Ventricular Conduction Velocity Research Articles

Mitochondrial Oxidative Stress and Cardiac Dysfunction in TERT Knockout Mice

Abstract Background Heart failure (HF) is a major cause of hospitalization in the elderly, with its incidence increasing due to aging and associated risk factors like hypertension, diabetes, and myocardial fibrosis. Aging-related myocardial fibrosis, characterized by alterations in the extracellular matrix and myocardial structure, impairs cardiac function. Mitochondrial oxidative stress plays a pivotal role in cardiac electrical and structural remodeling, necessitating a deeper understanding of its involvement in aging-related heart failure. Purpose The purpose of this study is to investigate the mechanisms of aging-related ventricular electrical and structural remodeling by constructing an aging mouse model. This research aims to provide potential therapeutic targets for heart failure associated with aging. Methods We developed a third-generation homozygous telomerase reverse transcriptase (TERT) knockout mouse model. Comparing third-generation homozygous TERT knockout mice (F3 group) with age-matched wild-type males (WT group). The study encompassed blood pressure measurement, electrocardiographic analysis, echocardiography, ELISA for cardiac biomarkers, ventricular tissue electrophysiology, fibrosis assessment through staining, transcriptomic profiling via RNA-seq, and electron microscopy of ventricular mitochondria. Results Compared to the WT group, the F3 group demonstrated increased P53 and P16 protein expression. Decreased LVEF and FS, along with increased interventricular septum thickness, left ventricular diameter, and left ventricular volume. Elevated serum NT-pro-BNP and troponin I (cTnI) levels, with prolonged QRS duration. Decreased left and right ventricular conduction velocity, accompanied by increased conduction dispersion. Notable elevation in COLI, TGFβ, and α-SMA gene transcription and protein expression in ventricular tissue. Significant differences in mitochondrial oxidative stress signal pathways via RNA-seq analysis. Elevated serum MDA levels and decreased SOD levels, with up-regulated Mn-SOD gene transcription and protein expression, and down-regulated MFN2 and Drp1 gene transcription and protein expression in ventricular tissue. Electron microscopy revealed mitochondrial abnormalities such as loose arrangement, decreased number, swelling, vacuolation, and myofilament disintegration or breakage in the F3 group. Conclusion Third-generation TERT-/- senescent mice exhibit notable cardiac impairments, including electrical remodeling, structural changes, and mitochondrial dysfunction. These findings suggest a potential link between mitochondrial oxidative stress and aging-related heart failure, indicating novel therapeutic opportunities targeting mitochondrial dysfunction for managing such conditions.

Omnipolar conduction velocity mapping for ventricular substrate characterization: Impact of CV estimation method and EGM type on in vivo conduction velocity measurements

BackgroundAreas of abnormal or heterogeneous conduction velocity (CV) are important ablation targets for ventricular tachycardias, yet precise assessment of CV in clinical contact mapping remains challenging. Numerous different CV estimation methods have been proposed. ObjectiveThis study aimed to compare the automated local activation time (LAT)–independent omnipolar-based CV estimation method termed wave speed (WS) with 4 established LAT-based methods to formally establish the quantitative differences between them. MethodsHigh-density contact maps in patients with structurally normal hearts during sinus rhythm (SR) and ventricular ectopy (VE) were retrospectively analyzed. CV was assessed and compared by 5 methods: omnipolar WS, gradient method, planar wavefront fitting, circular wavefront fitting, and radial basis function. CV variations based on electrogram (EGM) type (unipolar, bipolar, and omnipolar), catheter movement, and surrogate markers for catheter contact were analyzed. ResultsThe study included 23 patients (47.8% male; 45.7 ± 17.3 years) with 22 SR maps (11 left ventricle, 11 right ventricle) and 16 VE maps (9 left ventricle, 7 right ventricle). The WS algorithm yielded statistically significant higher CV estimates in SR (mean, 1.41 ± 0.18 m/s) and VE (mean, 1.23 ± 0.18 m/s) maps compared with all LAT-based estimation methods, with absolute differences ranging from 0.1 m/s to 0.81 m/s. Median pointwise differences in SR and VE between WS and LAT-based methods were high, ranging from 0.55 ± 0.15 m/s (WS vs planar wavefront fitting) to 0.67 ± 0.16 m/s (WS vs radial basis function). For LAT-based methods, use of unipolar EGMs yielded significantly higher CV estimates than bipolar or omnipolar EGMs in SR. ConclusionThe CV estimation method has an important, statistically significant impact on ventricular CV measurements. Future work will focus on how these differences affect identification of pathologic conduction slowing in scar-related substrate.

