Mechanisms of adaptive hypertrophic cardiac remodeling in a large animal model of premature ventricular contraction-induced cardiomyopathy.

Frequent premature ventricular contractions (PVCs) promoted eccentric cardiac hypertrophy and reduced ejection fraction (EF) in a large animal model of PVC-induced cardiomyopathy (PVC-CM), but the molecular mechanisms and markers of this hypertrophic remodeling remain unexplored. Healthy mongrel canines were implanted with pacemakers to deliver bigeminal PVCs (50% burden with 200-220 ms coupling interval). After 12 weeks, left ventricular (LV) free wall samples were studied from PVC-CM and Sham groups. In addition to reduced LV ejection fraction (LVEF), the PVC-CM group showed larger cardiac myocytes without evident ultrastructural alterations compared to the Sham group. Biochemical markers of pathological hypertrophy, such as store-operated Ca2+ entry, calcineurin/NFAT pathway, ß-myosin heavy chain, and skeletal type α-actin were unaltered in the PVC-CM group. In contrast, pro-hypertrophic and antiapoptotic pathways including ERK1/2 and AKT/mTOR were activated and/or overexpressed in the PVC-CM group, which appeared counterbalanced by an overexpression of protein phosphatase 1 and a borderline elevation of the anti-hypertrophic factor atrial natriuretic peptide. Moreover, the potent angiogenic and pro-hypertrophic factor VEGF-A and its receptor VEGFR2 were significantly elevated in the PVC-CM group. In conclusion, a molecular program is in place to keep this structural remodeling associated with frequent PVCs as an adaptive pathological hypertrophy.

Alterations of sarcoplasmic reticulum-mediated Ca2+ uptake in a model of premature ventricular contraction (PVC)-induced cardiomyopathy.

Premature ventricular contractions (PVCs) are the most frequent ventricular arrhythmias in the overall population. PVCs are known to acutely enhance contractility by the post-extrasystolic potentiation phenomenon, but over time persistent PVCs promote PVC-induced cardiomyopathy (PVC-CM), characterized by a reduction of the left ventricular (LV) ejection fraction. Ca2+ cycling in myocytes commands muscle contraction and in this process, SERCA2 leads the Ca2+ reuptake into the sarcoplasmic reticulum (SR) shaping cytosolic Ca2+ signal decay and muscle relaxation. Altered Ca2+ reuptake can contribute to the contractile dysfunction observed in PVC-CM. To better understand Ca2+ handling using our PVC-CM model (canines with 50% PVC burden for 12 weeks), SR-Ca2+ reuptake was investigated by measuring Ca2+ dynamics and analyzing protein expression. Kinetic analysis of Ca2+ reuptake in electrically paced myocytes showed a ~ 21 ms delay in PVC-CM compared to Sham in intact isolated myocytes, along with a ~ 13% reduction in SERCA2 activity assessed in permeabilized myocytes. Although these trends were not statistically significant between groups using hierarchical statistics, relaxation of myocytes following contraction was significantly slower in PVC-CM vs Sham myocytes. Western blot analyses indicate a 22% reduction in SERCA2 expression, a 23% increase in phospholamban (PLN) expression, and a 50% reduction in PLN phosphorylation in PVC-CM samples vs Sham. Computational analysis simulating a 20% decrease in SR-Ca2+ reuptake resulted in a ~ 22 ms delay in Ca2+ signal decay, consistent with the experimental result described above. In conclusion, SERCA2 and PLB alterations described above have a modest contribution to functional adaptations observed in PVC-CM.

Cardiac Kir2.1 and NaV1.5 Channels Traffic Together to the Sarcolemma to Control Excitability.

RATIONALE: In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. OBJECTIVE: The objective of this study was to test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. METHODS AND RESULTS: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wild-type (WT) NaV1.5WT in heterologous cells reduced inward sodium current compared with NaV1.5WT alone or coexpressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas coexpression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. CONCLUSIONS: The NaV1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

Post-extrasystolic potentiation as a predictor of premature ventricular contraction-cardiomyopathy in an animal model.

