Phase 2 Re-Entry Without Ito: Role of Sodium Channel Kinetics in Brugada Syndrome Arrhythmias.

BACKGROUND: In Brugada syndrome (BrS), phase 2 re-excitation/re-entry (P2R) induced by the transient outward potassium current (Ito) is a proposed arrhythmia mechanism; yet, the most common genetic defects are loss-of-function sodium channel mutations. OBJECTIVES: The authors used computer simulations to investigate how sodium channel dysfunction affects P2R-mediated arrhythmogenesis in the presence and absence of Ito. METHODS: Computer simulations were carried out in 1-dimensional cables and 2-dimensional tissue using guinea pig and human ventricular action potential models. RESULTS: In the presence of Ito sufficient to generate robust P2R, reducing sodium current (INa) peak amplitude alone only slightly potentiated P2R. When INa inactivation kinetics were also altered to simulate reported effects of BrS mutations and sodium channel blockers, however, P2R occurred even in the absence of Ito. These effects could be potentiated by delaying L-type calcium channel activation or increasing ATP-sensitive potassium current, consistent with experimental and clinical findings. INa-mediated P2R also accounted for sex-related, day and night-related, and fever-related differences in arrhythmia risk in BrS patients. CONCLUSIONS: Altered INa kinetics synergize powerfully with reduced INa amplitude to promote P2R-induced arrhythmias in BrS in the absence of Ito, establishing a robust mechanistic link between altered INa kinetics and the P2R-mediated arrhythmia mechanism.

Cardiac Alternans: From Bedside to Bench and Back.

Cardiac alternans arises from dynamical instabilities in the electrical and calcium cycling systems of the heart, and often precedes ventricular arrhythmias and sudden cardiac death. In this review, we integrate clinical observations with theory and experiment to paint a holistic portrait of cardiac alternans: the underlying mechanisms, arrhythmic manifestations and electrocardiographic signatures. We first summarize the cellular and tissue mechanisms of alternans that have been demonstrated both theoretically and experimentally, including 3 voltage-driven and 2 calcium-driven alternans mechanisms. Based on experimental and simulation results, we describe their relevance to mechanisms of arrhythmogenesis under different disease conditions, and their link to electrocardiographic characteristics of alternans observed in patients. Our major conclusion is that alternans is not only a predictor, but also a causal mechanism of potentially lethal ventricular and atrial arrhythmias across the full spectrum of arrhythmia mechanisms that culminate in functional reentry, although less important for anatomic reentry and focal arrhythmias.

R-on-T and the initiation of reentry revisited: Integrating old and new concepts.

Initiation of reentry requires 2 factors: (1) a triggering event, most commonly focal excitations such as premature ventricular complexes (PVCs); and (2) a vulnerable substrate with regional dispersion of refractoriness and/or excitability, such as occurs during the T wave of the electrocardiogram when some areas of the ventricle have repolarized and recovered excitability but others have not. When the R wave of a PVC coincides in time with the T wave of the previous beat, this timing can lead to unidirectional block and initiation of reentry, known as the R-on-T phenomenon. Classically, the PVC triggering reentry has been viewed as arising focally from 1 region and propagating into another region whose recovery is delayed, resulting in unidirectional conduction block and reentry initiation. However, more recent evidence indicates that PVCs also can arise from the T wave itself. In the latter case, the PVC initiating reentry is not a separate event from the T wave but rather is causally generated from the repolarization gradient that manifests as the T wave. We call the former an "R-to-T" mechanism and the latter an "R-from-T" mechanism, which are initiation mechanisms distinct from each other. Both are important components of the R-on-T phenomenon and need to be taken into account when designing antiarrhythmic strategies. Strategies targeting suppression of triggers alone or vulnerable substrate alone may be appropriate in some instances but not in others. Preventing R-from-T arrhythmias requires suppressing the underlying dynamic tissue instabilities responsible for producing both triggers and substrate vulnerability simultaneously. The same principles are likely to apply to supraventricular arrhythmias.

Why Is Only Type 1 Electrocardiogram Diagnostic of Brugada Syndrome? Mechanistic Insights From Computer Modeling.