The role of MAPRE2 and microtubules in Brugada syndrome pathogenesis

Abstract Background Brugada syndrome (BrS) is associated with loss-of-function variants in SCN5A (encoding NaV1.5), which are found in only ∼20% of patients. Recent genome-wide association studies identified a novel locus within an intron of MAPRE2 (encoding microtubule end-binding protein 2, EB2), implicating microtubule (MT) involvement in BrS. Purpose To understand the role of MAPRE2 and MT in BrS pathogenesis. Methods A mapre2 knock-out (KO) zebrafish model was generated using CRISPR/Cas9 and validated by western blot. Larval hearts at 5 day-post-fertilization (dpf) were isolated for voltage mapping and immunohistochemistry with confocal imaging. Morpholino targeting mapre2 or ttl (encoding tubulin tyrosine ligase) were injected into zebrafish embryos at the one-cell stage. A transgenic zebrafish line with cdh2 (encoding N-cadherin) tandem fluorescent timer (tFT) was used to study adherens junction. MT plus-end tracking experiments were performed in iPSC-derived cardiomyocytes (CM) with MAPRE2 knockdown (KD) using siRNA. ECG was recorded in adult fish. Results Voltage mapping of larval hearts (14 WTs vs. 19 KOs) showed a significant decrease in ventricular conduction velocity (17.4±1.5 vs. 13.4±0.8 mm/s; p<0.05) and action potential upstroke velocity (Vmax; 86.1±3.6 vs. 73.9±3.2 1/s; p<0.05), suggesting loss of cardiac NaV function. Correspondingly, ECG of adult fish (11 WTs vs. 10 KOs) showed a significant increase in QRS duration (27.8±0.5 vs. 32.3±0.7 ms; p<0.0001). Confocal imaging of KO hearts showed disorganized adherens junction based on N-cadherin staining. Similarly, morpholino KD of mapre2 in the cdh2 tFT line showed ∼40% mislocalization of mature N-cadherin relative to ZO-1 staining in larval ventricles (8 KDs vs 9 control hearts; p<0.05), suggesting disruption of adherens junction. In terms of MT, immunohistochemistry showed a 23% decrease of detyrosinated tubulin relative to total alpha tubulin in KO hearts (16 vs. 8 WT hearts; p<0.01). MAPRE2 KD in iPSC-CM (337 control vs. 285 KD MT in 4 sets of cells) showed an increase in MT velocity (7.9±0.2 vs. 8.8±0.2 µm/min; p<0.001), distance (5.8±0.2 vs. 7.1±0.2 µm; p<0.0001), and duration (46.2±1.4 vs. 52.2±1.6 s; p<0.001), suggesting changes to MT dynamics. Finally, morpholino KD of ttl in mapre2 KO embryos restored the fraction of detyrosinated tubulin (46% increase; 16 controls vs. 16 KDs; p<0.01) as well as Vmax (74.3±3.0 vs. 88.3±2.1 1/s; 13 controls vs. 9 KDs; p<0.01) to WT levels. Conclusion Genetic ablation of mapre2 led to a decrease in cardiac NaV function, a hallmark of BrS. This is associated with disruption of adherens junction, a decrease of detyrosinated tubulin as a marker of MT stability, and changes in MT dynamics. Restoration of detyrosinated tubulin fraction with ttl KD led to a rescue of NaV function in zebrafish larval hearts. Taken together, MAPRE2 loss-of-function may contribute to BrS pathogenesis by disruption of adherens junction via changes in MT dynamics.

Extracellular Perinexal Separation Is a Principal Determinant of Cardiac Conduction.

Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in the heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular resistance. By contrast, ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs such as the perinexus. Our objective was to determine the relationship between ventricular CV and structural changes to micro- and nanoscale extracellular spaces. Conduction and Cx43 (connexin43) protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with the osmotic agents albumin, mannitol, dextran 70 kDa, or dextran 2 MDa. Peak sodium current was quantified in isolated guinea pig ventricular myocytes. Extracellular resistance was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs. CV primarily in the transverse direction of propagation was significantly reduced by mannitol and increased by albumin and both dextrans. The combination of albumin and dextran 70 kDa decreased CV relative to albumin alone. Extracellular resistance was reduced by mannitol, unchanged by albumin, and increased by both dextrans. Cx43 expression and conductance and peak sodium currents were not significantly altered by the osmotic agents. In response to osmotic agents, perinexal width, in order of narrowest to widest, was albumin with dextran 70 kDa; albumin or dextran 2 MDa; dextran 70 kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. Cardiac conduction does not correlate with extracellular resistance but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.

Abstract P1183: Phosphorylation Signatures Of Human Heart Failure: Characterizing Dilated Cardiomyopathy-associated Signaling Pathways At The Cardiomyocyte Intercalated Disc

Heart failure remains a highly prevalent condition with diverse etiology, yet the underlying signaling mechanisms are not fully understood. Despite the profound effects of post-translational protein modifications on downstream signaling, limited studies have investigated the cardiac phosphoproteome in human heart failure. We hypothesized that a combined proteomic and phosphoproteomic analysis of human dilated (DCM) and ischemic (ICM) cardiomyopathy would reveal novel etiology-associated disease pathways. Integrative analyses of left ventricular explants from DCM patients ( n =4) vs. non-failing controls ( n =4), and left ventricular infarct vs. non-infarct, and peri-infarct vs. non-infarct regions of ICM patients ( n =4) identified 5,570 unique proteins with 13,624 corresponding phosphorylation sites. Each pair-wise comparison revealed shared and etiology-specific signatures, with a unique DCM-associated enrichment of cell-cell adhesion pathways. We focused our attention on αT-catenin (CTNNA3) as a cardiomyocyte intercalated disc candidate phosphoprotein with a unique cluster of 4 hyperphosphorylated sites in DCM hearts ( P <0.0001). Overexpression of non-phosphorylatable hCTNNA3 in ex vivo isolated adult mouse cardiomyocytes showed internalized protein expression and weaker cell-cell adhesion vs. wildtype (WT) and phospho-mimetic forms. We established an in vivo mouse model using recombinant adeno-associated virus 9 (rAAV9) harboring hCTNNA3-WT, hCTNNA3-phospho-null, or empty rAAV9 control. Phospho-null CTNNA3 mice developed left ventricular dilation and contractile dysfunction (% EF; 51.25±1.17 phospho-null vs. 62.07±1.20 WT vs. 66.76±1.42 empty; n ≥10) with impaired left ventricular conduction velocity (cm/s; 36.83±1.10 phospho-null vs. 47.74±2.04 WT; n =6), by echocardiography and ex vivo optical mapping. Loss of CTNNA3 phosphorylation led to intercalated disc remodeling with internalization and dissociation of CTNNA3, connexin 43, N-cadherin, β-catenin, and plakophilin 2 from the adherens junction, using high-resolution confocal imaging. Together these findings reveal a compensatory role for αT-catenin phosphorylation in maintaining cardiomyocyte intercalated disc organization in human DCM.