AIMS: High premature ventricular contractions (PVCs) burden does not always predict the development of PVC-cardiomyopathy (CM). We sought to evaluate post-extrasystolic potentiation (PESP) of left ventricular ejection fraction (LVEF) to predict the severity of PVC-CM in an animal model. METHODS AND RESULTS: Right ventricular apical bigeminal PVCs were introduced for 12 weeks in 11 canines to induce PVC-CM. Echocardiograms were performed to obtain LVEF without ectopy (Echo-1) and during PVCs (200 and 350 ms coupling intervals, Echo-2, and Echo-3, respectively), and premature atrial contractions (PACs) (Echo-4) at baseline and after 12 weeks of bigeminal PVCs. PESP was calculated as delta-LVEF between the sinus beat post-ectopy LVEF (Echo-2, -3, and -4, respectively) and LVEF without PVC (Echo-1) at baseline and 12 weeks of high PVC burden. A hyperdynamic LV function (LVEF > 70%) was noted in all animals only with early-coupled PVCs (LVEF at 200 ms: 74.4 ± 6%) at baseline. While PVC PESP at 200 ms had a strong significant correlation with the final 12-week LVEF (R = 0.8, P = 0.003), PVC PESP at 350 ms and PAC PESP had a positive but non-significant correlation (R = 0.53, P = 0.09, and R = 0.29, P = 0.34, respectively). Premature ventricular contraction PESP at 350 ms was significantly higher after PVC-CM had developed (delta-LVEF baseline 2.7 ± 2.9% vs. 12 weeks 18.6 ± 12.3% P < 0.001). CONCLUSION: Bigeminal early-coupled PVCs cause hyperdynamic left ventricular function in the structurally normal canine heart due to PESP. The degree of PESP at baseline is inversely proportional to the PVC-CM severity at 12 weeks and maybe a predictor of PVC-CM as it may assess the myocardial adaptation reserve to PVCs.

Constitutive Intracellular Na+ Excess in Purkinje Cells Promotes Arrhythmogenesis at Lower Levels of Stress Than Ventricular Myocytes From Mice With Catecholaminergic Polymorphic Ventricular Tachycardia.

BACKGROUND: In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca(2+) dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2(R4496C+/Cx40eGFP)), we tested whether PC intracellular Ca(2+) ([Ca(2+)]i) dysregulation results from a constitutive [Na(+)]i surplus relative to VMs. METHODS AND RESULTS: Simultaneous optical mapping of voltage and [Ca(2+)]i in CPVT hearts showed that spontaneous Ca(2+) release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca(2+) imaging, early and delayed afterdepolarizations trailed spontaneous Ca(2+) release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca(2+) load, measured by caffeine-induced Ca(2+) transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na(+)]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na(+)/Ca(2+) exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na(+)]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca(2+) release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na(+)]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca(2+) spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na(+)]i played a central role. CONCLUSIONS: In CPVT mice, the constitutive [Na(+)]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs.

Dominant frequency increase rate predicts transition from paroxysmal to long-term persistent atrial fibrillation.

BACKGROUND: Little is known about the mechanisms underlying the transition from paroxysmal to persistent atrial fibrillation (AF). In an ovine model of long-standing persistent AF we tested the hypothesis that the rate of electric and structural remodeling, assessed by dominant frequency (DF) changes, determines the time at which AF becomes persistent. METHODS AND RESULTS: Self-sustained AF was induced by atrial tachypacing. Seven sheep were euthanized 11.5±2.3 days after the transition to persistent AF and without reversal to sinus rhythm; 7 sheep were euthanized after 341.3±16.7 days of long-standing persistent AF. Seven sham-operated animals were in sinus rhythm for 1 year. DF was monitored continuously in each group. Real-time polymerase chain reaction, Western blotting, patch clamping, and histological analyses were used to determine the changes in functional ion channel expression and structural remodeling. Atrial dilatation, mitral valve regurgitation, myocyte hypertrophy, and atrial fibrosis occurred progressively and became statistically significant after the transition to persistent AF, with no evidence for left ventricular dysfunction. DF increased progressively during the paroxysmal-to-persistent AF transition and stabilized when AF became persistent. Importantly, the rate of DF increase correlated strongly with the time to persistent AF. Significant action potential duration abbreviation, secondary to functional ion channel protein expression changes (CaV1.2, NaV1.5, and KV4.2 decrease; Kir2.3 increase), was already present at the transition and persisted for 1 year of follow up. CONCLUSIONS: In the sheep model of long-standing persistent AF, the rate of DF increase predicts the time at which AF stabilizes and becomes persistent, reflecting changes in action potential duration and densities of sodium, L-type calcium, and inward rectifier currents.