BACKGROUND: Three types of characteristic ST-segment elevation are associated with Brugada syndrome but only type 1 is diagnostic. Why only type 1 ECG is diagnostic remains unanswered. METHODS: Computer simulations were performed in single cells, 1-dimensional cables, and 2-dimensional tissues to investigate the effects of the peak and late components of the transient outward potassium current (Ito), sodium current, and L-type calcium current (ICa,L) as well as other potassium currents on the genesis of ECG morphologies and phase 2 reentry (P2R). RESULTS: Although a sufficiently large peak Ito was required to result in the type 1 ECG pattern and P2R, increasing the late component of Ito converted type 1 ECG to type 2 ECG and suppressed P2R. Increasing the peak Ito promoted spiral wave breakup, potentiating the transition from tachycardia to fibrillation, but increasing the late Ito prevented spiral wave breakup by flattening the action potential duration restitution and preventing P2R. A sufficiently large ICa,L conductance was needed for P2R to occur, but once above the critical conductance, blocking ICa,L promoted P2R. However, selectively blocking the window and late components of ICa,L suppressed P2R, countering the effect of the late Ito. Blocking either the peak or late components of sodium current promoted P2R, with the late sodium current blockade having the larger effect. As expected, increasing other potassium currents potentiated P2R, with ATP-sensitive potassium current exhibiting a larger effect than rapid and slow component of the delayed rectifier potassium current. CONCLUSIONS: The peak Ito promotes type 1 ECG and P2R, whereas the late Ito converts type 1 ECG to type 2 ECG and suppresses P2R. Blocking the peak ICa,L and either the peak or the late sodium current promotes P2R, whereas blocking the window and late ICa,L suppresses P2R. These results provide important insights into the mechanisms of arrhythmogenesis and potential therapeutic targets for treatment of Brugada syndrome. Graphic Abstract: A graphic abstract is available for this article.

Suppression of ventricular arrhythmias by targeting late L-type Ca2+ current.

Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late ICa,L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak ICa,L, which is essential for proper excitation-contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late ICa,L with minimal effect on peak current. Scaling our investigation from a human CaV1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces ICa,L noninactivating component in a human CaV1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late ICa,L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late ICa,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late ICa,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of CaV1.2 channels rather than blocking their pore, largely preserving contractility.

Mechanisms of Arrhythmogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant I79N.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of TNNT2 (cardiac troponin T-TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of TNNT2 variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a TNNT2 variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca2+ sensitivity and Ca2+ dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca2+ sensitivity and decreases the Ca2+ off-rate constant (k off). To investigate further, a heterozygous I79N+/- TNNT2 variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N+/- hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage (V m) and cytosolic Ca2+ transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca2+ transients due to the enhanced cytosolic Ca2+ buffering. These changes in Ca2+ handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both V m and Ca2+ transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N+/- TNNT2 hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of NPPA (atrial natriuretic peptide), NPPB (brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N+/- vs. the isogenic control. This significant shift demonstrates that this missense in the TNNT2 transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression.

Simultaneous activation of the small conductance calcium-activated potassium current by acetylcholine and inhibition of sodium current by ajmaline cause J-wave syndrome in Langendorff-perfused rabbit ventricles.

BACKGROUND: Concomitant apamin-sensitive small conductance calcium-activated potassium current (IKAS) activation and sodium current inhibition induce J-wave syndrome (JWS) in rabbit hearts. Sudden death in JWS occurs predominantly in men at night when parasympathetic tone is strong. OBJECTIVE: The purpose of this study was to test the hypotheses that acetylcholine (ACh), the parasympathetic transmitter, activates IKAS and causes JWS in the presence of ajmaline. METHODS: We performed optical mapping in Langendorff-perfused rabbit hearts and whole-cell voltage clamp to determine IKAS in isolated ventricular cardiomyocytes. RESULTS: ACh (1 µM) + ajmaline (2 µM) induced J-point elevations in all (6 male and 6 female) hearts from 0.01± 0.01 to 0.31 ± 0.05 mV (P<.001), which were reduced by apamin (specific IKAS inhibitor, 100 nM) to 0.14 ± 0.02 mV (P<.001). More J-point elevation was noted in male than in female hearts (P=.037). Patch clamp studies showed that ACh significantly (P<.001) activated IKAS in isolated male but not in female ventricular myocytes (n=8). Optical mapping studies showed that ACh induced action potential duration (APD) heterogeneity, which was more significant in right than in left ventricles. Apamin in the presence of ACh prolonged both APD at the level of 25% (P<.001) and APD at the level of 80% (P<.001) and attenuated APD heterogeneity. Ajmaline further increased APD heterogeneity induced by ACh. Ventricular arrhythmias were induced in 6 of 6 male and 1 of 6 female hearts (P=.015) in the presence of ACh and ajmaline, which was significantly suppressed by apamin in the former. CONCLUSION: ACh activates ventricular IKAS. ACh and ajmaline induce JWS and facilitate the induction of ventricular arrhythmias more in male than in female ventricles.

Sex-specific IKAS activation in rabbit ventricles with drug-induced QT prolongation.

BACKGROUND: Female sex is a known risk factor for drug-induced long QT syndrome (diLQTS). We recently demonstrated a sex difference in apamin-sensitive small-conductance Ca2+-activated K+ current (IKAS) activation during ß-adrenergic stimulation. OBJECTIVE: The purpose of this study was to test the hypothesis that there is a sex difference in IKAS in the rabbit models of diLQTS. METHODS: We evaluated the sex difference in ventricular repolarization in 15 male and 22 female Langendorff-perfused rabbit hearts with optical mapping techniques during atrial pacing. HMR1556 (slowly activating delayed rectifier K+ current [IKs] blocker), E4031 (rapidly activating delayed rectifier K+ current [IKr] blocker) and sea anemone toxin (ATX-II, late Na+ current [INaL] activator) were used to simulate types 1-3 long QT syndrome, respectively. Apamin, an IKAS blocker, was then added to determine the magnitude of further QT prolongation. RESULTS: HMR1556, E4031, and ATX-II led to the prolongation of action potential duration at 80% repolarization (APD80) in both male and female ventricles at pacing cycle lengths of 300-400 ms. Apamin further prolonged APD80 (pacing cycle length 350 ms) from 187.8±4.3 to 206.9±7.1 (P=.014) in HMR1556-treated, from 209.9±7.8 to 224.9±7.8 (P=.003) in E4031-treated, and from 174.3±3.3 to 188.1±3.0 (P=.0002) in ATX-II-treated female hearts. Apamin did not further prolong the APD80 in male hearts. The Cai transient duration (CaiTD) was significantly longer in diLQTS than baseline but without sex differences. Apamin did not change CaiTD. CONCLUSION: We conclude that IKAS is abundantly increased in female but not in male ventricles with diLQTS. Increased IKAS helps preserve the repolarization reserve in female ventricles treated with IKs and IKr blockers or INaL activators.

Stabilizer Cell Gene Therapy: A Less-Is-More Strategy to Prevent Cardiac Arrhythmias.

BACKGROUND: In cardiac gene therapy to improve contractile function, achieving gene expression in the majority of cardiac myocytes is essential. In preventing cardiac arrhythmias, however, this goal may not be as important since transduction efficiencies as low as 40% suppressed ventricular arrhythmias in genetically modified mice with catecholaminergic polymorphic ventricular tachycardia. METHODS: Using computational modeling, we simulated 1-, 2-, and 3-dimensional tissue under a variety of conditions to test the ability of genetically engineered nonarrhythmogenic stabilizer cells to suppress triggered activity due to delayed or early afterdepolarizations. RESULTS: Due to source-sink relationships in cardiac tissue, a minority (20%-50%) of randomly distributed stabilizer cells engineered to be nonarrhythmogenic can suppress the ability of arrhythmogenic cells to generate delayed and early afterdepolarizations-related arrhythmias. Stabilizer cell gene therapy strategy can be designed to correct a specific arrhythmogenic mutation, as in the catecholaminergic polymorphic ventricular tachycardia mice studies, or more generally to suppress delayed or early afterdepolarizations from any cause by overexpressing the inward rectifier K channel Kir2.1 in stabilizer cells. CONCLUSIONS: This promising antiarrhythmic strategy warrants further testing in experimental models to evaluate its clinical potential.

Small-conductance Ca2+-activated K+ channels promote J-wave syndrome and phase 2 reentry.

BACKGROUND: Small-conductance Ca2+-activated potassium (SK) channels play complex roles in cardiac arrhythmogenesis. SK channels colocalize with L-type Ca2+ channels, yet how this colocalization affects cardiac arrhythmogenesis is unknown. OBJECTIVE: The purpose of this study was to investigate the role of colocalization of SK channels with L-type Ca2+ channels in promoting J-wave syndrome and ventricular arrhythmias. METHODS: We carried out computer simulations of single-cell and tissue models. SK channels in the model were assigned to preferentially sense Ca2+ in the bulk cytosol, subsarcolemmal space, or junctional cleft. RESULTS: When SK channels sense Ca2+ in the bulk cytosol, the SK current (ISK) rises and decays slowly during an action potential, the action potential duration (APD) decreases as the maximum conductance increases, no complex APD dynamics and phase 2 reentry can be induced by ISK. When SK channels sense Ca2+ in the subsarcolemmal space or junctional cleft, ISK can rise and decay rapidly during an action potential in a spike-like pattern because of spiky Ca2+ transients in these compartments, which can cause spike-and-dome action potential morphology, APD alternans, J-wave elevation, and phase 2 reentry. Our results can account for the experimental finding that activation of ISK induced J-wave syndrome and phase 2 reentry in rabbit hearts. CONCLUSION: Colocalization of SK channels with L-type Ca2+ channels so that they preferentially sense Ca2+ in the subsarcolemmal or junctional space may result in a spiky ISK, which can functionally play a similar role of the transient outward K+ current in promoting J-wave syndrome and ventricular arrhythmias.

A Spatiotemporal Ventricular Myocyte Model Incorporating Mitochondrial Calcium Cycling.

Intracellular calcium (Ca2+) cycling dynamics in cardiac myocytes are spatiotemporally generated by stochastic events arising from a spatially distributed network of coupled Ca2+ release units that interact with an intertwined mitochondrial network. In this study, we developed a spatiotemporal ventricular myocyte model that integrates mitochondria-related Ca2+ cycling components into our previously developed ventricular myocyte model consisting of a three-dimensional Ca2+ release unit network. Mathematical formulations of mitochondrial membrane potential, mitochondrial Ca2+ cycling, mitochondrial permeability transition pore stochastic opening and closing, intracellular reactive oxygen species signaling, and oxidized Ca2+/calmodulin-dependent protein kinase II signaling were incorporated into the model. We then used the model to simulate the effects of mitochondrial depolarization on mitochondrial Ca2+ cycling, Ca2+ spark frequency, and Ca2+ amplitude, which agree well with experimental data. We also simulated the effects of the strength of mitochondrial Ca2+ uniporters and their spatial localization on intracellular Ca2+ cycling properties, which substantially affected diastolic and systolic Ca2+ levels in the mitochondria but exhibited only a small effect on sarcoplasmic reticulum and cytosolic Ca2+ levels under normal conditions. We show that mitochondrial depolarization can cause Ca2+ waves and Ca2+ alternans, which agrees with previous experimental observations. We propose that this new, to our knowledge, spatiotemporal ventricular myocyte model, incorporating properties of mitochondrial Ca2+ cycling and reactive-oxygen-species-dependent signaling, will be useful for investigating the effects of mitochondria on intracellular Ca2+ cycling and action potential dynamics in ventricular myocytes.

Small-conductance calcium-activated potassium current modulates the ventricular escape rhythm in normal rabbit hearts.

BACKGROUND: The apamin-sensitive small-conductance calcium-activated K (SK) current IKAS modulates automaticity of the sinus node. IKAS blockade by apamin causes sinus bradycardia. OBJECTIVE: The purpose of this study was to test the hypothesis that IKAS modulates ventricular automaticity. METHODS: We tested the effects of apamin (100 nM) on ventricular escape rhythms in Langendorff-perfused rabbit ventricles with atrioventricular block (protocol 1) and on recorded transmembrane action potential of pseudotendons of superfused right ventricular endocardial preparations (protocol 2). RESULTS: All preparations exhibited spontaneous ventricular escape rhythms. In protocol 1, apamin decreased the atrial rate from 186.2 ± 18.0 bpm to 163.8 ± 18.7 bpm (N = 6; P = .006) but accelerated the ventricular escape rate from 51.5 ± 10.7 bpm to 98.2 ± 25.4 bpm (P = .031). Three preparations exhibited bursts of nonsustained ventricular tachycardia and pauses, resulting in repeated burst termination pattern. In protocol 2, apamin increased the ventricular escape rate from 70.2 ± 13.1 bpm to 110.1 ± 2.2 bpm (P = .035). Spontaneous phase 4 depolarization was recorded from the pseudotendons in 6 of 10 preparations at baseline and in 3 in the presence of apamin. There were no changes of phase 4 slope (18.37 ± 3.55 mV/s vs 18.93 ± 3.26 mV/s, N = 3; P = .231, ), but the threshold of phase 0 activation (mV) reduced from -67.97 ± 1.53 to -75.26 ± 0.28 (P = .034). Addition of JTV-519, a ryanodine receptor 2 stabilizer, in 5 preparations reduced escape rate back to baseline. CONCLUSION: Contrary to its bradycardic effect in the sinus node, IKAS blockade by apamin accelerates ventricular automaticity and causes repeated nonsustained ventricular tachycardia in normal ventricles. ryanodine receptor 2 blockade reversed the apamin effects on ventricular automaticity.