Renal denervation reverses ventricular structural and functional remodeling in failing rabbit hearts

Renal denervation (RDN) suppresses the activity of the renin–angiotensin–aldosterone system and inflammatory cytokines, leading to the prevention of cardiac remodeling. Limited studies have reported the effects of renal denervation on ventricular electrophysiology. We aimed to use optical mapping to evaluate the effect of RDN on ventricular structural and electrical remodeling in a tachycardia-induced cardiomyopathy rabbit model. Eighteen rabbits were randomized into 4 groups: sham control group (n = 5), renal denervation group receiving RDN (n = 5), heart failure group receiving rapid ventricular pacing for 1 month (n = 4), and RDN-heart failure group (n = 4). Rabbit hearts were harvested for optical mapping. Different cycle lengths were paced (400, 300, 250, 200, and 150 ms), and the results were analyzed. In optical mapping, the heart failure group had a significantly slower epicardial ventricular conduction velocity than the other three groups. The RDN-heart failure, sham control, and RDN groups had similar velocities. We then analyzed the 80% action potential duration at different pacing cycle lengths, which showed a shorter action potential duration as cycle length decreased (P for trend < 0.01), which was consistent across all groups. The heart failure group had a significantly longer action potential duration than the sham control and RDN groups. Action potential duration was shorter in the RDN-heart failure group than the heart failure group (P < 0.05). Reduction of conduction velocity and prolongation of action potential duration are significant hallmarks of heart failure, and RDN reverses these remodeling processes.

PO-02-153 THE ROLE OF MAPRE2 AND MICROTUBULES IN CARDIAC ELECTROPHYSIOLOGY AND BRUGADA SYNDROME

Brugada syndrome (BrS) is associated with loss-of-function variants in SCN5A (encoding NaV1.5), yet these are only found in ∼20% of probands. Recent genome-wide association studies identified a novel locus within an intron of MAPRE2 (encoding microtubule end-binding protein 2, EB2), which implicates microtubule (MT) involvement in BrS. To understand the role of MAPRE2 and MT in cardiac electrophysiology and BrS pathogenesis. Using CRISPR/Cas9, a mapre2 knock-out (KO) mutant was generated in zebrafish and validated by western blot. Morpholino targeting ttl (encoding tubulin tyrosine ligase) was injected into zebrafish embryos at the one-cell stage. Larval hearts at 5 day-post-fertilization (dpf) were isolated for voltage mapping and immunohistochemistry. MT plus-end tracking experiments were performed in iPSC-derived cardiomyocytes with MAPRE2 knockdown (KD) using siRNA. ECG was recorded in adult fish. Voltage mapping of larval hearts (14 WTs vs. 19 KOs) showed a significant decrease in ventricular conduction velocity (17.4±1.5 vs. 13.4±0.8 mm/s; p<0.05) and action potential upstroke velocity (Vmax; 86.1±3.6 vs. 73.9±3.2 1/s; p<0.05), suggesting loss of cardiac NaV function. Correspondingly, ECG of adult fish (11 WTs vs. 10 KOs) showed a significant increase in QRS duration (27.8±0.5 vs. 32.3±0.7 ms; p<0.0001). Immunohistochemistry showed a 23% decrease of detyrosinated (glu) tubulin relative to total alpha tubulin in KO hearts (16 vs. 8 WT hearts; p<0.01). MT plus-end tracking experiments (337 control vs. 285 KD MT in 4 sets of cells) showed an increase in MT velocity (7.9±0.2 vs. 8.8±0.2 μm/min; p<0.001), distance (5.8±0.2 vs. 7.1±0.2 μm; p<0.0001), and duration (46.2±1.4 vs. 52.2±1.6 s; p<0.001) with MAPRE2 KD, suggesting changes to MT dynamics. Finally, morpholino KD of ttl in mapre2 KO embryos restored the fraction of glu-tubulin (46% increase; 16 controls vs. 16 ttl KDs; p<0.01) and Vmax (74.3±3.0 vs. 88.3±2.1 1/s; 13 controls vs. 9 KDs; p<0.01) to WT levels. Genetic ablation of mapre2 led to a decrease in NaV function, a hallmark of BrS. This is associated with a decrease in glu-tubulin as a marker of MT stability and changes in MT dynamics. Restoration of glu-tubulin fraction with ttl KD led to a rescue of NaV function in zebrafish larval hearts. Taken together, MAPRE2 loss-of-function may contribute to BrS pathogenesis via a novel paradigm of disrupting MT stability and dynamics.

