilonD173 mutations. epsilonD59 mutations had large effects on metocurine and cisatracurium. deltaD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two delta subunits but no epsilon subunit. Differences in IC50 arose from differences in both dissociation and association rates. CONCLUSION: Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the alpha-epsilon interface. Pancuronium, vecuronium, and cisatracurium were selective for the alpha-delta interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.