Atrial Fibrillation Initiated by Early Afterdepolarization-Mediated Triggered Activity during Acute Oxidative Stress: Efficacy of Late Sodium Current Blockade.

BACKGROUND: The mechanism of Atrial Fibrillation (AF) that emerges spontaneously during acute oxidative stress is poorly defined and its drug therapy remains suboptimal. We hypothesized that oxidative activation of Ca-calmodulin dependent protein kinase (CaMKII) promotes Early Afterdepolarization-(EAD)-mediated triggered AF in aged fibrotic atria that is sensitive to late Na current (INa-L) blockade. METHOD AND RESULTS: High-resolution voltage optical mapping of the Left and Right Atrial (LA & RA) epicardial surfaces along with microelectrode recordings were performed in isolated-perfused male Fisher 344 rat hearts in Langendorff setting. Aged atria (23-24 months) manifested 10-fold increase in atrial tissue fibrosis compared to young/adult (2-4 months) atria (P<0001. Spontaneous AF arose in 39 out of 41 of the aged atria but in 0 out of 12 young/adult hearts (P<001) during arterial perfusion of with 0.1 mm of hydrogen peroxide (H2O2). Optical Action Potential (AP) activation maps showed that the AF was initiated by a focal mechanism in the LA suggestive of EAD-mediated triggered activity. Cellular AP recordings with glass microelectrodes from the LA epicardial sites showing focal activity confirmed optical AP recordings that the spontaneous AF was initiated by late phase 3 EAD-mediated triggered activity. Inhibition of CaMKII activity with KN-93 (1 µM) (N=6) or its downstream target, the enhanced INa-L with GS-967 (1 µM), a specific blocker of INa-L (N=6), potently suppressed the AF and prevented its initiation when perfused 15 min prior to H2O2 (n=6). CONCLUSIONS: Increased atrial tissue fibrosis combined with acute oxidative activation of CaMK II Initiate AF by EAD-mediated triggered activity. Specific block of the INa-L with GS-967 effectively suppresses the AF. Drug therapy of oxidative AF in humans with traditional antiarrhythmic drugs remains suboptimal; suppressing INa-L offers a potential new strategy for effective suppression of oxidative human AF that remains suboptimal.

Concomitant SK current activation and sodium current inhibition cause J wave syndrome.

The mechanisms of J wave syndrome (JWS) are incompletely understood. Here, we showed that the concomitant activation of small-conductance calcium-activated potassium (SK) current (IKAS) and inhibition of sodium current by cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) recapitulate the phenotypes of JWS in Langendorff-perfused rabbit hearts. CyPPA induced significant J wave elevation and frequent spontaneous ventricular fibrillation (SVF), as well as sinus bradycardia, atrioventricular block, and intraventricular conduction delay. IKAS activation by CyPPA resulted in heterogeneous shortening of action potential (AP) duration (APD) and repolarization alternans. CyPPA inhibited cardiac sodium current (INa) and decelerated AP upstroke and intracellular calcium transient. SVFs were typically triggered by short-coupled premature ventricular contractions, initiated with phase 2 reentry and originated more frequently from the right than the left ventricles. Subsequent IKAS blockade by apamin reduced J wave elevation and eliminated SVF. ß-Adrenergic stimulation was antiarrhythmic in CyPPA-induced electrical storm. Like CyPPA, hypothermia (32.0°C) also induced J wave elevation and SVF. It facilitated negative calcium-voltage coupling and phase 2 repolarization alternans with spatial and electromechanical discordance, which were ameliorated by apamin. These findings suggest that IKAS activation contributes to the development of JWS in rabbit ventricles.

The Ca2+ transient as a feedback sensor controlling cardiomyocyte ionic conductances in mouse populations.