Sophoridine manifests as a leading compound for anti-arrhythmia with multiple ion-channel blocking effects

BackgroundSophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively inadequate, which limits the development of SR as an antiarrhythmic candidate. PurposeTo facilitate the development process of SR as an antiarrhythmic candidate, we performed integrated studies on the electrophysiological properties of SR in vitro and ex vivo to gain more comprehensive insights into the multi-ion channel blocking effects of SR, which provided the foundation for the further drugability studies in antiarrhythmic and safety studies. Firstly, SR's electrophysiological properties and antiarrhythmic potentials were recorded and assessed at the cell and tissue levels by comprehensively integrating the patch clamp with the Electrical and Optical Mapping systems. Subsequently, the antiarrhythmic effects of SR were validated by aconitine and ouabain-induced arrhythmia in vivo. Finally, the safety of SR as an antiarrhythmic candidate compound was evaluated based on the guidelines of the Comprehensive in Vitro Proarrhythmia Assay (CiPA). Study designThe antiarrhythmic effect of SR was evaluated at the in vitro, ex vivo, and in vivo levels. MethodsIsolated primary cardiomyocytes and stable cell lines were prepared to explore the electrophysiologic properties of being a multiple ion-channel blocker in vitro by whole-cell patch clamp. Using electrical and optical mapping, the negative chronotropic effect of SR was determined in langendorff-perfused rat or guinea-pig hearts.The antiarrhythmic activity of SR was assessed by the ex vivo tachyarrhythmia models induced by left coronary artery ligation (LCAL) and isoproterenol (ISO). Canonical models of aconitine and ouabain-induced arrhythmia were used to verify the antiarrhythmic effects in vivo. Finally, the pro-arrhythmic risk of SR was detected in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hSCCMs) using a Microelectrode array (MEA). ResultsSingle-cell patch assay validated the multiple ion-channel blockers of SR in transient outward current potassium currents (Ito), l-type calcium currents (ICa-l), and rapid activation delayed rectifier potassium currents (IKr). SR ex vivo depressed heart rates (HR) and ventricular conduction velocity (CV) and prolonged Q-T intervals in a concentration-dependent manner. Consistent with the changes in HRs, SR extended the active time of hearts and increased the action potential duration measured at 90% repolarization (APD90). SR could also significantly lengthen the onset time and curtail the duration of spontaneous ventricular tachycardia (VT) in the ex vivo arrhythmic model induced by LCAL. Meanwhile, SR could also significantly upregulate the programmed electrical stimulation (PES) frequency after the ISO challenge in forming electrical alternans and re-entrant excitation. Furthermore, SR exerted antiarrhythmic effects in the tachyarrhythmia models induced by aconitine and ouabain in vivo. Notably, the pro-arrhythmic risk of SR was shallow for a moderate inhibition of the human ether-à-go-go-related gene (hERG) channel. Moreover, SR prolonged field potential duration (FPDc) of hSCCMs in a concentration-dependent manner without early after depolarization (EAD) and arrhythmia occurrence. ConclusionOur results indicated that SR manifested as a multiple ion-channel blocker in the electrophysiological properties and exerts antiarrhythmic effects ex vivo and in vivo. Meanwhile, due to the low pro-arrhythmic risk in the hERG inhibition assay and the induction of EAD, SR has great potential as a leading candidate in the treatment of ventricular tachyarrhythmia.

Overlapping and Distinct Features of Cardiac Pathology in Inherited Human and Murine Ether Lipid Deficiency.

Inherited deficiency in ether lipids, a subgroup of glycerophospholipids with unique biochemical and biophysical properties, evokes severe symptoms in humans resulting in a multi-organ syndrome. Mouse models with defects in ether lipid biosynthesis have widely been used to understand the pathophysiology of human disease and to study the roles of ether lipids in various cell types and tissues. However, little is known about the function of these lipids in cardiac tissue. Previous studies included case reports of cardiac defects in ether-lipid-deficient patients, but a systematic analysis of the impact of ether lipid deficiency on the mammalian heart is still missing. Here, we utilize a mouse model of complete ether lipid deficiency (Gnpat KO) to accomplish this task. Similar to a subgroup of human patients with rhizomelic chondrodysplasia punctata (RCDP), a fraction of Gnpat KO fetuses present with defects in ventricular septation, presumably evoked by a developmental delay. We did not detect any signs of cardiomyopathy but identified increased left ventricular end-systolic and end-diastolic pressure in middle-aged ether-lipid-deficient mice. By comprehensive electrocardiographic characterization, we consistently found reduced ventricular conduction velocity, as indicated by a prolonged QRS complex, as well as increased QRS and QT dispersion in the Gnpat KO group. Furthermore, a shift of the Wenckebach point to longer cycle lengths indicated depressed atrioventricular nodal function. To complement our findings in mice, we analyzed medical records and performed electrocardiography in ether-lipid-deficient human patients, which, in contrast to the murine phenotype, indicated a trend towards shortened QT intervals. Taken together, our findings demonstrate that the cardiac phenotype upon ether lipid deficiency is highly heterogeneous, and although the manifestations in the mouse model only partially match the abnormalities in human patients, the results add to our understanding of the physiological role of ether lipids and emphasize their importance for proper cardiac development and function.

