Higher Pacing Frequencies Research Articles

Distinct Electrogram Features and Ventricular Arrhythmia Induction Modes Between Repolarization and Conduction Heterogeneities

BackgroundRecent clinical studies have indicated the presence of localized electrical abnormalities in idiopathic ventricular fibrillation and J-wave syndrome patients. ObjectivesThis study aims to characterize the specific electrical signatures of localized repolarization and conduction heterogeneities and their respective role in vulnerability to arrhythmias. MethodsOptical mapping was performed in porcine right ventricles with local: 1) repolarization shortening; 2) conduction slowing; or 3) structural heterogeneity induced by locally perfusing: 1) pinacidil (20 μmol/L, n = 13); or 2) flecainide (2 μmol/L, n = 13) via an epicardial catheter; or 3) by local epicardial tissue destruction (9 radiofrequency lesions n = 12). Electrograms were recorded (n = 5 in each group) and spontaneous and induced arrhythmias were quantified and optically mapped. ResultsElectrograms were normal in (1) but showed local fragmentation in 40% of preparations in (2) with greater effects observed at high pacing frequencies dependent on the wavefront direction. In (3), the structural substrate alone increased the width and number of peaks in the electrograms, and addition of flecainide induced pronounced fragmentation (≥3 peaks and ≥70 ms) in all cases. Occurrence of spontaneous arrhythmias was significantly increased in (1) and (2) (P < 0.0001 and 0.05, respectively, vs baseline) and were triggered by ectopies. Vulnerability to arrhythmias at high pacing frequencies (≥2 Hz) was the lowest in (1) and greatest in (2). ConclusionsMicrostructural substrates have the most pronounced impact on electrograms, especially when combined with sodium channel blockers, whereas local action potential duration shortening does not lead to electrogram fragmentation even though it is associated with the highest prevalence of spontaneous arrhythmias.

Alterations of local Ca2+ signaling by high pacing frequency and shear stress in right and left atrial myocytes

Alterations of local Ca2+ signaling by high pacing frequency and shear stress in right and left atrial myocytes

Local Control Model of a Human Ventricular Myocyte: An Exploration of Frequency-Dependent Changes and Calcium Sparks.

Calcium (Ca2+) sparks are the elementary events of excitation-contraction coupling, yet they are not explicitly represented in human ventricular myocyte models. A stochastic ventricular cardiomyocyte human model that adapts to intracellular Ca2+ ([Ca2+]i) dynamics, spark regulation, and frequency-dependent changes in the form of locally controlled Ca2+ release was developed. The 20,000 CRUs in this model are composed of 9 individual LCCs and 49 RyRs that function as couplons. The simulated action potential duration at 1 Hz steady-state pacing is ~0.280 s similar to human ventricular cell recordings. Rate-dependence experiments reveal that APD shortening mechanisms are largely contributed by the L-type calcium channel inactivation, RyR open fraction, and [Ca2+]myo concentrations. The dynamic slow-rapid-slow pacing protocol shows that RyR open probability during high pacing frequency (2.5 Hz) switches to an adapted "nonconducting" form of Ca2+-dependent transition state. The predicted force was also observed to be increased in high pacing, but the SR Ca2+ fractional release was lower due to the smaller difference between diastolic and systolic [Ca2+]SR. Restitution analysis through the S1S2 protocol and increased LCC Ca2+-dependent activation rate show that the duration of LCC opening helps modulate its effects on the APD restitution at different diastolic intervals. Ultimately, a longer duration of calcium sparks was observed in relation to the SR Ca2+ loading at high pacing rates. Overall, this study demonstrates the spontaneous Ca2+ release events and ion channel responses throughout various stimuli.

Contractile function of fasting and digesting broad-snouted caiman (Caiman latirostris)

Feeding regimes are a critical process to ensure survival and reproduction, which modulate several behavioral and physiological patterns of ectotherms, especially cardiovascular adjustments. However, crocodilian myocardial contractile function after food intake remains understudied. We investigated the importance of ventricular myocardial contractility during digestion in the broad-snouted caiman, Caiman latirostris Daudin, 1801. Isometric ventricle preparations and Ca2+-handling protein expression were used to investigate the effects of prolonged fasting (30 days) and feeding (48 h after feeding on meals at 15% of body mass) on cardiac function. Upon feeding, there was no significant difference in relative ventricular mass. The cardiac response of the caiman to digestion was characterized by upregulation of the Na+/Ca2+ exchanger expression and an increase in myocardial contractility (positive inotropism and lusitropism) within the physiological range of heart rate. The digesting caiman's ventricular myocardium was able to maintain faster kinetics of contraction and relaxation, allowing for significantly increased cardiac pumping capacity at higher pacing frequencies. Our results demonstrated that the myocardium of broad-snouted caimans exhibits specializations in excitation–contraction coupling, which can provide a means to support high cardiovascular performance during digestion.