Intracellular [Na(+)] modulates synergy between Na(+)/Ca (2+) exchanger and L-type Ca (2+) current in cardiac excitation-contraction coupling during action potentials.

Excitation-contraction coupling (ECC) in cardiac myocytes involves triggering of Ca(2+) release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca(2+) entry via the Na(+)/Ca(2+) exchanger (NCX) to trigger SR Ca(2+) release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX to SR Ca(2+) release, independent of effects on SR Ca(2+) load, Ca(2+) release was determined by recording Ca(2+) spikes using confocal microscopy on patch-clamped rat ventricular myocytes with [Ca(2+)](i) fixed at 150 nmol/L. In response to AP clamps, normalized Ca(2+) spike amplitudes (ΔF/F (0)) increased sigmoidally and doubled as [Na(+)](i) was elevated from 0 to 20 mmol/L with an EC(50) of ~10 mmol/L. This [Na(+)](i)-dependence was independent of I (Na) as well as SR Ca(2+) load, which was unchanged under our experimental conditions. However, NCX inhibition using either KB-R7943 or XIP reduced ΔF/F (0) amplitude in myocytes with 20 mmol/L [Na(+)](i), but not with 5 mmol/L [Na(+)](i). SR Ca(2+) release was complete before the membrane repolarized to -15 mV, indicating Ca(2+) entry into the dyad (not reduced extrusion) underlies [Na(+)](i)-dependent enhancement of ECC. Because I (Ca,L) inhibition with 50 mmol/L Cd(2+) abolished Ca(2+) spikes, our results demonstrate that during cardiac APs, NCX enhances SR Ca(2+) release by synergistically increasing the efficiency of I (Ca,L)-mediated ECC.

A new electrocardiographic marker for sympathetic nerve stimulation: modulation of repolarization by stimulation of stellate ganglia.

Activation of cardiac sympathetic nerves alters ventricular repolarization; however, these changes remain poorly characterized. The goal of this study was to examine effects of sympathetic stimulation on repolarization to identify electrocardiographic markers of sympathetic activation. Pigs underwent median sternotomy and bilateral thoracotomy to expose the stellate ganglia. Changes in T-wave duration, amplitude, repolarization vector, and time from peak to end (Tp-Te) were continuously monitored. Within 15 seconds of unilateral left or right stellate ganglion (LSG/RSG) stimulation, T-wave amplitude increased 6- and 4.5-fold, respectively, in lead aVF. T-wave duration and Tp-Te both increased 200% during LSG stimulation but only 50% and 30%, respectively, with RSG stimulation. During LSG stimulation, frontal and horizontal T-wave vectors, respectively, changed from 1.9° ± 22.8° and 333.8° ± 9.7° at baseline to 83.4° ± 3.9° (inferiorly) and 306.7° ± 1.8° (posteriorly). During RSG stimulation, frontal and horizontal T-wave vectors changed from 348.2° ± 21.6° and 333.8° ± 10.3° before stimulation to 280.7° ± 4.6° (superiorly) and 118.1° ± 5.6° (anteriorly). During stellate stimulation, T-wave vectors are displaced to angles specific for LSG activation (posteroinferiorly) or RSG activation (anterosuperiorly); T-wave amplitude, duration, and Tp-Te increase; and ST-duration decreases. Displaced repolarization vector and changes in T-wave morphometrics provide a novel marker of unilateral sympathetic nerve stimulation.

Eccentric hypertrophy in an animal model of mid- and long-term premature ventricular contraction-induced cardiomyopathy.

BACKGROUND: Tachycardia and heart rate irregularity are proposed triggers of premature ventricular contraction-induced cardiomyopathy (PVC-cardiomyopathy). Bigeminal premature atrial and ventricular contractions (PACs and PVCs) increase heart rate and result in rhythm irregularities but differ in their effects on ventricular synchrony. Comparing chronic bigeminal PACs with PVCs would provide insights into mechanisms of PVC-cardiomyopathy. OBJECTIVE: To compare the impact of chronic PACs and PVCs on ventricular hemodynamics, structure, and function. METHODS: Pacemakers were implanted in 27 canines to reproduce atrial (PACs, n = 7) or ventricular bigeminy (PVCs, n = 11) for 12 weeks, and compared to sham-operated animals (n = 9). Four additional animals were exposed to long-term bigeminal PVCs (48 weeks). Hemodynamic changes were assessed using a pressure-transducing catheter at baseline and 12 weeks. Cardiac remodeling was monitored by transthoracic echocardiography throughout the 12- and 48-week protocols in the respective groups. RESULTS: PVC group demonstrated a significant decrease in left ventricular (LV) ejection fraction and contractility (max dP/dt), impaired LV lusitropy (min dP/dt), and increase in LV dimensions and LV mass at 12 weeks without further deterioration beyond 16 weeks. Despite increased LV mass, relative wall thickness decreased, consistent with eccentric hypertrophy. No significant cardiac remodeling was noted in either sham or PAC groups at 12 weeks. CONCLUSION: In contrast to bigeminal PACs, PVCs result in a cardiomyopathy characterized by reduced LV ejection fraction, LV dilation, and eccentric hypertrophy that plateaus between 12 and 16 weeks. The lack of remodeling in chronic PACs suggests that tachycardia and heart rate irregularity do not play a significant role on the development of PVC-cardiomyopathy.