Sanwei sandalwood decoction ameliorates acute ischemia[sbnd]reperfusion injury in rats by modulating myocyte electrophysiological characteristics

Sanwei sandalwood decoction (SWTX) is a classical Chinese medicine formula and clinically effective treatment for coronary heart disease, including myocardial ischemia/reperfusion (I/R) injury. Because the treatment mechanism of SWTX in I/R injury remains obscure, we intended to analyze the potential cardioprotective effects of SWTX in rats with myocardial I/R injury. Our research revealed that SWTX prolonged ventricular conduction time in a dose-dependent manner. While SWTX significantly delayed left ventricular signal conduction velocity, it had no effect on left atrial conduction velocity. Under sinus conditions, low SWTX concentrations reduced left ventricular conduction dispersion, while high concentrations increased conduction dispersion. SWTX also prolonged the QRS interval, APD30/50/90, and ERP. In whole-cell patch clamp experiments on myocytes, Ito and Ikr were inhibited by SWTX. While SWTX had no effect on INa, the activation curve for Nav1.5 was left-shifted. Finally, SWTX reduced the probability of ventricular fibrillation and suppressed early and late depolarization in an acute I/R injury rat model. These findings shed light on the mechanism by which SWTX alleviates myocardial I/R injury.

Abstract 12787: Microrna Administration Attenuates the Contraction Deterioration of Microrna-1 Deficient Heart But Not the Susceptibility of Arrhythmia

Introduction: Aged people are much more likely to suffer arrhythmias or develop heart failure. MicroRNA-1 (miR1), encode by two distinct genes in the mammalian genome, is an evolutionarily conserved, striated muscle-enriched microRNA with a critical role in regulating cardiac physiology. Full knockout of miR1 in mice causes postnatal lethal. Interestingly, miR1 expression was significantly decreased to the level of 12.5±4.0% in the heart of 18-month aged animals, in comparison with that in young mice (age 3-4 months). Aims: We hypothesize that miR1 deficiency is one of the major etiologies in diseased and/or aged hearts and study the therapeutic effect of miR1 administration. Methods and Results: We generated miR1-75% knockdown (75%KD) mice and confirmed that miR1 expression was 4-5 fold lower in 75%KD hearts than in WT hearts. Echocardiogram showed that 75%KD mice demonstrated impaired cardiac contractile function, evidenced by decreased ejection fraction, and fraction shortening. Surface ECG recording showed that 75%KD mice had significantly lower heart rates, shorter PR interval, prolonged QRS interval as well as premature ventricular contractions. Consistently, optical mapping of ex vivo hearts revealed that 75%KD hearts had slower ventricular conduction velocity. Whole transcriptome RNA-sequencing and Proteomic assays demonstrated cardiac electrical and metabolic remodeling in miR1-deficient hearts. Compared to WT cells, 75%KD ventricular cardiomyocytes showed more hyperpolarized RMPs with prolonged APD; the sarcomere shortening was significantly decreased at a 5Hz fast pacing rate. Structurally, 75%KD myocytes had disrupted mitochondria with a shorter length of sarcomeres. We delivered miR into the animals through intravenous tail vein injection and found that 12-week miR1 administration could significantly attenuate the decrease of ejection fraction of 75%KD hearts, however, miR1-treated hearts had significantly higher inducibility of arrhythmia than control-RNA treated hearts. Conclusions: MicroRNA-1 deficiency causes cardiac electrical and metabolic remodeling. Restoring miR1 prevents the contraction deterioration of miR1-deficient hearts but increases the risk of arrhythmia.

Abstract 11466: Deficiency of Microrna-1 Induces Cardiac Electrical and Metabolic Remodeling of the Heart

Introduction: MicroRNA-1 (miR1) is an evolutionarily conserved, striated muscle-enriched microRNA and plays critical roles in regulation of the development and physiology of the heart. It has been found that miR1 was downregulated in various types of cardiac diseases. We observed that, compared to that in young mice (age 3-4 months), the expression of miR1 was significantly decreased to the level of 12.5±4.0% in the heart of aged animals (age 18 months). Hypothesis: We hypothesized that miR1 deficiency is a major etiology in diseased and/or aged hearts. In the mammalian genome, two distinct genes, miR1-1 and miR1-2 that are located on two chromosomes, generate identical mature miR1. Methods and Results: To study the hypothesis, we generated miR1-75% knockdown (75%KD) mice by crossbreeding miR1-1 knockout (KO) and miR1-2 KO animals and found that miR1-75%KD mice could survive to adult age with a relatively higher mortality, while miR1-100%-KO is postnatal lethal. The ejection fraction and fraction shortening of 75%KD hearts was significantly decreased than that of WT animals. Electrocardiogram showed that, compared to the WT group, 75%KD hearts had significantly slower heart rates with shorter PR interval and prolonged QRS interval with observed premature ventricular contractions. Optical mapping of ex vivo hearts revealed significantly slower ventricular conduction velocity in 75%KD hearts. Compared to WT cells, 75%KD ventricular cardiomyocytes had hyperpolarized RMPs with prolonged action potentials, and showed significantly decreased sarcomere shortening. Proteomic assays found that proteins of mitochondrial ATP synthesis and electron transport were significantly downregulated in 75%KD hearts. Conclusions: We conclude that the deficiency of miR1 is a major etiological factor of cardiac electrical and metabolic remodeling in the heart. Restoring the normal level of miR1 in the heart could be a critical and effective therapeutic strategy to prevent and/or treat heart diseases.

Abstract 13171: The Role of Novel Brugada Syndrome Gene MAPRE2 in Maintaining Normal Cardiac Electrophysiology

Introduction: Brugada syndrome (BrS) is a significant cause of sudden cardiac death yet only ~20% of patients are found with a loss-of-function variant in SCN5A encoding the cardiac Na + channel, leaving ~80% genetically undiagnosed. A recent genome-wide association study found 12 loci associated with BrS, including one in MAPRE2 encoding microtubule end-binding protein 2 (EB2). Hypothesis: MAPRE2 loss-of-function contributes to BrS and is necessary in maintaining normal cardiac electrophysiology (EP). Methods: Using CRISPR/Cas9, we generated two mapre2 loss-of-function mutants in zebrafish: a full knock-out and a mutant lacking the unique N-terminus of EB2 (delN). Cardiac EP was assessed using optical mapping in larvae, surface ECG in adults, and patch clamping in ventricular myocytes. Cardiac structure was assessed using videomicroscopy, histology, and confocal microscopy. Transcriptional changes were assessed using RNA-seq. Results: No gross changes in cardiac structure were observed with mapre2 loss-of-function. However, in both mutants, voltage mapping showed decreased ventricular conduction velocity and action potential upstroke velocity (V max ) and ECGs showed prolonged QRS, P wave, and corrected QT. Patch clamp of delN ventricular myocytes revealed reduced Na + current density without changes in gating properties consistent with the reduced V max . RNA-seq of larvel hearts suggested disruption of cell adhesion, confirmed by confocal microscopy, whereas Ca 2+ imaging found an increase in Ca 2+ transient amplitude. RNA-seq of adult hearts showed disruption in the Wnt signaling pathway and treatment with GSK3β inhibitor SB217673 rescued mutant ECG abnormalities. Conclusions: Our study supports MAPRE2 as a novel contributing gene in BrS pathogenesis and conduction slowing in general. Beyond its effect on Na + channel function, mapre2 and in particularly its unique N-terminal segment may play a broader role in cellular organization via the microtubule network affecting cellular EP in multiple ways. Our study also implicates for the first time the Wnt signaling pathway in BrS, suggesting not only shared biology with arrhythmogenic cardiomyopathies, but also the possibility of GSK3β modulation as a novel therapeutic approach.

Transcription factor Meis1 act as a new regulator of ischemic arrhythmias in mice

IntroductionThe principal voltage-gated Na+ channel, NaV1.5 governs heart excitability and conduction. NaV1.5 dysregulation is responsible for ventricular arrhythmias and subsequent sudden cardiac death (SCD) in post-infarct hearts. The transcription factor Meis1 performs a significant role in determining differentiation fate and regenerative capability of cardiomyocytes. However, the functions of Meis1 in ischemic arrhythmias following myocardial infarction (MI) are still largely undefined. ObjectivesHere we aimed to study whether Meis1 could act as a key regulator to mediate cardiac Na+ channel and its underlying mechanisms. MethodsHeart-specific Meis1 overexpression was established by AAV9 virus injection in C57BL/6 mice. The QRS duration, the incidence of ventricular arrhythmias and cardiac conduction velocity were evaluated by ECG, programmed electrical stimulation and optical mapping techniques respectively. The conventional patch clamp technique was performed to explore the INa characteristics of isolated mouse ventricular myocytes. In vitro, Meis1 was also overexpressed in hypoxic-treated neonatal cardiomyocytes. The analysis of immunoblotting and immunofluorescence were used to detect the changes in the expression of NaV1.5 in each group. ResultsWe found that forced expression of Meis1 rescued the prolongation of QRS complex, produced anti-arrhythmic activity and improved epicardial conduction velocity in infarcted mouse hearts. In terms of mechanisms, cardiac electrophysiological changes of MI mice can be ameliorated by the recovery of Meis1, which is characterized by the restoration of INa current density and NaV1.5 expression level of cardiomyocytes in the marginal zone of MI mouse hearts. Furthermore, in vitro studies showed that Meis1 was also able to rescue hypoxia-induced decreased expression and dysfunction of NaV1.5 in ventricular myocytes. We further revealed that E3 ubiquitin ligase CDC20 led to the ubiquitination and degradation of Meis1, which blocked the transcriptional regulation of SCN5A by Meis1 and ultimately led to the electrophysiological remodeling in ischemic-hypoxic cardiomyocytes. ConclusionCDC20 mediates ubiquitination of Meis1 to govern the transcription of SCN5A and cardiac electrical conduction in mouse cardiomyocytes. This finding uncovers a new mechanism of NaV1.5 dysregulation in infarcted heart, and provides new therapeutic strategies for malignant arrhythmias and sudden cardiac death following MI.