Role of localized repolarization and conduction heterogeneities in ventricular arrhythmias and sudden cardiac death

Recent clinical studies have indicated the presence of localized tissue abnormalities in sudden cardiac death (SCD) survivors without apparent structural heart disease, including idiopathic ventricular fibrillation (VF) and J-wave Syndromes. The origin of these abnormalities, showing distinct electrical features on contact mapping, and mechanisms of associated arrhythmias are still a matter of debate. To determine the respective role of localized repolarization and conduction heterogeneities in vulnerability to arrhythmias and SCD, and to characterize their specific electrical signatures. Optical mapping was performed on porcine right ventricular models of either local (i) repolarization shortening, (ii) conduction slowing, or (iii) micro-structural heterogeneity which were created by locally perfusing (i) pinacidil (20 μM n = 13) or (ii) flecainide (2 μM n = 13) via an epicardial catheter (branch of right coronary artery), or (iii) by local epicardial tissue destruction through radiofrequency application (9 micro lesions 30W 1 sec n = 13). Pseudo-ECG were assessed and the number of induced and spontaneous arrhythmias were quantified and mapped in each preparation. An HD grid mapping catheter was used to record bipolar electrograms (n = 5 in each group). Electrograms were normal in model (i), but showed local fragmentation in 40% of preparations in model (ii) with greater effects observed at high pacing frequencies (3 Hz). In model (iii), the structural substrate alone increased the width and number of peaks in the electrograms, and addition of flecainide (2 μM) in this model induced pronounced fragmentation (≥ 3 peaks and ≥ 70ms duration) in 100% of the cases. Arrhythmogenicity was significantly increased in models (i) and (ii) which presented 82% and 45% more spontaneous arrhythmias (P < 0.0001 and 0.05 vs. baseline) and a reduced pacing frequency threshold to induce arrhythmias (−69% and −50%, P < 0.001 vs. baseline). In these models, the frequency of spontaneous activity was increased (+411% P < 0.01 and +181% P < 0.05 vs. baseline) and originated near the heterogeneous area. Microstructural substrates have the most pronounced impact on electrograms, especially when combined with sodium channel blockers, while repolarization heterogeneities do not lead to electrogram fragmentation but are associated with the highest prevalence of spontaneous arrhythmias. Both heterogeneities show increased ectopic activity.

A comprehensive framework for evaluation of high pacing frequency and arrhythmic optical mapping signals.

Introduction: High pacing frequency or irregular activity due to arrhythmia produces complex optical mapping signals and challenges for processing. The objective is to establish an automated activation time-based analytical framework applicable to optical mapping images of complex electrical behavior. Methods: Optical mapping signals with varying complexity from sheep (N = 7) ventricular preparations were examined. Windows of activation centered on each action potential upstroke were derived using Hilbert transform phase. Upstroke morphology was evaluated for potential multiple activation components and peaks of upstroke signal derivatives defined activation time. Spatially and temporally clustered activation time points were grouped in to wave fronts for individual processing. Each activation time point was evaluated for corresponding repolarization times. Each wave front was subsequently classified based on repetitive or non-repetitive events. Wave fronts were evaluated for activation time minima defining sites of wave front origin. A visualization tool was further developed to probe dynamically the ensemble activation sequence. Results: Our framework facilitated activation time mapping during complex dynamic events including transitions to rotor-like reentry and ventricular fibrillation. We showed that using fixed AT windows to extract AT maps can impair interpretation of the activation sequence. However, the phase windowing of action potential upstrokes enabled accurate recapitulation of repetitive behavior, providing spatially coherent activation patterns. We further demonstrate that grouping the spatio-temporal distribution of AT points in to coherent wave fronts, facilitated interpretation of isolated conduction events, such as conduction slowing, and to derive dynamic changes in repolarization properties. Focal origins precisely detected sites of stimulation origin and breakthrough for individual wave fronts. Furthermore, a visualization tool to dynamically probe activation time windows during reentry revealed a critical single static line of conduction slowing associated with the rotation core. Conclusion: This comprehensive analytical framework enables detailed quantitative assessment and visualization of complex electrical behavior.

