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 ...