Panoramic Endocardial Optical Mapping Demonstrates Serial Rotors Acceleration and Increasing Complexity of Activity During Onset of Cholinergic Atrial Fibrillation.

Background Activation during onset of atrial fibrillation is poorly understood. We aimed at developing a panoramic optical mapping system for the atria and test the hypothesis that sequential rotors underlie acceleration of atrial fibrillation during onset. Methods and Results Five sheep hearts were Langendorff perfused in the presence of 0.25 µmol/L carbachol. Novel optical system recorded activations simultaneously from the entire left and right atrial endocardial surfaces. Twenty sustained (>40 s) atrial fibrillation episodes were induced by a train and premature stimuli protocol. Movies obtained immediately (Initiation stage) and 30 s (Early Stabilization stage) after premature stimulus were analyzed. Serial rotor formation was observed in all sustained inductions and none in nonsustained inductions. In sustained episodes maximal dominant frequency increased from (mean±SD) 11.5±1.74 Hz during Initiation to 14.79±1.30 Hz at Early Stabilization (P<0.0001) and stabilized thereafter. At rotor sites, mean cycle length (CL) during 10 prerotor activations increased every cycle by 0.53% (P=0.0303) during Initiation and 0.34% (P=0.0003) during Early Stabilization. In contrast, CLs at rotor sites showed abrupt decreases after the rotors appearances by a mean of 9.65% (P<0.0001) during both stages. At Initiation, atria-wide accelerations and decelerations during rotors showed a net acceleration result whereby post-rotors atria-wide minimal CL (CLmin) were 95.5±6.8% of the prerotor CLmin (P=0.0042). In contrast, during Early Stabilization, there was no net acceleration in CLmin during accelerating rotors (prerotor=84.9±11.0% versus postrotor=85.8±10.8% of Initiation, P=0.4029). Levels of rotor drift distance and velocity correlated with atria-wide acceleration. Nonrotor phase singularity points did not accelerate atria-wide activation but multiplied during Initiation until Early Stabilization. Increasing number of singularity points, indicating increased complexity, correlated with atria-wide CLmin reduction (P<0.0001). Conclusions Novel panoramic optical mapping of the atria demonstrates shortening CL at rotor sites during cholinergic atrial fibrillation onset. Atrial fibrillation acceleration toward Early Stabilization correlates with the net result of atria-wide accelerations during drifting rotors activity.

Transcriptome and proteome mapping in the sheep atria reveal molecular featurets of atrial fibrillation progression.

AIMS: Atrial fibrillation (AF) is a progressive cardiac arrhythmia that increases the risk of hospitalization and adverse cardiovascular events. There is a clear demand for more inclusive and large-scale approaches to understand the molecular drivers responsible for AF, as well as the fundamental mechanisms governing the transition from paroxysmal to persistent and permanent forms. In this study, we aimed to create a molecular map of AF and find the distinct molecular programmes underlying cell type-specific atrial remodelling and AF progression. METHODS AND RESULTS: We used a sheep model of long-standing, tachypacing-induced AF, sampled right and left atrial tissue, and isolated cardiomyocytes (CMs) from control, intermediate (transition), and late time points during AF progression, and performed transcriptomic and proteome profiling. We have merged all these layers of information into a meaningful three-component space in which we explored the genes and proteins detected and their common patterns of expression. Our data-driven analysis points at extracellular matrix remodelling, inflammation, ion channel, myofibril structure, mitochondrial complexes, chromatin remodelling, and genes related to neural function, as well as critical regulators of cell proliferation as hallmarks of AF progression. Most important, we prove that these changes occur at early transitional stages of the disease, but not at later stages, and that the left atrium undergoes significantly more profound changes than the right atrium in its expression programme. The pattern of dynamic changes in gene and protein expression replicate the electrical and structural remodelling demonstrated previously in the sheep and in humans, and uncover novel mechanisms potentially relevant for disease treatment. CONCLUSIONS: Transcriptomic and proteomic analysis of AF progression in a large animal model shows that significant changes occur at early stages, and that among others involve previously undescribed increase in mitochondria, changes to the chromatin of atrial CMs, and genes related to neural function and cell proliferation.