Melatonin receptor blockade slows ventricular conduction by IK1 downregulation

Abstract Background Melatonin has been reported to prevent life-threatening arrhythmias in myocardial ischemia and reperfusion. The recent study from our group demonstrated that the antiarrhythmic action of melatonin was associated with enhanced recovery of resting membrane potential (RMP) and shortened activation time delay in the affected myocardium. This effect was independent of melatonin antioxidative properties, which raises a question about the involvement of melatonin receptor-dependent signaling in the electrophysiological action of melatonin. The aim of this study was to evaluate a role of MT1/MT2 melatonin receptors in the regulation of RMP and ventricular conduction velocity (CV). Methods Anesthetized male rats underwent coronary occlusion (5 min) and reperfusion (5 min). 5 min before ischemia induction the animals were intravenously injected with saline (control, n=7), melatonin (4 mg/kg, n=8), luzindole (MT1/MT2 receptor blocker, 0.4 mg/kg, n=6), and melatonin/luzindole combination (4 and 0.4 mg/kg, respectively, n=7). CV was measured in baseline and reperfusion by epicardial 64-lead mapping under epicardial pacing. In the enzymatically isolated ventricular myocytes (n=20 cells), voltage- and current-clamp modes of whole cell patch-clamp were used for measurements of IK1 and INa currents, and RMP, respectively. Melatonin (10 uM), luzindole (1 uM) and their combination were added to the bath solution. Results Luzindole application decreased baseline longitudinal CV as compared to placebo (52±3 vs 42±2 cm/s, p=0.028, respectively). Paired comparisons demonstrated that longitudinal CV decreased at reperfusion vs baseline in the control group (35±2 vs 53±6 cm/s, p=0.018) and in the luzindole group (33±4 vs 38±3 cm/s, p=0.039), whereas no significant differences were observed in the melatonin and melatonin/luzindole groups (40±5 vs 51±3 cm/s, p=0.114 and 39±3 vs 43±3 cm/s, p=0.498, respectively). Neither melatonin nor luzindole changed INa current characteristics (Figure, panels a and b). The average amplitude of IK1 current was 2.08±0.92 at −50 mV and −16.11±4.70 pA/pF at −120 mV in the normal Tyrode solution. Luzindole significantly reduced IK1 amplitude to 0.82±0.48 (p=0.028) and −5.6±1.6 (p=0.008) pA/pF at −50 and −120 mV, respectively. Addition of melatonin to the luzindole-containing solution partly abolished the luzindole effect (Figure, panel c). Luzindole also reduced RMP from −63±10 mV to −37±12 mV (p=0.013) and caused an increase in APD100 duration from 35±6 ms to 170±43 ms (p=0.003) (Figure, panel d). Conclusion Thus, MT1/MT2 receptor block with luzindole did not affect INa but led to the down-regulation of IK1 and depolarization of RMP. These effects can reduce accessibility of sodium channels and be the basis for the observed luzindole-dependent CV slowing. The obtained data support the significance of melatonin receptor-dependent signaling in the realization of antiarrhythmic effects of melatonin. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Russian Science Foundation

Effects of post-treatment electroacupuncture on ventricular monophasic action potential and cardiac function in a rat model of ischemia/reperfusion injury.

To determine the effects of post-treatment electroacupuncture (EA) on the electrophysiological properties of ventricular muscle in rats with ischemia/reperfusion (IR) injury. Male Sprague-Dawley (SD) rats were randomly assigned into sham-operated (SH), IR and IR + EA groups (n = 8 each). The IR model was generated by ligation of the left anterior descending (LAD) coronary artery for 30 min. After establishing the IR model, EA was administered at PC6 for 30 min while opening the coronary artery and allowing reperfusion for 30 min. Heart rate (HR), mean arterial pressure and monophasic action potential (MAP) of cardiac muscle in the outer membrane of the antetheca of the left ventricle before coronary artery ligation (T0), after coronary artery ligation for 30 min (T1) and after reperfusion for 30 min (T2) were recorded. At the same time, ventricular electrophysiological parameters including ventricular effective refractory period (ERP), conduction velocity (CV) and ventricular fibrillation threshold (VFT) were measured. Then, the cardiac function and the levels of creatine kinase-muscle/brain (CK-MB) and cardiac troponin I (cTnI) were monitored. Based on these data, monophasic action potential amplitude (MAPA), the maximum depolarization velocity (Vmax) and the MAP durations at 50% and 90% repolarization (MAPD50 and MAPD90) were calculated to determine the incidence of arrhythmia during reperfusion. Compared with the SH group, the IR group showed an obviously decreased HR as well as reduced mean arterial pressure, Vmax, CV, ERP and MAPA. All indices of cardiac function except left ventricular end-diastolic pressure (LVEDP) decreased (i.e. ventricular systolic pressure (LVSP), left ventricular ejection fraction (LVEF), fractional shortening (FS) and rate of the ventricular pressure rise/drop (±dp/dt)). Furthermore, the MAPD50 and MAPD90 were prolonged, and the levels of CK-MB and cTnI increased (p < 0.05). In comparison to the IR group, HR and the mean arterial pressure were increased. All indices of cardiac function except LVEDP increased (LVSP, LVEF, FS and ±dp/dt). Vmax, CV, ERP and MAPA were also increased in the IR + EA group. However, MAPD50 and MAPD90 were distinctly shortened, and the levels of CK-MB and cTnI decreased (p < 0.05). There were no statistically significant differences in VFT between the three groups (p > 0.05). EA post-treatment can relieve prolongation of repolarization and slowed depolarization of ventricular muscle during IR, thus decreasing the rate of incidence of reperfusion arrhythmia.

Targeting the Microtubule EB1-CLASP2 Complex Modulates NaV1.5 at Intercalated Discs.