A Mathematical Model for Electrical Activity in Pig Atrial Tissue.

State of the art mathematical models are currently used to bridge the gap between basic research conducted in the laboratory and preclinical research conducted on large animals, which ultimately paves the way for clinical translation. In this regard, there is a great need for models that can be used alongside experiments for in-depth investigation and validation. One such experimental model is the porcine atrium, which is commonly used to study the mechanisms of onset and control of atrial fibrillation in the context of its surgical management. However, a mathematical model of pig atria is lacking. In this paper, we present the first ionically detailed mathematical model of porcine atrial electrophysiology, at body temperature. The model includes 12 ionic currents, 4 of which were designed based on experimental patch-clamp data directly obtained from literature. The formulations for the other currents are adopted from the human atrial model, and modified for porcine specificity based on our measured restitution data for different action potential characteristics: resting membrane potential, action potential amplitude, maximum upstroke velocity and action potential duration and different levels of membrane voltage repolarization. The intracellular Ca2+ dynamics follows the Luo-Rudy formulation for guinea pig ventricular cardiomyocytes. The resulting model represents “normal” cells which are formulated as a system of ordinary differential equations. We extend our model to two dimensions to obtain plane wave propagation in tissue with a velocity of 0.58 m/s and a wavelength of 8 cm. The wavelength reduces to 5 cm when the tissue is paced at 200 ms. Using S1-S2 cross-field protocol, we demonstrate in an 11.26 cm square simulation domain, the ability to initiate single spiral waves (rotation period ≃ 180 ms) that remain stable for more than 40 s. The spiral tip exhibits hypermeander. In agreement with previous experimental results using pig atria, our model shows that early repolarization is primarily driven by a calcium-mediated chloride current, IClCa, which is completely inactivated at high pacing frequencies. This is a condition that occurs only in porcine atria. Furthermore, the model shows spatiotemporal chaos with reduced repolarization.

Abstract 14180: RyR2 Hyperactivity Contributes to Arrhythmogenic Triggers in the ex vivo Human Right Ventricle

Introduction: Sudden cardiac death (SCD) caused by VT/VF is a major cause of mortality and morbidity, accounting for up to 20% of all deaths in the United States. Novel antiarrhythmic targets and relevant translational models of SCD are urgently needed for basic and translational arrhythmia research, and ex vivo donor human hearts are invaluable resources for such pharmacological studies. It was the goal of this study to investigate the occurrence of VT/VF in excised human hearts and to test a novel pharmacological agent to suppress arrhythmias. Hypothesis: We assessed the hypothesis that RyR2 hyperactivity contributes to arrhythmogenic triggers and substrates in the human RV, and we analyzed the antiarrhythmic effects of pharmacological RyR2 suppression. Methods: Adult human hearts (n = 10; 3F, 7M) were procured from the Washington Regional Transplant Community. The entire RV free wall was isolated, coronary perfused, and placed in an oxygenated, 37°C bath of Tyrode’s solution for optical mapping with the addition of Di-4-ANBDQBS and blebbistatin. Pseduo ECGs were recorded, and action potentials (APs) were spatiotemporally mapped using high-speed CMOS cameras. Optical recordings were taken at baseline and after exposure to the adrenergic agonist isoproterenol (ISO, 250 nM) and the adenosine antagonist caffeine (CAF, 200 μM) to elicit RyR2 hyperactivity. We also evaluated pharmacological RyR2 suppression (2 μM dantrolene) on arrhythmia inhibition. Results: ISO and ISO+CAF significantly shortened tissue APDs. ISO+CAF increased the incidence of PVCs at low pacing frequencies as well as increased the incidence of VT/VF at high pacing frequencies. We observed non-sustained arrhythmias in roughly a third of the hearts examined. No significant differences in conduction velocities were observed. While dantrolene did not return APDs to baseline conditions, it did significantly reverse the effect of caffeine on inducing PVCs. Conclusions: In conclusion, caffeine contributes to arrhythmogenic triggers in the ex vivo human right ventricle, and dantrolene may be used as a pharmacological tool to suppress RyR2 hyperactivity and thus prevent SCD.

