Crystal M. Ripplinger

Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca2+ handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca2+ concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca2+ release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.

A Computational Model to Predict the Effects of Class I Anti-Arrhythmic Drugs on Ventricular Rhythms

Two- and three-dimensional models of cardiac excitability based on sodium channel kinetics can predict the adverse effects of class I anti-arrhythmic drugs. Crowdsourcing the Heart for Drug Screening The old way: Consult a specialist to answer your question. The new way: Consult a crowd of generalists who in the aggregate can come up with a better answer. The old way—testing drugs on single cardiac cells in vitro—has not worked well for screening out potential anti-arrhythmia agents that can occasionally block conduction in the heart or exacerbate arrhythmia, serious problems that cause sudden death in treated patients. Instead, Moreno et al. have called on the crowd by building a model of heart tissue that includes many cardiac cells and their interactions. When anti-arrhythmia drugs are “applied” to the model’s beating heart tissue—but not when they are applied to the single cardiac cells that make up the model—the drugs that cause side effects, and the concentrations at which they do so, are revealed, results that the authors were able to validate experimentally. The model starts with the detailed kinetics of the heart’s sodium channels, first in the context of a single cell, then in two- and three-dimensional cardiac tissue. The authors compared the action of lidocaine, a class 1B anti-arrhythmic drug not known to cause conduction block, and flecainide, a prototypical class 1C drug that carries a warning from the Food and Drug Administration. In the modeled analyses of single cardiac cells, both drugs slowed excitability at concentrations that matched those used in patients, but the cells retained the ability to generate action potentials. But when the model incorporated coupled groups of cells, the behavior of the drugs diverged. Lidocaine lowered excitability without causing block, but at the higher concentrations (used clinically), flecainide caused serious conduction block when heart rates reached 160 beats per minute. Experiments in rabbit heart confirmed the results of the model. In scaled-up, 500 by 500 groups of cells, the authors’ model could also successfully predict the tendency of flecainide, but not lidocaine, to make the heart extra sensitive to heartbeats occurring too early or too late, an effect that causes even more severe arrhythmias in patients when they take anti-arrhythmia drugs. Again, experiments in rabbit hearts replicated the model’s predictions, as did simulations of anatomically accurate human hearts derived from magnetic resonance imaging images. The ability of this sophisticated model of living cardiac tissue to replicate the clinical adverse effects of lidocaine and flecainide is promising, but it will be necessary to validate its performance with other drugs to understand how to deploy it most effectively. Ideally, such models will be useful for screening out potential arrhythmic drugs that promote conduction block or exacerbate arrhythmias. Such a view of how drugs affect the collective activity of cardiac cells should help in these situations in which the cure proves more deadly than the disease. A long-sought, and thus far elusive, goal has been to develop drugs to manage diseases of excitability. One such disease that affects millions each year is cardiac arrhythmia, which occurs when electrical impulses in the heart become disordered, sometimes causing sudden death. Pharmacological management of cardiac arrhythmia has failed because it is not possible to predict how drugs that target cardiac ion channels, and have intrinsically complex dynamic interactions with ion channels, will alter the emergent electrical behavior generated in the heart. Here, we applied a computational model, which was informed and validated by experimental data, that defined key measurable parameters necessary to simulate the interaction kinetics of the anti-arrhythmic drugs flecainide and lidocaine with cardiac sodium channels. We then used the model to predict the effects of these drugs on normal human ventricular cellular and tissue electrical activity in the setting of a common arrhythmia trigger, spontaneous ventricular ectopy. The model forecasts the clinically relevant concentrations at which flecainide and lidocaine exacerbate, rather than ameliorate, arrhythmia. Experiments in rabbit hearts and simulations in human ventricles based on magnetic resonance images validated the model predictions. This computational framework initiates the first steps toward development of a virtual drug-screening system that models drug-channel interactions and predicts the effects of drugs on emergent electrical activity in the heart.

Local β-Adrenergic Stimulation Overcomes Source-Sink Mismatch to Generate Focal Arrhythmia

