Abstract-Structural and functional cardiac anisotropy varies with the development, location, and pathophysiology in the heart. The goal of this study was to design a cell culture model system in which the degree, change in fiber direction, and discontinuity of anisotropy can be controlled over centimeter-size length scales. Neonatal rat ventricular myocytes were cultured on fibronectin on 20-mm diameter circular cover slips. Structure-function relationships were assessed using immunostaining and optical mapping. Cell culture on microabraded cover slips yielded cell elongation and coalignment in the direction of abrasion, and uniform, macroscopically continuous, elliptical propagation with point stimulation. Coarser microabrasion (wider and deeper abrasion grooves) increased longitudinal (23.5 to 37.2 cm/s; rϭ0.66) and decreased transverse conduction velocity (18.1 to 9.2 cm/s; rϭϪ0.84), which resulted in increased longitudinal-to-transverse velocity anisotropy ratios (1.3 to 3.7, nϭ61). A thin transition zone between adjacent uniformly anisotropic areas with 45°or 90°difference in fiber orientation acted as a secondary source during 2ϫ threshold field stimulus. Cell culture on cover slips micropatterned with 12-or 25-m wide fibronectin lines and previously coated with decreasing concentrations of background fibronectin yielded transition from continuous to discontinuous anisotropic architecture with longitudinally oriented intercellular clefts, decreased transverse velocity (16.9 to 2.6 cm/s; rϭϪ0.95), increased velocity anisotropy ratios (1.6 to 5.6, nϭ70), and decreased longitudinal velocity (36.4 to 14.6 cm/s; rϭϪ0.85) for anisotropy ratios Ͼ3.5. Cultures of cardiac myocytes with controlled degree, uniformity and continuity of structural, and functional anisotropy may enable systematic 2-dimensional in vitro studies of macroscopic structure-related mechanisms of reentrant arrhythmias.

Objectives We sought to test whether c-Src tyrosine kinase mediates connexin 43 (Cx43) reduction and sudden cardiac death in a transgenic mouse model of cardiac-restricted overexpression of angiotensin-converting enzyme (ACE8/8). Background Renin-angiotensin system (RAS) activation is associated with an increased risk of arrhythmia and sudden cardiac death; however, that mechanism is not well understood. The upregulation of c-Src by angiotensin II may result in the reduction of Cx43, which impairs gap junction function and provides a substrate for arrhythmia. Method Wild-type and ACE8/8 mice with and without treatment with the c-Src inhibitor PP1 were studied. Telemetry monitoring, in vivo electrophysiology studies, Western blot analyses for total and phosphorylated c-Src and Cx43, immunohistochemistry staining for Cx43, and functional assessment of Cx43 with fluorescent dye diffusion were performed. Results The majority of the arrhythmic deaths resulted from ventricular tachycardia denegerating to ventricular fibrillation (83%). Levels of total and phosphorylated c-Src were increased and Cx43 reduced in ACE8/8 mice. PP1 reduced total and phospho c-Src levels, increased the Cx43 level by 2.1-fold (P < 0.005), increased Cx43 at the gap junctions (immunostaining), improved gap junctional communication (dye spread), and reduced ventricular tachycardia inducibility and sudden cardiac death. The survival rate increased from 11% to 86% with four weeks of PP1 treatment (P < 0.005). Treatment with an inactive analog did not change survival or Cx43 levels. Conclusion RAS activation is associated with c-Src upregulation, Cx43 loss, reduced myocyte coupling, and arrhythmic sudden death, which can be prevented by c-Src inhibition. This suggests that an increase in c-Src activity may help mediate RAS-induced arrhythmias and that c-Src inhibitors might exert antiarrhythmic activity.

The role of the renin-angiotensin-aldosterone system (RAAS) in many cardiovascular disorders, including hypertension, cardiac hypertrophy, and atherosclerosis is well established, whereas its relationship with cardiac arrhythmias is a new area of investigation. Atrial fibrillation and malignant ventricular tachyarrhythmias, especially in the setting of cardiac hypertrophy or failure, appear to be examples of RAAS-related arrhythmias, since treatment with RAAS modulators, including angiotensin converting enzyme inhibitors, angiotensin receptor blockers and mineralocorticoid receptor blockers, reduces the incidence of these arrhythmias. RAAS has a multitude of electrophysiological effects and can potentially cause arrhythmia through a variety of mechanisms. We review new experimental results that suggest RAAS has pro-arrhythmic effects on membrane and sarcoplasmic reticulum ion channels and that increased oxidative stress is likely contributing to the increased arrhythmic incidence. A summary of ongoing clinical trials that will address the clinical usefulness of RAAS modulators for prevention or treatment of arrhythmias is presented.