Altered spatio-specific CaMKII activation in autophagy deficient mice

Abstract Background Autophagy is linked to preventing the development of cardiac hypertrophy and failure. While aberrant activation of Ca2+/calmodulin-dependent kinase II (CaMKII) promotes myocardial remodeling, the role of autophagy in maintaining cardiac Ca2+ homeostasis and regulating CaMKII signaling is unknown. Objective To test whether loss of autophagy promotes subcellular alterations in CaMKII activation in early myocardial remodelling, and whether compromised in vivo cardiac function parallels those changes. Methods Young (10–15 weeks) cardiomyocyte-specific autophagy protein 5-deficient mice (Atg5−/−) mice and their littermate controls (Atg5+/+) underwent comprehensive in vivo phenotyping using echocardiography, exercise tolerance and hemodynamic stress testing. In vitro assessment included gravimetry, qPCR of hypertrophy marker genes and cellular and nuclear dimensions of isolated ventricular myocytes. CaMKII activation was studied by immunocytochemistry in cardiomyocytes upon exposure to basal (1Hz) or high (4Hz) pacing frequency. Autophosphorylated CaMKII (pT286) signal was evaluated in different subcellular spaces (i.e. cytoplasm, nucleoplasm and nuclear envelope). Results Before symptomatic cardiac dysfunction occurred, Atg5−/− mice showed compromised cardiac reserve in response to β-adrenergic stimulation (dp/dt max: 9475±126 vs 7364±496 mmHg/s, N=4–5; p=0.041), despite similar maximum heart rate. Consequently, effort intolerance (distance run: 251±22 vs 152±13 m, N=8; p=0.03) and maximal oxygen consumption (2093±66 vs 1763±131 ml/h/kg, N=8; p=0.04) were reduced during treadmill exercise tolerance testing. Increased heart-to-body weight ratio (8.1±0.5 vs 10.2±0.8 N=9; p=0.017) was associated with elevated mRNA expression of hypertrophy marker NppB (278% of Atg5+/+, N=5; p=0.016) in Atg5−/− mice, which showed enlarged cardiomyocytes and nuclei, as width-to-length ratio. Because Atg5−/− cardiomyocytes exhibit elevated nuclear Ca2+ levels at high pacing frequency, we now measured subcellular CaMKII activation under the same experimental conditions. Interestingly, at 1Hz, p-CaMKII was increased specifically at the nuclear envelope (154% of Atg5+/+, N=5 mice, 153–159 cells; p=0.029), but not in the cytoplasm or nucleoplasm. Increasing pacing frequency to 4Hz did not alter p-CaMKII levels in Atg5+/+ cells. However, p-CaMKII was increased by ∼30% and ∼20% in the cytoplasm and nucleoplasm of Atg5−/− cells respectively (N=5 mice, 153–155 cells). Conclusion Loss of ATG5-dependent autophagy causes cardiac hypertrophy and impaired cardiac reserve upon acute stress, which involves CaMKII activation, likely through the imbalance of nuclear Ca2+ load. Although, selective increase in p-CaMKII at the nuclear envelope in Atg5−/− mice may temporarily protect from nuclear Ca2+ overload, excessive CaMKII activation in the cytoplasm and the nucleoplasm upon increased workload, likely drives hypertrophic signalling toward heart failure in autophagy-defective mice. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund (FWF)

Autophagy-related gene 5 (Atg5) is required for maintaining subcellular calcium homeostasis during acute cardiac stress

Abstract Background Autophagy exerts protective effects during cardiac stress inflicted by β-adrenergic stimulation and pressure overload. Contrarily, deletion of the cardiac-specific Atg5 gene contributes to left ventricular hypertrophy and contractile dysfunction, resulting in dilated cardiomyopathy. Heart failure is characterized by perturbations of excitation-contraction coupling (ECC) underlying alterations in intracellular calcium signaling. Purpose In this study, we aimed to understand whether loss of basal autophagy due to Atg5 deletion specifically in cardiomyocytes contributes directly to disturbances in subcellular calcium cycling. Methods Cardiac-specific Atg5−/− and Atg5+/+ mice were used to isolate ventricular cardiomyocytes, which were loaded with 8 μM Ca2+ indicator Fluo4/AM. Isolated cells were electrically stimulated and subjected to elevated workload by increasing pacing frequencies from 1 to 4 Hz. Cytosolic and nuclear calcium transients (CaTs) were recorded in line-scan mode using a laser scanning confocal microscope. Pharmacological inhibition of autophagic flux was performed by an intraperitoneal injection of 40 mg/kg leupeptin to Atg5+/+ mice. Results At baseline (1 Hz) stimulation the amplitude and kinetics of calcium transients were comparable between Atg5−/− and Atg5+/+ cells. However, after progressive increase of the pacing rate from 1 Hz to 4 Hz, Atg5−/− cardiomyocytes displayed reduced cytoplasmic and nuclear CaT amplitude, but increased nuclear time-averaged CaTs compared to controls. Such increase in nuclear calcium load stimulates Ca2+-dependent transcriptional activity, and likely involves CaMKII-mediated pro-hypertrophic gene program thus contributing to cardiac remodelling. In addition, we observed increased occurrence of arrhythmic events in Atg5−/− hearts at high pacing frequency, indicative of intracellular calcium overload and disturbed ECC. Acute pharmacological autophagy inhibition failed to elicit direct changes in time-averaged CaTs in the cytoplasm and nucleoplasm, suggesting that loss of Atg5-dependent autophagy seems to induce adverse cardiac remodelling that involves increased CaMKII activity due to the imbalances in nuclear Ca2+ levels. Conclusion Specific loss of ATG5 protein impairs calcium cycling during acute stress exposure by reducing CaT amplitudes, promoting arrhythmia, and increasing nuclear calcium load, which may induce hypertrophic gene expression. Further work is needed to identify key components mediating autophagy-related cardioprotection to develop strategies against cardiac remodeling and its progression to heart failure. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund (FWF)