Rationale: &bgr;-Adrenergic receptor stimulation produces sarcoplasmic reticulum Ca2+ overload and delayed afterdepolarizations in isolated ventricular myocytes. How delayed afterdepolarizations are synchronized to overcome the source-sink mismatch and produce focal arrhythmia in the intact heart remains unknown. Objective: To determine whether local &bgr;-adrenergic receptor stimulation produces spatiotemporal synchronization of delayed afterdepolarizations and to examine the effects of tissue geometry and cell-cell coupling on the induction of focal arrhythmia. Methods and Results: Simultaneous optical mapping of transmembrane potential and Ca2+ transients was performed in normal rabbit hearts during subepicardial injections (50 &mgr;L) of norepinephrine (NE) or control (normal Tyrodes solution). Local NE produced premature ventricular complexes (PVCs) from the injection site that were dose-dependent (low-dose [30–60 &mgr;mol/L], 0.45±0.62 PVCs per injection; high-dose [125–250 &mgr;mol/L], 1.33±1.46 PVCs per injection; P<0.0001) and were inhibited by propranolol. NE-induced PVCs exhibited abnormal voltage–Ca2+ delay at the initiation site and were inhibited by either sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibition or reduced perfusate [Ca2+], which indicates a Ca2+-mediated mechanism. NE-induced PVCs were more common at right ventricular than at left ventricular sites (1.48±1.50 versus 0.55±0.89, P<0.01), and this was unchanged after chemical ablation of endocardial Purkinje fibers, which suggests that source-sink interactions may contribute to the greater propensity to right ventricular PVCs. Partial gap junction uncoupling with carbenoxolone (25 &mgr;mol/L) increased focal activity (2.18±1.43 versus 1.33±1.46 PVCs per injection, P<0.05), which further supports source-sink balance as a critical mediator of Ca2+-induced PVCs. Conclusions: These data provide the first experimental demonstration that localized &bgr;-adrenergic receptor stimulation produces spatiotemporal synchronization of sarcoplasmic reticulum Ca2+ overload and release in the intact heart and highlight the critical nature of source-sink balance in initiating focal arrhythmias.

Resolution of Established Cardiac Hypertrophy and Fibrosis and Prevention of Systolic Dysfunction in a Transgenic Rabbit Model of Human Cardiomyopathy Through Thiol-Sensitive Mechanisms

Background— Cardiac hypertrophy, the clinical hallmark of hypertrophic cardiomyopathy (HCM), is a major determinant of morbidity and mortality not only in HCM but also in a number of cardiovascular diseases. There is no effective therapy for HCM and generally for cardiac hypertrophy. Myocardial oxidative stress and thiol-sensitive signaling molecules are implicated in pathogenesis of hypertrophy and fibrosis. We posit that treatment with N-acetylcysteine, a precursor of glutathione, the largest intracellular thiol pool against oxidative stress, could reverse cardiac hypertrophy and fibrosis in HCM. Methods and Results— We treated 2-year-old β-myosin heavy-chain Q403 transgenic rabbits with established cardiac hypertrophy and preserved systolic function with N-acetylcysteine or a placebo for 12 months (n=10 per group). Transgenic rabbits in the placebo group had cardiac hypertrophy, fibrosis, systolic dysfunction, increased oxidized to total glutathione ratio, higher levels of activated thiol-sensitive active protein kinase G, dephosphorylated nuclear factor of activated T cells (NFATc1) and phospho-p38, and reduced levels of glutathiolated cardiac α-actin. Treatment with N-acetylcysteine restored oxidized to total glutathione ratio, normalized levels of glutathiolated cardiac α-actin, reversed cardiac and myocyte hypertrophy and interstitial fibrosis, reduced the propensity for ventricular arrhythmias, prevented cardiac dysfunction, restored myocardial levels of active protein kinase G, and dephosphorylated NFATc1 and phospho-p38. Conclusions— Treatment with N-acetylcysteine, a safe prodrug against oxidation, reversed established cardiac phenotype in a transgenic rabbit model of human HCM. Because there is no effective pharmacological therapy for HCM and given that hypertrophy, fibrosis, and cardiac dysfunction are common and major predictors of clinical outcomes, the findings could have implications in various cardiovascular disorders.

Importance of Geometry and Refractory Period in Sustaining Atrial Fibrillation Testing the Critical Mass Hypothesis

Background—The critical mass hypothesis for atrial fibrillation (AF) was proposed in 1914. There has never been a systematic investigation defining the relationship between tissue geometry and AF. The purpose of this study was to determine the association among the probability of maintaining AF and the width, area, weight, effective refractory period (ERP), and wavelength in atrial tissue. Methods and Results—Isolated canine atria (n=20) were perfused with Krebs-Henseleit solution. Baseline ERPs were obtained with and without acetylcholine (10E-3.5 mol/L) using single extra-stimulus pacing while unipolar electrograms were recorded from 250 sites. The tissue was then partitioned using bipolar radiofrequency ablation, and the ERPs were measured again with and without acetylcholine. Any section of tissue that maintained AF was divided until the arrhythmia was no longer inducible. ERPs and conduction velocities were measured in all of the sections after each ablation, and the wavelengths were calculated. The probability of AF was found to be correlated with increasing tissue areas, widths, and weights (P<0.001). The probability of AF was significantly associated with the length of the ERP and the wavelength (P<0.001). With shorter ERPs and shorter wavelengths, there was an increased probability of sustained AF. Conclusions—The probability of sustained AF was significantly associated with increasing tissue area, width, and weight and decreasing ERPs and wavelengths. These data may lead to a better understanding of the mechanism of AF and, thus, help to design more-effective interventional procedures in the future.

Optical Mapping of Sarcoplasmic Reticulum Ca2+ in the Intact Heart: Ryanodine Receptor Refractoriness During Alternans and Fibrillation

Rationale: Sarcoplasmic reticulum (SR) Ca2+ cycling is key to normal excitation–contraction coupling but may also contribute to pathological cardiac alternans and arrhythmia. Objective: To measure intra-SR free [Ca2+] ([Ca2+]SR) changes in intact hearts during alternans and ventricular fibrillation (VF). Methods and Results: Simultaneous optical mapping of Vm (with RH237) and [Ca2+]SR (with Fluo-5N AM) was performed in Langendorff-perfused rabbit hearts. Alternans and VF were induced by rapid pacing. SR Ca2+ and action potential duration (APD) alternans occurred in-phase, but SR Ca2+ alternans emerged first as cycle length was progressively reduced (217±10 versus 190±13 ms; P<0.05). Ryanodine receptor (RyR) refractoriness played a key role in the onset of SR Ca2+ alternans, with SR Ca2+ release alternans routinely occurring without changes in diastolic [Ca2+]SR. Sensitizing RyR with caffeine (200 &mgr;mol/L) significantly reduced the pacing threshold for both SR Ca2+ and APD alternans (188±15 and 173±12 ms; P<0.05 versus baseline). Caffeine also reduced the magnitude of spatially discordant SR Ca2+ alternans, but not APD alternans, the pacing threshold for discordance, or threshold for VF. During VF, [Ca2+]SR was high, but RyR remained nearly continuously refractory, resulting in minimal SR Ca2+ release throughout VF. Conclusions: In intact hearts, RyR refractoriness initiates SR Ca2+ release alternans that can be amplified by diastolic [Ca2+]SR alternans and lead to APD alternans. Sensitizing RyR suppresses spatially concordant but not discordant SR Ca2+ and APD alternans. Despite increased [Ca2+]SR during VF, SR Ca2+ release was nearly continuously refractory. This novel method provides insight into SR Ca2+ handling during cardiac alternans and arrhythmia.Rationale: Sarcoplasmic reticulum (SR) Ca2+ cycling is key to normal excitation–contraction coupling but may also contribute to pathological cardiac alternans and arrhythmia. Objective: To measure intra-SR free [Ca2+] ([Ca2+]SR) changes in intact hearts during alternans and ventricular fibrillation (VF). Methods and Results: Simultaneous optical mapping of Vm (with RH237) and [Ca2+]SR (with Fluo-5N AM) was performed in Langendorff-perfused rabbit hearts. Alternans and VF were induced by rapid pacing. SR Ca2+ and action potential duration (APD) alternans occurred in-phase, but SR Ca2+ alternans emerged first as cycle length was progressively reduced (217±10 versus 190±13 ms; P <0.05). Ryanodine receptor (RyR) refractoriness played a key role in the onset of SR Ca2+ alternans, with SR Ca2+ release alternans routinely occurring without changes in diastolic [Ca2+]SR. Sensitizing RyR with caffeine (200 μmol/L) significantly reduced the pacing threshold for both SR Ca2+ and APD alternans (188±15 and 173±12 ms; P <0.05 versus baseline). Caffeine also reduced the magnitude of spatially discordant SR Ca2+ alternans, but not APD alternans, the pacing threshold for discordance, or threshold for VF. During VF, [Ca2+]SR was high, but RyR remained nearly continuously refractory, resulting in minimal SR Ca2+ release throughout VF. Conclusions: In intact hearts, RyR refractoriness initiates SR Ca2+ release alternans that can be amplified by diastolic [Ca2+]SR alternans and lead to APD alternans. Sensitizing RyR suppresses spatially concordant but not discordant SR Ca2+ and APD alternans. Despite increased [Ca2+]SR during VF, SR Ca2+ release was nearly continuously refractory. This novel method provides insight into SR Ca2+ handling during cardiac alternans and arrhythmia. # Novelty and Significance {#article-title-40}

Enhanced transmural fiber rotation and connexin 43 heterogeneity are associated with an increased upper limit of vulnerability in a transgenic rabbit model of human hypertrophic cardiomyopathy