Mechanisms by Which Ranolazine Terminates Paroxysmal but Not Persistent Atrial Fibrillation.

BACKGROUND: Ranolazine inhibits Na+ current (INa), but whether it can convert atrial fibrillation (AF) to sinus rhythm remains unclear. We investigated antiarrhythmic mechanisms of ranolazine in sheep models of paroxysmal (PxAF) and persistent AF (PsAF). METHODS: PxAF was maintained during acute stretch (N=8), and PsAF was induced by long-term atrial tachypacing (N=9). Isolated, Langendorff-perfused sheep hearts were optically mapped. RESULTS: In PxAF ranolazine (10 µmol/L) reduced dominant frequency from 8.3±0.4 to 6.2±0.5 Hz (P<0.01) before converting to sinus rhythm, decreased singularity point density from 0.070±0.007 to 0.039±0.005 cm-2 s-1 (P<0.001) in left atrial epicardium (LAepi), and prolonged AF cycle length (AFCL); rotor duration, tip trajectory, and variance of AFCL were unaltered. In PsAF, ranolazine reduced dominant frequency (8.3±0.5 to 6.5±0.4 Hz; P<0.01), prolonged AFCL, increased the variance of AFCL, had no effect on singularity point density (0.048±0.011 to 0.042±0.016 cm-2 s-1; P=ns) and failed to convert AF to sinus rhythm. Doubling the ranolazine concentration (20 µmol/L) or supplementing with dofetilide (1 µmol/L) failed to convert PsAF to sinus rhythm. In computer simulations of rotors, reducing INa decreased dominant frequency, increased tip meandering and produced vortex shedding on wave interaction with unexcitable regions. CONCLUSIONS: PxAF and PsAF respond differently to ranolazine. Cardioversion in the former can be attributed partly to decreased dominant frequency and singularity point density, and prolongation of AFCL. In the latter, increased dispersion of AFCL and likely vortex shedding contributes to rotor formation, compensating for any rotor loss, and may underlie the inefficacy of ranolazine to terminate PsAF.

Eplerenone Reduces Atrial Fibrillation Burden Without Preventing Atrial Electrical Remodeling.

BACKGROUND: The aldosterone inhibitor eplerenone (EPL) has been shown to reduce the incidence of atrial fibrillation (AF) in patients with systolic heart failure, but the mechanism is unknown. OBJECTIVES: This study hypothesized that by reducing atrial dilation and fibrosis in the absence of heart failure, EPL also reduces AF burden and prevents AF perpetuation. METHODS: The authors conducted a randomized controlled study in 34 sheep that were atrially tachypaced (13 ± 1 week). They compared daily oral EPL (n = 19) versus sugar pill (SP) treatment (n = 15) from the start of tachypacing. The endpoint was a continuous 7-day stretch of persistent AF (n = 29) or completion of 23 weeks tachypacing (n = 5). RESULTS: EPL significantly reduced the rate of left atrial dilation increase during AF progression. Atria from EPL-treated sheep had less smooth muscle actin protein, collagen-III expression, interstitial atrial fibrosis, and cell hypertrophy than SP-treated sheep atria did. However, EPL did not modify the AF-induced increase in the rate of dominant frequency and ion channel densities seen under SP treatment, but rather prolonged the time to persistent AF in 26% of animals. It also reduced the degree of fibrillatory conduction, AF inducibility, and AF burden. CONCLUSIONS: In the sheep model, EPL mitigates fibrosis and atrial dilation, modifies AF inducibility and AF complexity, and prolongs the transition to persistent AF in 26% of animals, but it does not prevent AF-induced electrical remodeling or AF persistence. The results highlight structural remodeling as a central upstream target to reduce AF burden, and the need to prevent electrical remodeling to avert AF perpetuation.