Altered atrial restitution dynamics and refractoriness in metabolic syndrome due to up-regulation of potassium repolarizing currents increases susceptibility to atrial fibrillation

Abstract Background Metabolic alterations, such as Metabolic Syndrome (MS), describe an association of factors including diabetes, hypertension, obesity and dyslipidemia, linked to higher risk and prevalence of overall cardiovascular disease, arrhythmogenesis and sudden cardiac death. Obese and diabetic patients have shown an increased risk for developing atrial fibrillation (AF). However, underlying mechanisms are not understood. Purpose To study the effects of MS and obesity remodeling in atrial restitution dynamics, frequency-dependent adaptation, refractoriness and its potential susceptibility to AF. Methods Electrophysiological experimental data from High-fat (HF-O, standard rabbit chow with an additional 15% fat) and Hig-fat High-Sucrose Metabolic Syndrome (HFHS-MS, 10% hydrogenated coconut oil and 5% lard, 15% high-sucrose dissolved in water) rabbit models were used to adjust computational models atrial electrophysiology remodeling under each condition. Additionally, isoproterenol and AF conditions were considered to challenge both in-silico models. Validation and sensitivity analysis were performed for each model parameters. Computational simulations in conditions of pacing at different pacing cycle lengths was assessed at 100, 125, 150, 200, 250, 350, 450, 500, 650, 750, 850, 1000 ms. Restitution dynamics were automatically determined and analyzed, as well as restitution slopes and presence of automaticity, early after-depolarizations, alternans and cardiac arrhythmia induction. Results Shortening of action potential duration and refractoriness in the left atrium was observed under HFHS-MS. Upstroke velocity, maximum excitability and sodium availability were altered both in HF-O and HFHS-MS. HF-O remodeling showed presence of alternans at high pacing frequencies. Repolarization restitution was shortened in conditions of ISO and MS-AF. Restitution slopes were &amp;gt;1 in HF-O and HFHS-MS, which was correlated to higher susceptibility to AF, and further increased in MS-AF. Under MS-AF, abbreviation in APD in both atria, resulted in increased reentrant frequencies in AF, which was exacerbated under IK1 up-regulation, increasing atrial vulnerability. Conclusions HFHS-MS underlies modifications in atrial electrophysiology including shorter refractoriness in HFHS-MS, as well as modifications in atrial excitability, which may be pro-arrhythmic mainly at high frequency rates. This could be explained in part by an up-regulation of outward potassium channels. These results could partially explain increased susceptibility for AF in MS. Funding Acknowledgement Type of funding sources: None.

Effects of change in temperature on the cardiac contractility of broad-snouted caiman (Caiman latirostris) during digestion.

In many reptiles, digestion has been associated with the selection of higher body temperatures, the so-called post-prandial thermophilic response. This study aimed to investigate the excitation-contraction (E-C) coupling in postprandial broad-snouted caimans (Caiman latirostris) in response to acute warming within a preferred body temperature range of crocodiles. Isometric preparations subjected to a temperature transition from 25°C to 30°C were used to investigate myocardial contractility of postprandial caimans, that is, 48 h after the animals ingested a rodent meal corresponding to 15% of body mass. The caiman heart exhibits a negative force-frequency relationship that is independent of the temperature. At 25°C, cardiac muscle was able to maintain a constant force up to 36 bpm, above which it decreased significantly, reaching minimum values at the highest frequency of 84 bpm. Moreover, E-C coupling is predominantly dependent on transsarcolemmal Ca2+ transport denoted by the lack of significant ryanodine effects on force generation. On the contrary, ventricular strips at 30°C were able to sustain the cardiac contractility at higher pacing frequencies (from 12 to 144 bpm) due to an important role of Na+ /Ca2+ exchanger in Ca2+ cycling, as indicated by the decay of the post-rest contraction, and a significant contribution of the sarcoplasmic reticulum above 72 bpm. Our results demonstrated that the myocardium of postprandial caimans exhibits a significant degree of thermal plasticity of E-C coupling during acute warming. Therefore, myocardial contractility can be maximized when postprandial broad-snouted caimans select higher body temperatures (preferred temperature zone) following feeding.

CaMKII and PKA-dependent phosphorylation co-regulate nuclear localization of HDAC4 in adult cardiomyocytes

Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII—but not PKA, PKC or PKD—raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

Balance Between Rapid Delayed Rectifier K+ Current and Late Na+ Current on Ventricular Repolarization: An Effective Antiarrhythmic Target?

Rapid delayed rectifier K+ current (IKr) and late Na+ current (INaL) significantly shape the cardiac action potential (AP). Changes in their magnitudes can cause either long or short QT syndromes associated with malignant ventricular arrhythmias and sudden cardiac death. Physiological self AP-clamp was used to measure INaL and IKr during the AP in rabbit and porcine ventricular cardiomyocytes to test our hypothesis that the balance between IKr and INaL affects repolarization stability in health and disease conditions. We found comparable amount of net charge carried by IKr and INaL during the physiological AP, suggesting that outward K+ current via IKr and inward Na+ current via INaL are in balance during physiological repolarization. Remarkably, IKr and INaL integrals in each control myocyte were highly correlated in both healthy rabbit and pig myocytes, despite high overall cell-to-cell variability. This close correlation was lost in heart failure myocytes from both species. Pretreatment with E-4031 to block IKr (mimicking long QT syndrome 2) or with sea anemone toxin II to impair Na+ channel inactivation (mimicking long QT syndrome 3) prolonged AP duration (APD); however, using GS-967 to inhibit INaL sufficiently restored APD to control in both cases. Importantly, INaL inhibition significantly reduced the beat-to-beat and short-term variabilities of APD. Moreover, INaL inhibition also restored APD and repolarization stability in heart failure. Conversely, pretreatment with GS-967 shortened APD (mimicking short QT syndrome), and E-4031 reverted APD shortening. Furthermore, the amplitude of AP alternans occurring at high pacing frequency was decreased by INaL inhibition, increased by IKr inhibition, and restored by combined INaL and IKr inhibitions. Our data demonstrate that IKr and INaL are counterbalancing currents during the physiological ventricular AP and their integrals covary in individual myocytes. Targeting these ionic currents to normalize their balance may have significant therapeutic potential in heart diseases with repolarization abnormalities. Visual Overview: A visual overview is available for this article.

End-diastolic force pre-activates cardiomyocytes and determines contractile force: role of titin and calcium.

Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control the length of titin. We hypothesized that together with diastolic [Ca2+], titin‐based stretch pre‐activates cardiomyocytes during diastole and is a major determinant of force production in the subsequent contraction. Through this mechanism titin would play an important role in active force development and length‐dependent activation. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) result in expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work loops that mimic the cardiac cycle in wild‐type (WT) and heterozygous (HET) RBM20‐deficient rats. In addition, we studied the role of diastolic [Ca2+] in membrane‐permeabilized WT and HET cardiomyocytes. Intact cardiomyocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT) at low pacing frequencies, but this difference disappeared at high pacing frequencies. Length‐dependent activation (force–sarcomere length relationship) was blunted in HET cardiomyocytes, but the force–end‐diastolic force relationship was not different between HET and WT cardiomyocytes. To delineate the effects of diastolic [Ca2+] and titin pre‐activation on force generation, measurements were performed in detergent‐permeabilized cardiomyocytes. Cardiac twitches were simulated by transiently exposing permeabilized cardiomyocytes to 2 µm Ca2+. Increasing diastolic [Ca2+] from 1 to 80 nm increased force development twofold in WT. Higher diastolic [Ca2+] was needed in HET. These findings are consistent with our hypothesis that pre‐activation increases active force development. Highly compliant titin allows cells to function at higher diastolic [Ca2+].

Early Afterdepolarizations and Alternans are the Underlying Mechanism to Cause Arrhythmogenic Disorder in the Mutant Calsequestrin 2 (CASQ2)

Calsequestrin 2 (CASQ2) is high capacity and low-affinity Ca2+-binding protein expressed in the sarcoplasmic reticulum (SR) of cardiac myocyte. Mutation that occurs in CASQ2 expressing gene have been linked to arrhythmia and possibly sudden cardiac death (SCD) with acute emotional stress or exercise. This inherited genetic disorder is known as catecholamine polymorphic ventricular tachycardia (CPVT). Understanding the subcellular mechanisms of CPVT is experimentally challenging because the arrhythmias are rare and the development of SCD are even rarer. The underlying causes of CPVT have been described as delayed afterdepolarizations (DADs), early afterdepolarizations (EADs) or Ca2+ signaling alternans. To gain insight into the nature of this rare disease, we have developed a local control stochastic model of the cardiac myocyte to investigate how the mutant CASQ2s may be responsible for the development of an arrhythmogenic episode under the condition of beta-adrenergic stimulation or in the pauses afterward. We choose a long action potential model to contrast with our previous studies with the short action potential rat model. Experimental observation indicate that the mutation is accompanied by increased ryanodine receptor open probability and increased junctional sarcoplasmic reticulum volume. The model simulates EADs at low pacing frequency. At high pacing frequency, alternans occurs not due to spark restitution as seen in the rat model, but by the accumulated inactivation of the sodium channel. These alternans do not require changes in jSR volume which were important in the spark restitution-based mechanism. The model offers novel insights into how mechanisms can vary between a long- and short- action potential models of arrhythmia.

Beta1-adrenoceptor antagonist, metoprolol attenuates cardiac myocyte Ca2+ handling dysfunction in rats with pulmonary artery hypertension

Right heart failure is the major cause of death in Pulmonary Artery Hypertension (PAH) patients but is not a current, specific therapeutic target. Pre-clinical studies have shown that adrenoceptor blockade can improve cardiac function but the mechanisms of action within right ventricular (RV) myocytes are unknown. We tested whether the β1–adrenoceptor blocker metoprolol could improve RV myocyte function in an animal model of PAH, by attenuating adverse excitation-contraction coupling remodeling. PAH with RV failure was induced in rats by monocrotaline injection. When PAH was established, animals were given 10 mg/kg/day metoprolol (MCT + BB) or vehicle (MCT). The median time to the onset of heart failure signs was delayed from 23 days (MCT), to 31 days (MCT + BB). At 23 ± 1 days post-injection, MCT + BB showed improved in vivo cardiac function, measured by echocardiography. RV hypertrophy was reduced despite persistent elevated afterload. RV myocyte contractility during field stimulation was improved at higher pacing frequencies in MCT + BB. Preserved t-tubule structure, more uniform evoked Ca2+ release, increased SERCA2a expression and faster ventricular repolarization (measured in vivo by telemetry) may account for the improved contractile function. Sarcoplasmic reticulum Ca2+ overload was prevented in MCT + BB myocytes resulting in fewer spontaneous Ca2+ waves, with a lower pro-arrhythmic potential. Our novel finding of attenuation of defects in excitation contraction coupling by β1–adrenoceptor blockade with delays in the onset of HF, identifies the RV as a promising therapeutic target in PAH. Moreover, our data suggest existing therapies for left ventricular failure may also be beneficial in PAH induced RV failure.

Pre-Activation of Cardiomyocytes Determines Contractile Force and Speed of Contraction; Role of Titin and Calcium

Cardiomyocytes have, through splicing, an exquisite control over the length of titin, enabling it to regulate passive stiffness. As titin sets the preload of the cardiomyocyte, we hypothesized that together with diastolic Ca2+ it pre-activates the cardiomyocyte during diastole and that this is a major determinant of force production in the subsequent systolic phase. Through this mechanism titin would play an important role in active force development and length-dependent activation. Mutations in the splicing factor RNA binding motif protein (RBM20) results in the expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work-loops that mimic the cardiac cycle in wildtype (WT) and heterozygous (HET) RBM20 deficient rats, with long compliant titin. In addition we studied membrane-permeabilized cardiomyocytes with different amounts of diastolic calcium. At low pacing frequencies, myocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT), but this difference disappeared when diastolic calcium increased at high pacing frequencies. HET myocytes operated at higher SL to achieve the same level of work (2.10 µm vs. 1.94 µm at 6 Hz). To delineate the effects of diastolic Ca2+ and titin pre-activation on force generation, measurements were performed in detergent-permeabilized cardiomyocytes isolated from rat hearts. In these cells cardiac twitches were simulated by transiently (1 s) exposing the cell to a Ca2+-concentration of 2 µM. Increasing pre-activation by pre-stretching the myocyte increased the kinetics of force development and total force development. Increasing diastolic Ca2+ did increase force development, but only within a small concentration range. These findings are consistent with our hypothesis that pre-activation can increase force development. Highly compliant titin allows cells to function at higher diastolic Ca2+, but this is expected to result in limited contractile reserve.