[Figure: see text].

Analysis of Resibufogenin on Cardiac conduction reveals a species difference in the cardiac electrophysiology: Rats versus guinea pigs.

Resibufogenin (RBG) is a chemical ingredient of Chan Su. In our research, we found RBG affected cardiac rhythm in a negative chronotropic way in vivo. The cardiac Mapping system ex vivo and the patch clamp in vitro were used to explore how RBG influenced the cardiac electrophysiological properties. The negative chronotropic action of RBG at 100 μM might be attribute to prolongation in the atrioventricular conduction time and reduction in the ventricular conduction velocity. Using whole-cell patch clamp in ventricular myocytes of adult rats, we found that RBG prolonged the action potential duration (APD) in APD20, APD50, and APD90 at 100 μM and inhibited calcium currents (ICa), total outward potassium currents (IK), and transient outward potassium current (Ito) in a concentration-dependent manner, but not on the inward rectifying potassium current (IK1). Notably, RBG had a potent proarrhythmic action ex vivo in the isolated perfused guinea pig hearts at 10 μM, but not in rats. To avoid the potential cardiotoxicity derived from the distributional differences of ion channels among species, the effect of RGB on IKr in hERG-HEK293 cells was detected. The IC50 of RGB on IKr was more than 100 μM. In summary, all these results indicated that the negative chronotropic action of RBG relied on the blocking activities on multiple ion channels, and the species-difference of proarrhythmic effects might result from lack of the Ito on the myocardial membrane of guinea pigs. Anyhow, the cardiotoxicity observed in guinea pigs required further detailed studies to mitigate the potential risks in the clinical application of Chan Su.

Transcription&amp;nbsp;Factor&amp;nbsp;Meis1&amp;nbsp;Act&amp;nbsp;As&amp;nbsp;a&amp;nbsp;New&amp;nbsp;Regulator&amp;nbsp;Of&amp;nbsp;Ischemic&amp;nbsp;Arrhythmias&amp;nbsp;In&amp;nbsp;Mice

Background: The principal voltage-gated Nam+ channel, NaV1.5 governs heart excitability and conduction. NaV1.5 dysregulation is responsible for ventricular arrhythmias and subsequent sudden cardiac death in post-infarct hearts. The transcription factor Meis1 plays an important role in determining differentiation fate and regenerative capability of cardiomyocytes. However, the functions of Meis1 in ischemic arrhythmias following myocardial infarction (MI) are still largely undefined. Here we set out to study whether Meis1 could act as a key regulator to mediate cardiac Na+ channels and arrhythmogenesis and its underlying mechanisms. Methods: Heart-specific Meis1 overexpression was established by AAV9 virus injection in C57BL/6 mice. The QTc interval, the incidence of ventricular arrhythmias and cardiac conduction velocity were evaluated by ECG, programmed electrical stimulation and optical mapping techniques respectively. The conventional patch clamp technique was used to study the I Na characteristics of isolated mouse ventricular myocytes. Findings: We found that forced expression of Meis1 rescued the prolongation of heart rate corrected QT (QTc) interval, produced anti-arrhythmic activity and improved epicardial conduction velocity in infarcted mouse hearts. Mechanistically, Meis1 may ameliorate cardiac electrophysiological remodeling as characterized by the decrease of INa current density and NaV1.5 expression in marginal zone of infarcted mouse hearts. Furthermore, in vitro studies showed that E3 ubiquitin ligase CDC20 led to the ubiquitination and degradation of Meis1, which blocked the transcriptional regulation of SCN5A. Interpretation: This study demonstrates that Meis1 overexpression can effectively inhibit the ventricular arrhythmia by regulating the transcription of SCN5A and cardiac electrical conductance in mouse ischemic heart, which provides new therapeutic strategies for malignant arrhythmias and sudden cardiac death following MI. Funding: This work was supported by the National Nature Science Foundation of China [Grant No. 81573425 and 81773733 to NW]. Declaration of Interest: None to declare. Ethical Approval: All animal experiments were performed along with the NIH guidelines (Guide for the care and use of laboratory animals, NIH Publication No. 85-23, revised 1996) and were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Harbin Medical University.

The bradycardic agent ivabradine decreases conduction velocity in the AV node and in the ventricles in-vivo

Ivabradine blocks hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels, thereby lowering the heart rate, an action that is used clinically for the treatment of heart failure and angina pectoris. We and others have shown previously that ivabradine, in addition to its HCN channel blocking activity, also inhibits voltage-gated Na channels in vitro at concentrations that may be clinically relevant. Such action may reduce conduction velocity in cardiac atria and ventricles.Here, we explore the effect of administration of ivabradine on parameters of ventricular conduction and repolarization in the surface ECG of anesthetized mice. We found that 5 min after i.p. administration of 10 mg/kg ivabradine spontaneous heart rate had declined by ~13%, which is within the range observed in human clinical studies. At the same time a significant increase in QRS duration by ~18% was observed, suggesting a reduction in ventricular conduction velocity. During transesophageal pacing at heart rates between 100 and 220 beats/min there was no obvious rate-dependence of ivabradine–induced QRS prolongation. On the other hand, ivabradine produced substantial rate-dependent slowing of AV nodal conduction. We conclude that ivabradine prolongs conduction in the AV-node and in the ventricles in vivo.