Electrical Pacing Research Articles

The glutamic acid-rich–long C-terminal extension of troponin T has a critical role in insect muscle functions

The troponin complex regulates the Ca2+ activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500-700 million years ago during early animal evolution. Troponin T (TnT) is the thin-filament-anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and they also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly-diverged structure consisting of a long glutamic acid-rich C-terminal extension of ∼70 residues with unknown function. We found here that C-terminally truncated Drosophila TnT (TpnT-CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnTCD70 gene residing on the X chromosome resulted in lethality in male flies. We demonstrate that this X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific mAb indicated expression of TpnT-CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and EM examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT-CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca2+ overloading induced by high-frequency electrical pacing. Our findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca2+ buffer/reservoir and is potentially critical to the high-frequency asynchronous contraction of flight muscles.

Effectiveness and safety of lidocaine use (review)

Cardiac arrhythmias are complications of many cardiovascular diseases. They quite often acquire an unpredictable course and can result in the death. According to epidemiology data, heart rhythm disorders lead to the sudden death of about 200 thousand people in the Russian Federation annually. Sudden cardiac death overtakes about 400 thousand outpatients and hospital patients in the USA and about 280 thousand patients in Europe. The unpredictability of the occurrence and the transience of cardiac arrhythmias require urgent measures, including both non-pharmacological (electrical pacing, electrical shock therapy) and pharmacotherapeutic methods of treatment. This review is devoted to the study of the clinical features of the lidocaine infusions in patients with ventricular arrhythmias caused by an increase of the conducting cardiomyocytes automatism. The article presents the results of comparative effectiveness and safety studies of lidocaine prescribed for the treatment of ventricular tachycardia and ventricular fibrillation (in the absence of the effect of electro-impulse therapy). Lidocaine is compared with other antiarrhythmic drugs such as amiodarone, sotalol, bretylium tosylate and procainamide. Recommendations of ILCOR (International Committee for Interaction in the Field of Resuscitation), AHA (American Cardiology Association), ECS (European Cardiology Society) and European Society for Resuscitation for the treatment of refractory forms of ventricular arrhythmias and ventricular fibrillation are also discussed.

Examining Cardiomyocyte Dysfunction Using Acute Chemical Induction of an Ageing Phenotype

Much effort is focussed on understanding the structural and functional changes in the heart that underlie age-dependent deterioration of cardiac performance. Longitudinal studies, using aged animals, have pinpointed changes occurring to the contractile myocytes within the heart. However, whilst longitudinal studies are important, other experimental approaches are being advanced that can recapitulate the phenotypic changes seen during ageing. This study investigated the induction of an ageing cardiomyocyte phenotypic change by incubation of cells with hydroxyurea for several days ex vivo. Hydroxyurea incubation has been demonstrated to phenocopy age- and senescence-induced changes in neurons, but its utility for ageing studies with cardiac cells has not been examined. Incubation of neonatal rat ventricular myocytes with hydroxyurea for up to 7 days replicated specific aspects of cardiac ageing including reduced systolic calcium responses, increased alternans and a lesser ability of the cells to follow electrical pacing. Additional functional and structural changes were observed within the myocytes that pointed to ageing-like remodelling, including lipofuscin granule accumulation, reduced mitochondrial membrane potential, increased production of reactive oxygen species, and altered ultrastructure, such as mitochondria with disrupted cristae and disorganised myofibres. These data highlight the utility of alternative approaches for exploring cellular ageing whilst avoiding the costs and co-morbid factors that can affect longitudinal studies.

Glial cells influence cardiac permittivity as evidenced through in vitro and in silico models