Increased passive stiffness promotes diastolic dysfunction despite improved Ca2+ handling during left ventricular concentric hypertrophy.

AimsConcentric hypertrophy following pressure-overload is linked to preserved systolic function but impaired diastolic function, and is an important substrate for heart failure with preserved ejection fraction. While increased passive stiffness of the myocardium is a suggested mechanism underlying diastolic dysfunction in these hearts, the contribution of active diastolic Ca2+ cycling in cardiomyocytes remains unclear. In this study, we sought to dissect contributions of passive and active mechanisms to diastolic dysfunction in the concentrically hypertrophied heart following pressure-overload.Methods and resultsRats were subjected to aortic banding (AB), and experiments were performed 6 weeks after surgery using sham-operated rats as controls. In vivo ejection fraction and fractional shortening were normal, confirming preservation of systolic function. Left ventricular concentric hypertrophy and diastolic dysfunction following AB were indicated by thickening of the ventricular wall, reduced peak early diastolic tissue velocity, and higher E/e’ values. Slowed relaxation was also observed in left ventricular muscle strips isolated from AB hearts, during both isometric and isotonic stimulation, and accompanied by increases in passive tension, viscosity, and extracellular collagen. An altered titin phosphorylation profile was observed with hypophosphorylation of the phosphosites S4080 and S3991 sites within the N2Bus, and S12884 within the PEVK region. Increased titin-based stiffness was confirmed by salt-extraction experiments. In contrast, isolated, unloaded cardiomyocytes exhibited accelerated relaxation in AB compared to sham, and less contracture at high pacing frequencies. Parallel enhancement of diastolic Ca2+ handling was observed, with augmented NCX and SERCA2 activity and lowered resting cytosolic [Ca2+].ConclusionIn the hypertrophied heart with preserved systolic function, in vivo diastolic dysfunction develops as cardiac fibrosis and alterations in titin phosphorylation compromise left ventricular compliance, and despite compensatory changes in cardiomyocyte Ca2+ homeostasis.

Pre-Activation of Cardiomyocytes Determines Speed of Contraction: Role of Titin

The giant myofilament protein titin has an extendable region that functions as a molecular spring. Cardiomyocytes have exquisite control over the length of titin, through splicing, enabling it to regulate passive stiffness. We hypothesized that titin as it sets the preload on the cardiomyocyte when stretched, together with diastolic Ca2+ pre-activates the cardiomyocyte during diastole and that this pre-activation is a major determinant for force production in the subsequent systolic phase. Via this route titin is thought to play an important role in active force development. Mutations in the splicing factor RNA binding motif protein (RBM20) results in the expression of large, highly compliant titin isoforms. In the present study we aimed to investigate the effect of long, highly compliant titin on the contractile properties of single cardiomyocytes. We measured single cardiomyocyte work-loops that mimic the cardiac cycle, in wildtype (WT) and heterozygous (HET) RBM20 deficient rats. In addition we studied detergent-permeabilized human patient samples that had known variations in titin based passive stiffness. At low pacing frequencies, myocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT), but this difference disappeared when diastolic calcium increased at high pacing frequencies (>6 Hz). HET myocytes operated at higher SL to achieve the same level of work (2.1µm vs. 1.94µm at 6 Hz). In detergent-permeabilized cardiomyocytes isolated from human and rat heart we simulated cardiac twitches by transiently (0.5 s) exposing the cell to a physiological calcium concentration of pCa 5.7. Increasing pre-activation by bathing the cells in pCa 6.7 or pre-stretching the myocyte increased the kinetics of force development and thus the total force development within a transient activation. This is consistent with our hypothesis that pre-activation can increase force development in a time limited contraction such as a cardiac twitch. Pre-activation was pre-load dependent as the sarcomere length to which the myocytes had to be stretched for equivalent levels of pre-activation varied with the compliance of titin.