Modulation of Ca(2+) release in cardiac myocytes by changes in repolarization rate: role of phase-1 action potential repolarization in excitation-contraction coupling.

The early rate of action potential (AP) repolarization varies in the mammalian heart regionally, during development, and in disease. We used confocal microscopy to assess the effects of changes in repolarization rate on spatially resolved sarcoplasmic reticulum (SR) Ca(2+) release. The kinetics and peak amplitude of Ca(2+) transients were reduced, and the amplitude, frequency, and temporal synchronization of Ca(2+) spikes decreased as the rate of repolarization was slowed. The first latencies and temporal dispersion of Ca(2+) spikes tracked closely with the time to peak and the width of the L-type Ca(2+) current (I(Ca,L)), suggesting that the effects of repolarization on excitation-contraction coupling occur primarily via changes in I(Ca,L). Next, we examined the effect of changes in the rapid early repolarization rate (phase 1) of a model human AP on SR Ca(2+) release by varying the amount of transient outward K(+) current. Slowing of phase-1 repolarization also caused a loss of temporal synchrony and recruitment of Ca(2+)-release events, associated with a reduced amplitude and lengthened time to peak of I(Ca,L). Isoproterenol application enhanced and largely resynchronized SR Ca(2+) release, while it increased the magnitude and shortened the time to peak of I(Ca,L). Our data demonstrate that membrane repolarization modulates the recruitment and synchronization of SR Ca(2+) release via I(Ca,L) and illustrate a physiological role for the phase-1 notch of the AP in optimizing temporal summation and recruitment of Ca(2+)-release events. The effects of slowing phase-1 repolarization can be overcome by beta-adrenergic stimulation.

Galectin-3 Regulates Atrial Fibrillation Remodeling and Predicts Catheter Ablation Outcomes.

OBJECTIVES: To determine whether Gal-3 mediates sustained atrial fibrillation (AF)-induced atrial structural and electrical remodeling and contributes to AF perpetuation. BACKGROUND: Galectin-3 (Gal-3) mediates extracellular matrix remodeling in heart failure, but its role in AF progression remains unexplored. METHODS: We examined intracardiac blood samples from patients with AF (N=55) to identify potential biomarkers of AF recurrence. In a sheep model of tachypacing-induced AF (N=20), we tested the effects of Gal-3 inhibition during AF progression. RESULTS: In patients, intracardiac serum Gal-3 levels were greater in persistent than paroxysmal AF and independently predicted atrial tachyarrhythmia recurrences after a single ablation procedure. In the sheep model, both Gal-3 and TGF-ß1 were elevated in the atria of persistent AF animals. The Gal-3 inhibitor GM-CT-01 (GMCT) reduced both Gal-3 and TGF-ß1-induced sheep atrial fibroblast migration and proliferation in vitro. GMCT (12 mg/kg twice/week) prevented the increase in serum procollagen type III N-terminal peptide seen during progression to persistent AF, and also mitigated atrial dilatation, myocyte hypertrophy, fibrosis, and the expected increase in dominant frequency of excitation. Atria of GMCT-treated animals had significantly less TGF-ß1-Smad2/3 signaling pathway activation and expression of α-smooth muscle actin and collagen than saline-treated animals. Ex-vivo hearts from GMCT-treated animals had significantly longer action potential durations and fewer rotors and wavebreaks during AF, and myocytes had lower functional expression of inward rectifier K+ channel (Kir2.3) than saline-treated animals. Importantly, GMCT increased the probability of spontaneous AF termination, decreased AF inducibility and reduced overall AF burden. CONCLUSIONS: Inhibiting Gal-3 during AF progression might be useful as an adjuvant treatment to improve outcomes of catheter ablation for persistent AF. Gal-3 inhibition may be a potential new upstream therapy for prevention of AF progression.

The phosphoinositide 3-kinase inhibitor LY294002 enhances cardiac myocyte contractility via a direct inhibition of Ik,slow currents.