Excitation–contraction (EC) coupling in the heart has, until recently, been solely accredited to cardiomyocytes. The inherent complexities of the heart make it difficult to examine non-muscle contributions to contraction in vivo, and conventional in vitro models fail to capture multiple features and cellular heterogeneity of the myocardium. Here, we report on the development of a 3D cardiac μTissue to investigate changes in the cellular composition of native myocardium in vitro. Cells are encapsulated within micropatterned gelatin-based hydrogels formed via visible light photocrosslinking. This system enables spatial control of the microarchitecture, perturbation of the cellular composition, and functional measures of EC coupling via video microscopy and a custom algorithm to quantify beat frequency and degree of coordination. To demonstrate the robustness of these tools and evaluate the impact of altered cell population densities on cardiac μTissues, contractility and cell morphology were assessed with the inclusion of exogenous non-myelinating Schwann cells (SCs). Results demonstrate that the addition of exogenous SCs alter cardiomyocyte EC, profoundly inhibiting the response to electrical pacing. Computational modeling of connexin-mediated coupling suggests that SCs impact cardiomyocyte resting potential and rectification following depolarization. Cardiac μTissues hold potential for examining the role of cellular heterogeneity in heart health, pathologies, and cellular therapies.

Adult human cardiac stem cell supplementation effectively increases contractile function and maturation in human engineered cardiac tissues

BackgroundDelivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium.MethodsHuman induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0 Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel.ResultsThe hCSC-supplemented hECTs displayed a twofold higher developed force vs. hCM-only controls by day 6, with approximately threefold higher developed stress and maximum rates of contraction and relaxation during pacing at 0.75 Hz. The spontaneous beat rate characteristics were similar between groups, and hCSC supplementation did not adversely impact beat rate variability. The increased contractility persisted through days 8 and 10, albeit with some decrease in the magnitude of the difference of the force by day 10, but with developed stress still significantly higher in hCSC-supplemented hECT; these findings were confirmed with multiple hCSC and hCM cell lines. The force-frequency relationship, while negative for both, control (− 0.687 Hz− 1; p = 0.013 vs. zero) and hCSC-supplemented (− 0.233 Hz− 1;p = 0.067 vs. zero) hECTs, showed a significant rectification in the regression slope in hCSC-supplemented hECT (p = 0.011 vs. control). Targeted gene exploration (59 genes) identified a total of 14 differentially expressed genes, with increases in the ratios of MYH7/MHY6, MYL2/MYL7, and TNNI3/TNNI1 in hCSC-supplemented hECT versus controls.ConclusionsFor the first time, hCSC supplementation was shown to significantly improve human cardiac tissue contractility in vitro, without evidence of proarrhythmic effects, and was associated with increased expression of markers of cardiac maturation. These findings provide new insights about adult cardiac stem cells as contributors to functional improvement of human myocardium.

Genomics of Early Cardiac Dysfunction and Mortality in Obese Drosophila melanogaster.

In experimental evolution, we impose functional demands on laboratory populations of model organisms using selection. After enough generations of such selection, the resulting populations constitute excellent material for physiological research. An intense selection regime for increased starvation resistance was imposed on 10 large outbred Drosophila populations. We observed the selection responses of starvation and desiccation resistance, metabolic reserves, and heart robustness via electrical pacing. Furthermore, we sequenced the pooled genomes of these populations. As expected, significant increases in starvation resistance and lipid content were found in our 10 intensely selected SCO populations. The selection regime also improved desiccation resistance, water content, and glycogen content among these populations. Additionally, the average rate of cardiac arrests in our 10 obese SCO populations was double the rate of the 10 ancestral CO populations. Age-specific mortality rates were increased at early adult ages by selection. Genomic analysis revealed a large number of single nucleotide polymorphisms across the genome that changed in frequency as a result of selection. These genomic results were similar to those obtained in our laboratory from less direct selection procedures. The combination of extensive genomic and phenotypic differentiation between these 10 populations and their ancestors makes them a powerful system for the analysis of the physiological underpinnings of starvation resistance.

Cardiac overexpression of perilipin 2 induces atrial steatosis, connexin 43 remodeling, and atrial fibrillation in aged mice