Previous studies of reentrant arrhythmias in the heart have been performed in computer models and tissue experiments. We hypothesized that confluent monolayers of cardiac cells can provide a simple, controlled, and reproducible experimental model of reentry. Neonatal rat ventricular cells were cultured on 22-mm-diameter coverslips and stained with the voltage-sensitive dye RH-237. Recordings of transmembrane potentials were obtained from 61 sites with the use of a contact fluorescence imaging system. An electrical field stimulus, followed by a point stimulus, induced 39 episodes of sustained reentry and 21 episodes of nonsustained reentry. Sustained reentry consisted of single-loop (n ϭ 18 monolayers) or figure-of-eight (n ϭ 4) patterns. The cycle length, action potential duration at 80% repolarization, and conduction velocity were (in means Ϯ SE) 358 Ϯ 33 ms, 118 Ϯ 12 ms, and 12.9 Ϯ 1.0 cm/s for single loop and 311 Ϯ 78 ms, 137 Ϯ 18 ms, and 7.8 Ϯ 1.3 cm/s for figure-of-eight, respectively. Electrical termination by 6-to 13-V/cm field pulses or 15-to 20-V point stimuli was successful in 60% of the attempts. In summary, highly stable reentry can be induced, sustained for extensive periods of time, and electrically terminated in monolayers of cultured neonatal rat cardiac myocytes. arrhythmia; cardiac electrophysiology; voltage-sensitive dye; optical mapping UNIDIRECTIONAL CONDUCTION block and formation of wave breaks during propagation in heart muscle can result in self-sustained propagation of electrical and mechanical activity, in which an activation wave front can reenter the same area after propagating around a fixed anatomic or functional obstacle (9). Functional reentry is generally accepted to be the major mechanism underlying many monomorphic and polymorphic tachycardias, which can evolve into irregular electrical activity such as atrial or ventricular flutter and fibrillation. Although the initiation and cardioversion of functional reentry have been subjects of numerous theoretical and experimental studies (2-4, 12, 23, 24, 27, 31, 33), there still exists a need for the development of new model systems that can aid in the understanding and treatment of these life-threatening arrhythmias.The use of cultured monolayers of cardiac cells as a simplified model for the study of functional cardiac electrophysiology offers many advantages, including: 1) control of the cell microenvironment, 2) elimination of excitation-contraction decouplers that are used for optical mapping but may alter the electrophysiological properties of the cells (17, 18), 3) removal of large scale tissue heterogeneities such as blood vessels, connective tissue, or rotational anisotropy, and 4) the certainty that the electrophysiological signals are produced from a known layer of cells, thus enabling a one-to-one correspondence with two-dimensional computer simulations and nonlinear dynamic theory. Currently, there is a gap between the computer simulations, which assume an ideal homogeneous excitable media, and tissue experiments, where ...

(RAS) system activation is associated with an increased risk of sudden death. Previously, we used cardiac-restricted angiotensin-converting enzyme (ACE) overexpression to construct a mouse model of RAS activation. These ACE 8/8 mice die prematurely and abruptly. Here, we have investigated cardiac electrophysiological abnormalities that may contribute to early mortality in this model. In ACE 8/8 mice, surface ECG voltages are reduced. Intracardiac electrograms showed atrial and ventricular potential amplitudes of 11% and 24% compared with matched wild-type (WT) controls. The atrioventricular (AV), atrioHisian (AH), and Hisian-ventricular (HV) intervals were prolonged 2.8-, 2.6-, and 3.9-fold, respectively, in ACE 8/8 vs. WT mice. Various degrees of AV nodal block were present only in ACE 8/8 mice. Intracardiac electrophysiology studies demonstrated that WT and heterozygote (HZ) mice were noninducible, whereas 83% of ACE 8/8 mice demonstrated ventricular tachycardia with burst pacing. Atrial connexin 40 (Cx40) and connexin 43 (Cx43) protein levels, ventricular Cx43 protein level, atrial and ventricular Cx40 mRNA abundances, ventricular Cx43 mRNA abundance, and atrial and ventricular cardiac Na ϩ channel (Scn5a) mRNA abundances were reduced in ACE 8/8 compared with WT mice. ACE 8/8 mice demonstrated ventricular Cx43 dephosphorylation. Atrial and ventricular L-type Ca 2ϩ channel, Kv4.2 K ϩ channel ␣-subunit, and Cx45 mRNA abundances and the peak ventricular Na ϩ current did not differ between the groups. In isolated heart preparations, a connexin blocker, 1-heptanol (0.5 mM), produced an electrophysiological phenotype similar to that seen in ACE 8/8 mice. Therefore, cardiac-specific ACE overexpression resulted in changes in connexins consistent with the phenotype of low-voltage electrical activity, conduction defects, and induced ventricular arrhythmia. These results may help explain the increased risk of arrhythmia in states of RAS activation such as heart failure.peptidyl-dipeptidase A; angiotensin II; heart block ARRHYTHMIC SUDDEN DEATH is a common terminal event in various cardiomyopathies and end-stage heart failure. Upregulation of the renin-angiotensin system (RAS) has been implicated in risk of sudden death in these conditions. A critical component of this system is angiotensin-converting enzyme (ACE), which produces the eight-amino acid peptide angiotensin II (ANG II), a major effector peptide of the RAS. In humans, increased ANG II levels are associated with an increased risk of arrhythmia (2), which is reduced by use of ACE inhibitors or ANG II receptor blockers (4,13,20,23,27,30,49).A number of ion-handling protein changes have been posited to underlie the increase in risk of arrhythmia in states of RAS activation, and ANG II is known to act on a number of these proteins (3, 41). For example, ANG II has been implicated in Na ϩ -K ϩ pump regulation (24). Furthermore, ANG II inhibits the Ca 2ϩ -activated K ϩ current in vascular smooth muscle cells (51) and the delayed rectifier K ϩ currents in heart and...