Human hypertrophic cardiomyopathy, characterized by cardiac hypertrophy and myocyte disarray, is the most common cause of sudden cardiac death in the young. Hypertrophic cardiomyopathy is often caused by mutations in sarcomeric genes. We sought to determine arrhythmia propensity and underlying mechanisms contributing to arrhythmia in a transgenic (TG) rabbit model (&bgr;-myosin heavy chain–Q403) of human hypertrophic cardiomyopathy. Langendorff-perfused hearts from TG (n=6) and wild-type (WT) rabbits (n=6) were optically mapped. The upper and lower limits of vulnerability, action potential duration (APD) restitution, and conduction velocity were measured. The transmural fiber angle shift was determined using diffusion tensor MRI. The transmural distribution of connexin 43 was quantified with immunohistochemistry. The upper limit of vulnerability was significantly increased in TG versus WT hearts (13.3±2.1 versus 7.4±2.3 V/cm; P=3.2e−5), whereas the lower limits of vulnerability were similar. APD restitution, conduction velocities, and anisotropy were also similar. Left ventricular transmural fiber rotation was significantly higher in TG versus WT hearts (95.6±10.9° versus 79.2±7.8°; P=0.039). The connexin 43 density was significantly increased in the mid-myocardium of TG hearts compared with WT (5.46±2.44% versus 2.68±0.77%; P=0.024), and similar densities were observed in the endo- and epicardium. Because a nearly 2-fold increase in upper limit of vulnerability was observed in the TG hearts without significant changes in APD restitution, conduction velocity, or the anisotropy ratio, we conclude that structural remodeling may underlie the elevated upper limit of vulnerability in human hypertrophic cardiomyopathy.

Quantification of cardiac fiber orientation using optical coherence tomography

Heterogeneity in cardiac tissue microstructure is a potential mechanism for the generation and maintenance of arrhythmias. Abnormal changes in fiber orientation increase the likelihood of arrhythmia. We present optical coherence tomography (OCT) as a method to image myofibers in excised intact heart preparations. Three-dimensional (3-D) image sets were gathered from the rabbit right ventricular free wall (RVFW) using a microscope-integrated OCT system. An automated algorithm for fiber orientation quantification in the plane parallel to the wall surface was developed. The algorithm was validated by comparison with manual measurements. Quantifying fiber orientation in the plane parallel to the wall surface from OCT images can be used to help understand the conduction system of the specific sample being imaged.

The crossroads of inflammation, fibrosis, and arrhythmia following myocardial infarction

Optimal healing of damaged tissue following myocardial infarction (MI) requires a coordinated cellular response that can be divided into three phases: inflammatory, proliferative/reparative, and maturation. The inflammatory phase, characterized by rapid influx of cytokines, chemokines, and immune cells, is critical to the removal of damaged tissue. The onset of the proliferative/reparative phase is marked by increased proliferation of myofibroblasts and secretion of collagen to replace dead tissue. Lastly, crosslinking of collagen fibers and apoptosis of immune cells marks the maturation phase. Excessive inflammation or fibrosis has been linked to increased incidence of arrhythmia and other MI-related pathologies. This review describes the roles of inflammation and fibrosis in arrhythmogenesis and prospective therapies for anti-arrhythmic treatment.

Panoramic imaging reveals basic mechanisms of induction and termination of ventricular tachycardia in rabbit heart with chronic infarction: implications for low-voltage cardioversion.

BACKGROUND Sudden cardiac death due to arrhythmia in the settings of chronic myocardial infarction (MI) is an important clinical problem. Arrhythmic risk post-MI continues indefinitely even if heart failure and acute ischemia are not present due to the anatomic substrate of the scar and border zone (BZ) tissue. OBJECTIVE The purpose of this study was to determine mechanisms of arrhythmia initiation and termination in a rabbit model of chronic MI. METHODS Ligation of the lateral division of the left circumflex artery was performed 72 +/- 29 days before acute experiments (n = 11). Flecainide (2.13 +/- 0.64 microM) was administered to promote sustained arrhythmias, which were induced with burst pacing or a multiple shock protocol (four pulses, 140-200 ms coupling interval). RESULTS Panoramic optical mapping with blebbistatin (5 microM) revealed monomorphic ventricular tachycardia (VT) maintained by a single mother rotor (cycle length [CL] = 174.7 +/- 38.4 ms) as the primary mechanism of arrhythmia. Mother rotors were anchored to the scar or BZ for 16 of the 19 rotor locations recorded. Cardioversion thresholds (CVTs) were determined at various phases throughout the VT CL from external shock electrodes. CVTs were found to be phase dependent, and the maximum versus minimum CVT was 7.8 +/- 1.9 vs. 4.1 +/- 1.6 V/cm, respectively (P = .005). Antitachycardia pacing was found to be effective in only 2.7% of cases in this model. CONCLUSIONS These results indicate that scar and BZ tissue heterogeneity provide the substrate for VT by attracting and stabilizing rotors. Additionally, a significant reduction in CVT may be achieved by appropriately timed shocks in which the shock-induced virtual electrode polarization interacts with the rotor to destabilize VT.