Conductances of ion channels and transporters controlling cardiac excitation may vary in a population of subjects with different cardiac gene expression patterns. However, the amount of variability and its origin are not quantitatively known. We propose a new conceptual approach to predict this variability that consists of finding combinations of conductances generating a normal intracellular Ca2+ transient without any constraint on the action potential. Furthermore, we validate experimentally its predictions using the Hybrid Mouse Diversity Panel, a model system of genetically diverse mouse strains that allows us to quantify inter-subject versus intra-subject variability. The method predicts that conductances of inward Ca2+ and outward K+ currents compensate each other to generate a normal Ca2+ transient in good quantitative agreement with current measurements in ventricular myocytes from hearts of different isogenic strains. Our results suggest that a feedback mechanism sensing the aggregate Ca2+ transient of the heart suffices to regulate ionic conductances.

Sex-specific activation of SK current by isoproterenol facilitates action potential triangulation and arrhythmogenesis in rabbit ventricles.

KEY POINTS: It is unknown if a sex difference exists in cardiac apamin-sensitive small conductance Ca2+ -activated K+ (SK) current (IKAS ). There is no sex difference in IKAS in the basal condition. However, there is larger IKAS in female rabbit ventricles than in male during isoproterenol infusion. IKAS activation by isoproterenol leads to action potential triangulation in females, indicating its abundant activation at early phases of repolarization. IKAS activation in females induces negative Ca2+ -voltage coupling and promotes electromechanically discordant phase 2 repolarization alternans. IKAS is important in the mechanisms of ventricular fibrillation in females during sympathetic stimulation. ABSTRACT: Sex has a large influence on cardiac electrophysiological properties. Whether sex differences exist in apamin-sensitive small conductance Ca2+ -activated K+ (SK) current (IKAS ) remains unknown. We performed optical mapping, transmembrane potential, patch clamp, western blot and immunostaining in 62 normal rabbit ventricles, including 32 females and 30 males. IKAS blockade by apamin only minimally prolonged action potential (AP) duration (APD) in the basal condition for both sexes, but significantly prolonged APD in the presence of isoproterenol in females. Apamin prolonged APD at the level of 25% repolarization (APD25 ) more prominently than APD at the level of 80% repolarization (APD80 ), consequently reversing isoproterenol-induced AP triangulation in females. In comparison, apamin prolonged APD to a significantly lesser extent in males and failed to restore the AP plateau during isoproterenol infusion. IKAS in males did not respond to the L-type calcium current agonist BayK8644, but was amplified by the casein kinase 2 (CK2) inhibitor 4,5,6,7-tetrabromobenzotriazole. In addition, whole-cell outward IKAS densities in ventricular cardiomyocytes were significantly larger in females than in males. SK channel subtype 2 (SK2) protein expression was higher and the CK2/SK2 ratio was lower in females than in males. IKAS activation in females induced negative intracellular Ca2+ -voltage coupling, promoted electromechanically discordant phase 2 repolarization alternans and facilitated ventricular fibrillation (VF). Apamin eliminated the negative Ca2+ -voltage coupling, attenuated alternans and reduced VF inducibility, phase singularities and dominant frequencies in females, but not in males. We conclude that ß-adrenergic stimulation activates ventricular IKAS in females to a much greater extent than in males. IKAS activation plays an important role in ventricular arrhythmogenesis in females during sympathetic stimulation.

A personalized, multiomics approach identifies genes involved in cardiac hypertrophy and heart failure.

A traditional approach to investigate the genetic basis of complex diseases is to identify genes with a global change in expression between diseased and healthy individuals. However, population heterogeneity may undermine the effort to uncover genes with significant but individual contribution to the spectrum of disease phenotypes within a population. Here we investigate individual changes of gene expression when inducing hypertrophy and heart failure in 100 + strains of genetically distinct mice from the Hybrid Mouse Diversity Panel (HMDP). We find that genes whose expression fold-change correlates in a statistically significant way with the severity of the disease are either up or down-regulated across strains, and therefore missed by a traditional population-wide analysis of differential gene expression. Furthermore, those "fold-change" genes are enriched in human cardiac disease genes and form a dense co-regulated module strongly interacting with the cardiac hypertrophic signaling network in the human interactome. We validate our approach by showing that the knockdown of Hes1, predicted as a strong candidate, induces a dramatic reduction of hypertrophy by 80-90% in neonatal rat ventricular myocytes. Our results demonstrate that individualized approaches are crucial to identify genes underlying complex diseases as well as to develop personalized therapies.