BACKGROUND Early during the current coronavirus disease 19 (COVID-19) pandemic, hydroxychloroquine (HCQ) received a significant amount of attention as a potential antiviral treatment, such that it became one of the most commonly prescribed medications for COVID-19 patients. However, not only has the effectiveness of HCQ remained questionable, but mainly based on preclinical and a few small clinical studies, HCQ is known to be potentially arrhythmogenic, especially as a result of QT prolongation.OBJECTIVE The purpose of this study was to investigate the arrhythmic effects of HCQ, as the heightened risk is especially relevant to COVID-19 patients, who are at higher risk for cardiac complications and arrhythmias at baseline. METHODSAn optical mapping technique utilizing voltagesensitive fluorescent dyes was used to determine the arrhythmic effects of HCQ in ex vivo guinea pig and rabbit hearts perfused with the upper therapeutic serum dose of HCQ (1000 ng/mL).RESULTS HCQ markedly increased action potential dispersion, resulted in development of repolarization alternans, and initiated polymorphic ventricular tachycardia.CONCLUSION The study results further highlight the proarrhythmic effects of HCQ.

Renin-angiotensin system (RAS) activation is associated with arrhythmias. We investigated the effects of RAS inhibition in cardiac-specific angiotensin-converting enzyme (ACE) overexpression (ACE 8/8) mice, which exhibit proclivity to ventricular tachycardia (VT) and sudden death because of reduced connexin43 (Cx43). ACE 8/8 mice were treated with an ACE inhibitor (captopril) or an angiotensin receptor type-1 blocker (losartan). Subsequently, electrophysiological studies were performed, and the hearts were extracted for Cx43 quantification using immunoblotting, immunohistochemistry, fluorescent dye spread method, and sodium current quantification using whole cell patch clamping. VT was induced in 12.5% of captopril-treated ACE 8/8 and in 28.6% of losartan-treated mice compared to 87.5% of untreated mice (P<0.01). Losartan and captopril treatment increased total Cx43 2.4-fold (P=0.01) and the Cx43 phosphorylation ratio 2.3-fold (P=0.005). Treatment was associated with a recovery of gap junctional conductance. Survival in treated mice improved to 0.78 at 10 weeks (95% confidence interval 0.64 to 0.92), compared to the expected survival of less than 0.50. In a model of RAS activation, arrhythmic risk was correlated with reduced Cx43 amount and phosphorylation. RAS inhibition resulted in increased total and phosphorylated Cx43, decreased VT inducibility, and improved survival.

BackgroundIt is important to understand the relationship between electrical and mechanical ventricular activation in CRT patients. By measuring local electrical activation at multiple locations within the coronary veins and myocardial contraction at the same locations in the left ventricle, we determined the relationship between electrical and mechanical activation at potential left ventricular pacing locations.MethodsIn this study, mechanical contraction times were computed using high temporal resolution cine cardiovascular magnetic resonance (CMR) data, while electrical activation times were derived from intra-procedural local electrograms.ResultsIn our cohort, there was a strong correlation between electrical and mechanical delay times within each patient (R2 = 0.78 ± 0.23). Additionally, the latest electrically activated location corresponded with the latest mechanically contracting location in 91% of patients.ConclusionsThis study provides initial evidence that our method of obtaining non-invasive mechanical activation patterns accurately reflects the underlying electromechanical substrate of intraventricular dyssynchrony.