Atrial fibrillation (AF) is prevalent in patients with obesity and diabetes, and such patients often exhibit cardiac steatosis. Since the role of cardiac steatosis per se in the induction of AF has not been elucidated, the present study was designed to explore the relation between cardiac steatosis and AF. Transgenic (Tg) mice with cardiac-specific overexpression of perilipin 2 (PLIN2) were housed in the laboratory for more than 12 mo before the study. Electron microscopy of the atria of PLIN2-Tg mice showed accumulation of small lipid droplets around mitochondrial chains, and five- to ninefold greater atrial triacylglycerol (TAG) content compared with wild-type (WT) mice. Electrocardiography showed significantly longer RR intervals in PLIN2-Tg mice than in WT mice. Transesophageal electrical burst pacing resulted in significantly higher prevalence of sustained (>5 min) AF (69%) in PLIN2-Tg mice than in WT mice (24%), although it was comparable in younger (4-mo-old) mice. Connexin 43 (Cx43), a gap junction protein, was localized at the intercalated disks in WT atria but was heterogeneously distributed on the lateral side of cardiomyocytes in PLIN2-Tg atria. Langendorff-perfused hearts using the optical mapping technique showed slower and heterogeneous impulse propagation in PLIN2-Tg atria compared with WT atria. Cardiac overexpression of hormone-sensitive lipase in PLIN2-Tg mice resulted in atrial TAG depletion and amelioration of AF susceptibility. The results suggest that PLIN2-induced steatosis is associated with Cx43 remodeling, impaired conduction propagation, and higher incidence of AF in aged mice. Therapies targeting cardiac steatosis could be potentially beneficial against AF in patients with obesity or diabetes.

Modular design of a tissue engineered pulsatile conduit using human induced pluripotent stem cell-derived cardiomyocytes

Single ventricle heart defects (SVDs) are congenital disorders that result in a variety of complications, including increased ventricular mechanical strain and mixing of oxygenated and deoxygenated blood, leading to heart failure without surgical intervention. Corrective surgery for SVDs are traditionally handled by the Fontan procedure, requiring a vascular conduit for completion. Although effective, current conduits are limited by their inability to aid in pumping blood into the pulmonary circulation. In this report, we propose an innovative and versatile design strategy for a tissue engineered pulsatile conduit (TEPC) to aid circulation through the pulmonary system by producing contractile force. Several design strategies were tested for production of a functional TEPC. Ultimately, we found that porcine extracellular matrix (ECM)-based engineered heart tissue (EHT) composed of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and primary cardiac fibroblasts (HCF) wrapped around decellularized human umbilical artery (HUA) made an efficacious basal TEPC. Importantly, the TEPCs showed effective electrical and mechanical function. Initial pressure readings from our TEPC in vitro (0.68 mmHg) displayed efficient electrical conductivity enabling them to follow electrical pacing up to a 2 Hz frequency. This work represents a proof of principle study for our current TEPC design strategy. Refinement and optimization of this promising TEPC design will lay the groundwork for testing the construct's therapeutic potential in the future. Together this work represents a progressive step toward developing an improved treatment for SVD patients. Statement of significanceSingle Ventricle Cardiac defects (SVD) are a form of congenital disorder with a morbid prognosis without surgical intervention. These patients are treated through the Fontan procedure which requires vascular conduits to complete. Fontan conduits have been traditionally made from stable or biodegradable materials with no pumping activity. Here, we propose a tissue engineered pulsatile conduit (TEPC) for use in Fontan circulation to alleviate excess strain in SVD patients. In contrast to previous strategies for making a pulsatile Fontan conduit, we employ a modular design strategy that allows for the optimization of each component individually to make a standalone tissue. This work sets the foundation for an in vitro, trainable human induced pluripotent stem cell based TEPC.

Chronic intermittent tachypacing by an optogenetic approach induces arrhythmia vulnerability in human engineered heart tissue.

AimsChronic tachypacing is commonly used in animals to induce cardiac dysfunction and to study mechanisms of heart failure and arrhythmogenesis. Human induced pluripotent stem cells (hiPSC) may replace animal models to overcome species differences and ethical problems. Here, 3D engineered heart tissue (EHT) was used to investigate the effect of chronic tachypacing on hiPSC-cardiomyocytes (hiPSC-CMs).Methods and resultsTo avoid cell toxicity by electrical pacing, we developed an optogenetic approach. EHTs were transduced with lentivirus expressing channelrhodopsin-2 (H134R) and stimulated by 15 s bursts of blue light pulses (0.3 mW/mm2, 30 ms, 3 Hz) separated by 15 s without pacing for 3 weeks. Chronic optical tachypacing did not affect contractile peak force, but induced faster contraction kinetics, shorter action potentials, and shorter effective refractory periods. This electrical remodelling increased vulnerability to tachycardia episodes upon electrical burst pacing. Lower calsequestrin 2 protein levels, faster diastolic depolarization (DD) and efficacy of JTV-519 (46% at 1 µmol/L) to terminate tachycardia indicate alterations of Ca2+ handling being part of the underlying mechanism. However, other antiarrhythmic compounds like flecainide (69% at 1 µmol/L) and E-4031 (100% at 1 µmol/L) were also effective, but not ivabradine (1 µmol/L) or SEA0400 (10 µmol/L).ConclusionWe demonstrated a high vulnerability to tachycardia of optically tachypaced hiPSC-CMs in EHT and the effective termination by ryanodine receptor stabilization, sodium or hERG potassium channel inhibition. This new model might serve as a preclinical tool to test antiarrhythmic drugs increasing the insight in treating ventricular tachycardia.

P6593Comparison of the termination effects between multi-segment continuous illumination and anti-tachycardia pacing on myocardial infarction ventricular arrhythmia

Abstract Introduction Anti-tachycardia pacing (ATP) therapy is an important treatment for current ICD termination of ventricular arrhythmia (VA). But there was concern about excessive frequency and voltage can cause arrhythmia, pain and damage. Optogenetic allowed modulation of electrical excitation with light in myocardium over-expressing light-sensitive protein, and optical illumination developed a non-contact and pain-free approach to research and treatment of cardiac disorder. Purpose Explore the effects of optogenetic termination and electric termination of myocardial infarction VA in vivo. Methods Rats (N=10) accepted jugular vein injection with 1×1012 vector genomes of (AAV-CAG-hChR2(H134R)-mcherry) to achieve cardiac transgenic expression of light sensitive protein Channelrhodopsin-2 (ChR2). After thoracotomy, myocardial infarction was induced by ligation of the left anterior descending coronary artery, and then VA was induced by electrical burst stimulation (10 v, 50 Hz, 2 s). Before the myocardial infarction operation, every heart was illuminated by 473 nm laser or electrical stimulated on the right ventricle in a train of 30 pulses at 8 HZ to test the threshold of light intensity or electrical pacing voltage. The VA termination process during 20 s after burst stimulation was investigated in different performance: optical termination, 1 s constant illumination repeated in 4 episodes with 1 s interval (470 nm, 20 times threshold intensity); electrical termination, ATP in 8 pulses of 8hz with 2ms duration in 4 times pacing threshold; natural recovery from VA, without optical or electrical intervention. Recovery time was defined as the time from the end of the burst stimulation to the recovery of sinus rhythm, and the termination rate was the percentage of sinus rhythm recovery among 11s after the end of burst stimulation with or without any intervention. Results We analyzed the recovery time and the termination rate (N=10, n=127 episodes per mode). The sequence of recovery time was optical termination (7.328±0.3623 s) < electric termination (10.31±0.4482 s) < natural recovery (12.97±0.3834 s) (figure D). We confirmed that the termination rate of optical illumination (86.14±4.145%) was higher than those of ATP (63.5±6.371%) and natural recovery (47.71±5.476%) (figure E), and there was no significant difference between the last two process (one-way of ANOVA; p=0.124). Comparison of three modes Conclusions Optogenetic manipulation shortened the recovery time and increased the termination rate in myocardial infarction ventricular arrhythmia, which may provide a potential approach for translational arrhythmia therapy. Acknowledgement/Funding The national natural science foundation of China (81772044)

Automated detection of shockable and non-shockable arrhythmia using novel wavelet-based ECG features

Malignant arrhythmia can lead to sudden cardiac death (SCD). Shockable arrhythmia can be terminated with device electrical shock therapies. Ventricular-tachycardia (VT) and ventricular fibrillation (VF) are responsive to electrical anti-tachycardia pacing therapy and defibrillation which help to restore normal electrical and mechanical function of the heart. In contrast, non-shockable arrhythmia like asystole and bradycardia are not responsive to electric shock therapy. Distinguishing between shockable and non-shockable arrhythmia is an important diagnostic challenge that has practical clinical relevance. It is difficult to accurately differentiate between these two types of arrhythmia by manual inspection of electrocardiogram (ECG) segments within the short time duration before triggering the device for electrical therapy. Automated defibrillators are equipped with automatic shockable arrhythmia detection algorithms based on ECG morphological features, which may possess variable diagnostic performance depending on machine models. In our work, we have designed a robust system using wavelet decomposition filter banks for extraction of features from the ECG signal and then classifying the features. We believe this method will improve the accuracy of discriminating between shockable and non-shockable arrhythmia compared with existing conventional algorithms. We used a novel three channel orthogonal wavelet filter bank, which extracted features from ECG epochs of duration 2 s to distinguish between shockable and non-shockable arrhythmia. The fuzzy, Renyi and sample entropies are extracted from the various wavelet coefficients and fed to support vector machine (SVM) classifier for automated classification. We have obtained an accuracy of 98.9%, sensitivity and specificity of 99.08% and 97.11.9%, respectively, using 10-fold cross validation. The area under the receiver operating characteristic has been found to be 0.99 with F1-score of 0.994. The system developed is more accurate than the existing algorithms. Hence, the proposed system can be employed in automated defibrillators inside and outside hospitals for emergency revival of patients suffering from SCD. These automated defibrillators can also be implanted inside the human body for automatic detection of potentially fatal shockable arrhythmia and to deliver an appropriate electric shock to the heart.

Esophageal electrode model and simulation of directed transesophageal electrical pacing of the spinal cord

Abstract Spinal cord stimulation (SCS) is the most commonly used technique of neurostimulation. It involves the stimulation of the spinal cord and is therefore used to treat chronic pain. The existing esophageal catheters are used for temperature monitoring during an electrophysiology study with ablation and transesophageal echocardiography. The aim of the study was to model the spine and new esophageal electrodes for the transesophageal electrical pacing of the spinal cord, and to integrate them in the Offenburg heart rhythm model for the static and dynamic simulation of transesophageal neurostimulation. The modeling and simulation were both performed with the electromagnetic and thermal simulation software CST (Computer Simulation Technology, Darmstadt). Two new esophageal catheters were modelled as well as a thoracic spine based on the dimensions of a human skeleton. The simulation of directed transesophageal neurostimulation is performed using the esophageal balloon catheter with an electric pacing potential of 5 V and a trapezoidal signal. A potential of 4.33 V can be measured directly at the electrode, 3.71 V in the myocardium at a depth of 2 mm, 2.68 V in the thoracic vertebra at a depth of 10 mm, 2.1 V in the thoracic vertebra at a depth of 50 mm and 2.09 V in the spinal cord at a depth of 70 mm. The relation between the voltage delivered to the electrodes and the voltage applied to the spinal cord is linear. Virtual heart rhythm and catheter models as well as the simulation of electrical pacing fields and electrical sensing fields allow the static and dynamic simulation of directed transesophageal electrical pacing of the spinal cord. The 3D simulation of the electrical sensing and pacing fields may be used to optimize transesophageal neurostimulation.

IL-1β Plays an Important Role in Pressure Overload-Induced Atrial Fibrillation in Mice.

Hypertension is one risk for atrial fibrillation (AF) and induces cardiac inflammation. Recent evidence indicates that pressure overload-induced ventricular structural remodeling is associated with the activation of nucleotide binding-oligomerization domain (NOD)-like receptor P3 (NLRP3) inflammasomes, including an apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We hypothesized that NLRP3 inflammasomes are an initial sensor for danger signals in pressure overload-induced atrial remodeling, leading to AF. Transverse aortic constriction (TAC) or a sham procedure was performed in mice deficient for ASC-/- and interleukin-1β (IL-1β-/-). One week after the procedure, electrical left atrial burst pacing from the esophagus was performed for 30 s to induce AF. IL-1β, monocyte chemotactic protein 1 (MCP-1), connective tissue growth factor (CTGF), and collagen 1 gene expression were also examined. The electrical burst pacing induced AF in TAC-operated wild-type (WT) (p < 0.001) and ASC-/- (p < 0.05) mice, compared to no AF in the sham-operated WT and ASC-/- mice, respectively. In contrast, the number of mice in which sustained AF was induced was similar between TAC-operated IL-1β-/- and sham-operated IL-1β-/- mice (p > 0.05). The expression of all genes tested was increased in TAC-operated WT and ASC-/- mice compared with sham-operated WT and ASC-/- mouse atria, respectively. CTGF and collagen 1, but not MCP-1, gene expressions were increased in TAC-operated IL-1β-/- mouse atria compared with sham-operated WT and IL-1β-/- mouse atria. In contrast, the IL-1β gene was not detected in either TAC-operated or sham-operated IL-1β-/- mouse atria. These results suggest that an IL-1β activation pathway, different from NLRP3 inflammasomes, plays an important role in pressure overload-induced sustained AF.

Hemodynamic Responses to Ventricular Pacing in the Fish Heart Depend on Repolarization Duration at the Paced Regions

IntroductionSpatiotemporal distribution of myocardial action potential durations is heterogeneous but a role of these heterogeneities is largely unknown. Heart ventricular contraction needs muscle mechanical heterogeneity, namely activation spread from “slow” to “fast” muscle elements, which are expected to have longer and shorter action potentials, respectively. We hypothesized that the longer repolarization duration at the primary activation area provides the better pump function. The objective of the present study was to evaluate an association between the repolarization duration in the area of initial activation and aortic blood flow under electrical pacing of different ventricular regions. Having a single ventricle and primitive, if any, conduction system, a fish heart presents a useful model for assessing the functional significance of electrophysiological nonuniformities.MethodsExperiments were performed in wild caught Atlantic cod (n = 18). Ventricular unipolar electrograms were led from an epicardial sock and intramural plunge electrodes (24–64 leads simultaneously) connected to a custom‐made mapping system. In each lead, activation times (ATs) and repolarization times (RTs) were determined as dV/dt min during QRS complex and dV/dt max during T wave, respectively. Activation‐repolarization intervals (ARIs, surrogate for action potential durations) were calculated as ARI = RT‐AT. Aortic blood flow was measured with transit time flow meter.ResultsIn sinus rhythm, the posterior wall near the atrioventricular junction was earliest to be activated (median ATs 9 [IQR 3–15] ms) and had the longest median ARIs (516 [IQR 430–580] ms). The most remote from this area regions had expectedly longer ATs (heart apex: 33 [IQR 29–44] ms; anterior base 33 [IQR 25–43] ms; both p&lt;0.05 vs posterior wall) and also demonstrated shorter ARIs (heart apex: 482 [IQR 427–544] ms; anterior base 416 [IQR 390–497]; both p&lt;0.05 vs posterior wall). Posterior wall pacing yielded a highest median aortic flow 47.3 [IQR 40.4–74.4] mL/min/kg. Apical and anterior basal pacing gave lower median aortic flow values (40.7 [IQR 29.0–49.2] and 29.7 [29.3–39.1] mL/min/kg, respectively; both p&lt;0.05 vs posterior wall pacing).ConclusionPump function of the Atlantic cod heart ventricle is associated with activation‐to‐repolarization relationship: the longer repolarization at the site of activation origin provides the higher cardiac output.Support or Funding InformationThe study was supported by by the Ural Branch of the Russian Academy of Sciences (Project AAAA‐A18‐118012290365‐2) and the Program for Fundamental Research of RAS (AAAA‐A17‐117012310154‐6).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glutathione-related substances maintain cardiomyocyte contractile function in hypoxic conditions

Severe hypoxia leads to decline in cardiac contractility and induces arrhythmic events in part due to oxidative damage to cardiomyocyte proteins including ion transporters. This results in compromised handling of Ca2+ ions that trigger heart contractile machinery. Here, we demonstrate that thiol-containing compounds such as N-acetylcysteine (NAC), glutathione ethyl ester (et-GSH), oxidized tetraethylglutathione (tet-GSSG), oxidized glutathione (GSSG) and S-nitrosoglutathione (GSNO) are capable of reducing negative effects of hypoxia on isolated rat cardiomyocytes. Preincubation of cardiomyocytes with 0.1 mM GSNO, 0.5 mM et-GSH, GSSG, tet-GSSG or with 10 mM NAC allows cells 5-times longer tolerate the hypoxic conditions and elicit regular Ca2+ transients in response to electric pacing. The shape of Ca2+ transients generated in the presence of GSNO, et-GSH and NAC was similar to that observed in normoxic control cardiomyocytes. The leader compound, GSNO, accelerated by 34% the recovery of normal contractile function of isolated rat heart subjected to ischemia-reperfusion. GSNO increased glutathionylation of Na,K-ATPase alpha-2 subunit, the principal ion-transporter of cardiac myocyte sarcolemma, which prevents irreversible oxidation of Na,K-ATPase and regulates its function to support normal Ca2+ ion handling in hypoxic cardiomyocytes. Altogether, GSNO appears effective cardioprotector in hypoxic conditions worth further studies toward its cardiovascular application.

A Novel Gastric Pacing Device to Modulate Slow Waves and Assessment by High-Resolution Mapping.

The use of electrical pacing in the gastrointestinal field continues to advance clinical and basic science; however, the efficacy and effectiveness of gastric stimulation and pacing remains limited. In the stomach, rhythmic bioelectrical events, known as slowwaves, coordinate the muscular contractions that aid digestion. A range of slow wave abnormalities have been shown to be associated with functional motility disorders, such as gastroparesis, chronic unexplained nausea and vomiting, and functional dyspepsia. Pacing is an attractive therapeutic approach to revert slow wave abnormalities. However, there are currently no clinical gastric pacing devices in active use. In this study, a novel battery-powered pacing device was developed, implementing wireless control and tissue electrical parameter monitoring. The pacing device was applied in five pigs in vivo along with high-resolution (HR) mapping to validate the device and to elucidate the pacing response of slow waves. The pacing leads were placed in the middle of the HR array to determine any changes to the propagation pattern. The pacing period range was 14-30 s. In all studies, the novel pacing device initiated slow wave activation from a location near the pacing leads at the specified period. Slow wave propagation speed increased after pacing (from 6.4 ± 2.0 to 8.1 ± 3.2mm/s; P<0.001), commensurate with induction of paced anisotropic propagation. This study introduces a novel gastric pacing system suitable for clinical trials, achieving reliable induction of slow wave pacing at specific location and periods. The device is now available to be trialed as a therapeutic application for motility disorders and obesity.

Directed Self-Organization of Human and Non-Human Primate Heart Muscle Organoids from Pluripotent Stem Cells

Pluripotent stem cells (PSCs) can differentiate into all somatic cell lineages, but fail to form functional macro-scale heart muscle organoids in monolayer, suspension, or aggregate cultures. Here we show that human and non-human primate (NHP) PSCs have the capacity to self-organize into bioengineered heart muscle (BHM) if exposed to a serum-free cardio-instructive collagen-hydrogel environment with defined temporarily restricted growth factor, small molecule, and mechanical stimulation. BHMs traversed through distinct developmental stages with molecular signatures representative for: (i) mesoderm induction (3 days), (ii) cardiac specification (+10 days), and (iii) cardiac maturation (+9 days). Matured BHM responded to electrical pacing, preloading, inotropic stimuli and pharmacological agents similar to bona fide myocardium. We further demonstrate the applicability of BHM in modelling of Noonan Syndrome associated with Raf1 mutations. Collectively, we provide proof-of-concept for directed assembly of human and NHP PSCs into force-generating macro-scale myocardial organoids for applications in simulations of heart development and disease.

A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promiseto revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues withchamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to8months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

Diverse relaxation rates exist among rat cardiomyocytes isolated from a single myocardial region.

Prior studies have shown variation in the functional properties of cardiomyocytes isolated from different regions of the left ventricular myocardium. We found that these region-dependent variations vanish below a tissue volume of ∼7 mm3 in the adult rat myocardium, revealing a fixed level of intrinsic relaxation rate heterogeneity that is independent of tissue volume. Within these microscopically varying cell populations, fast-relaxing cells were shown to have elevated phosphorylated troponin I compared to slow-relaxing cells. Relaxation rate was also correlated with cardiomyocyte length, in that slow-relaxing cells were longer than fast-relaxing cells. These results show a new relationship between cardiomyocyte morphology and myofilament relaxation, and suggest that functional diversity among individual myocytes at the microscale may contribute to bulk relaxation of the myocardium. The mean contractility and calcium handling properties of cardiomyocytes isolated from different regions of the ventricular myocardium are known to vary significantly. We designed experiments to quantify the variance in contractile properties among cells within the same myocardial region. Longitudinal strips of myocardial tissue were excised from the epicardial left ventricular free walls of adult Sprague-Dawley rats and then treated with collagenase to isolate individual myocytes. Cardiomyocytes were characterized by measuring sarcomere length changes and calcium transients during electrical pacing. Variance of the time from peak sarcomere shortening to 50% re-lengthening (RT50 ) was assessed in each cell population. Isolating cells from progressively shorter strips allowed an estimate of the myocardial volume below which regional variation vanished and only microscale heterogeneity remained (∼7 mm3 ). The SD of RT50 within this myocardial volume was 28% of the mean. In a series of follow-up experiments, RT50 was shown to correlate significantly with resting myocyte length, suggesting a connection between cell morphology and intrinsic relaxation behaviour. To explore the mechanistic basis of varying RT50 , a novel single-cell aspirator was employed to collect small batches of cardiomyocytes grouped according to their relaxation rates (fast or slow). Western blot analysis of the two groups revealed significantly elevated troponin I phosphorylation in fast-relaxing cells. Our observations suggest that cell-to-cell heterogeneity of active contractile properties is substantial, with implications for how we understand myocardial relaxation and design drug therapies intended to alter relaxation rate.

STATINS MEDIATE RECOVERY FROM CHEMOTHERAPHY-INDUCED CARDIOTOXICITY

Doxorubicin (Dox) is a frequently used chemotherapeutic agent with heart failure as a major adverse event. While several studies have demonstrated prevention of Dox-induced cardiomyopathy (Dox-CM) in rodents, no translatable therapies have emerged to reverse Dox-CM. Statins have potential as a widely-prescribed, well-tolerated class of drugs with pleiotropic effects and a limited side-effect profile compared to current Dox-CM standard treatment strategies. To determine the potential of statins to reverse Dox-CM, C57BL/6 mice were administered Dox (10mg/kg bolus, IP), leading to a wide derangement of the cardiac transcriptome within 7 days , and reduced fractional shortening 14 days post Dox (27.4%±1.1 vs control: 40%±0.4). Dox-CM mice showed no evidence of cardiac hypertrophy or fibrosis, although there was significant activation of the DNA damage response pathway (γH2AX, ATM, and acetyl-p53) compared to controls. Interestingly, Dox-induced apoptosis was limited to non-cardiomyocyte cells. In vitro analysis of isolated neonatal rat cardiomyocytes shows a significant decrease in spontaneous contractile rate following Dox-treatment, while electrical pacing thresholds required to induce cardiac alternans were reduced in whole heart preparations from Dox-CM mice compared to controls. Accordingly, SERCA-2 protein expression was decreased while phospholamban and HAX-1 are upregulated in Dox-CM hearts vs control. Together, this data suggests that Dox-CM is a result of defective calcium handling. Dox-CM mice that then received daily rosuvastatin (10μg or 100μg/d, IP; figure 1) or atorvastatin treatment (20 μg/d, IP) showed significant improvement in fractional shortening (37.7%±1.8) after two weeks compared to vehicle controls (27.75%±1.9). Statin treatment significantly improved spontaneous cardiomyocyte contractile rate and increased the pacing threshold required to induce cardiac alternans. Analysis of whole heart lysates by Western blotting revealed activation of HSP27-Akt signaling along with resolution of Dox-induced reduction in SERCA-2 protein expression. Furthermore, statin treated mice show fewer CD68 positive cells in heart cross-sections compared to animals treated with Dox alone. Inflammatory pathways appeared to be differentially regulated in the Dox-CM and statin post-treatment groups as noted by increased expression of C5a, CXCL12, IL-7, IL-13, and IL-16 in the statin treated mice compared to Dox-CM. Our detailed analysis of Dox-CM suggests that contractile impairment is related to reversible changes in calcium handling and inflammation, as opposed to the long-held theory of cardiomyocyte loss. Accordingly, the recovery of heart function through statin treatment represents a major step forward in the treatment of Dox-CM and is a readily translatable therapy for cancer survivors suffering from heart failure.