OBJECTIVE: Phosphoinositide 3-kinase (PI3K) is a key component in regulating myocardial growth, survival and contractility. LY294002 and wortmannin are two PI3K inhibitors used widely to establish the role of PI3K. The goal of this study was to examine the effects of acute application of LY294002 and wortmannin on cardiac myocyte contractility and underlying mechanisms. METHODS: Patch-clamp, indo-1 epifluorescence and video-edge detection techniques were used to measure outward K(+) currents, action potentials (AP), Ca(2+) transients and shortening of myocytes isolated from mouse left ventricular free wall. RESULTS: In field-stimulated myocytes, LY294002 (10 micromol/l) increased Ca(2+) transient amplitude by 23%, and cell shortening amplitude by 60% in the absence or presence of wortmannin, while wortmannin alone had no effect. LY294002 (but not wortmannin) prolonged AP duration by specifically inhibiting slowly inactivating K(+) currents (i.e., the 4-aminopyrydine-sensitive I(k,slow1) and the tetraethylammonium-sensitive I(k,slow2)), leading to an increase in sarcoplasmic reticular Ca(2+) levels. It appeared that the AP prolongation was responsible for elevated contractility since AP-clamp of myocytes with prolonged APs (recorded in LY294002-treated myocytes) induced a 29% increase in cell shortening compared with control APs, while LY294002 application did not increase contractility in voltage-clamp studies using either step or AP depolarizations. CONCLUSIONS: The putative PI3K inhibitor LY294002 increases Ca(2+) release and myocyte contractility via direct inhibition of cardiac I(k,slow) and AP prolongation, thus limiting the usefulness of this agent in the analyses of the role of PI3K in heart function.

Cell-selective arrhythmia ablation for photomodulation of heart rhythm.

Heart disease, a leading cause of death in the developed world, is overwhelmingly correlated with arrhythmias, where heart muscle cells, myocytes, beat abnormally. Cardiac arrhythmias are usually managed by electric shock intervention, antiarrhythmic drugs, surgery, and/or catheter ablation. Despite recent improvements in techniques, ablation procedures are still limited by the risk of complications from unwanted cellular damage, caused by the nonspecific delivery of ablative energy to all heart cell types. We describe an engineered nanoparticle containing a cardiac-targeting peptide (CTP) and a photosensitizer, chlorin e6 (Ce6), for specific delivery to myocytes. Specificity was confirmed in vitro using adult rat heart cell and human stem cell-derived cardiomyocyte and fibroblast cocultures. In vivo, the CTP-Ce6 nanoparticles were injected intravenously into rats and, upon laser illumination of the heart, induced localized, myocyte-specific ablation with 85% efficiency, restoring sinus rhythm without collateral damage to other cell types in the heart, such as fibroblasts. In both sheep and rat hearts ex vivo, upon perfusion of CTP-Ce6 particles, laser illumination led to the formation of a complete electrical block at the ablated region and restored the physiological rhythm of the heart. This nano-based, cell-targeted approach could improve ablative technologies for patients with arrhythmias by reducing currently encountered complications.

Elevated heart rate triggers action potential alternans and sudden death. translational study of a homozygous KCNH2 mutation.

BACKGROUND: Long QT syndrome (LQTS) leads to arrhythmic events and increased risk for sudden cardiac death (SCD). Homozygous KCNH2 mutations underlying LQTS-2 have previously been termed "human HERG knockout" and typically express severe phenotypes. We studied genotype-phenotype correlations of an LQTS type 2 mutation identified in the homozygous index patient from a consanguineous Turkish family after his brother died suddenly during febrile illness. METHODS AND RESULTS: Clinical work-up, DNA sequencing, mutagenesis, cell culture, patch-clamp, in silico mathematical modelling, protein biochemistry, confocal microscopy were performed. Genetic analysis revealed a homozygous C-terminal KCNH2 mutation (p.R835Q) in the index patient (QTc ∼506 ms with notched T waves). Parents were I° cousins - both heterozygous for the mutation and clinically unremarkable (QTc ∼447 ms, father and ∼396 ms, mother). Heterologous expression of KCNH2-R835Q showed mildly reduced current amplitudes. Biophysical properties of ionic currents were also only nominally changed with slight acceleration of deactivation and more negative V50 in R835Q-currents. Protein biochemistry and confocal microscopy revealed similar expression patterns and trafficking of WT and R835Q, even at elevated temperature. In silico analysis demonstrated mildly prolonged ventricular action potential duration (APD) compared to WT at a cycle length of 1000 ms. At a cycle length of 350 ms M-cell APD remained stable in WT, but displayed APD alternans in R835Q. CONCLUSION: Kv11.1 channels affected by the C-terminal R835Q mutation display mildly modified biophysical properties, but leads to M-cell APD alternans with elevated heart rate and could precipitate SCD under specific clinical circumstances associated with high heart rates.

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers.