The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes, make fluorescence resonance energy transfer (FRET) a powerful technique for studying molecular interactions inside living cells with improved spatial (angstrom) and temporal (nanosecond) resolution, distance range, and sensitivity and a broader range of biological applications.

Background-Mesenchymal stem cells (MSCs) are bone marrow stromal cells that are in phase 1 clinical studies of cellular cardiomyoplasty. However, the electrophysiological effects of MSC transplantation have not been studied. Although improvement of ventricular function would represent a positive outcome of MSC transplantation, focal application of stem cells has the potential downside of creating inhomogeneities that may predispose the heart to reentrant arrhythmias. In the present study we use an MSC and neonatal rat ventricular myocyte (NRVM) coculture system to investigate potential proarrhythmic consequences of MSC transplantation into the heart. Methods and Results-Human MSCs were cocultured with NRVMs in ratios of 1:99, 1:9, and 1:4 and optically mapped.We found that conduction velocity was decreased in cocultures compared with controls, but action potential duration (APD 80 ) was not affected. Reentrant arrhythmias were induced in 86% of cocultures containing 10% and 20% MSCs (nϭ36) but not in controls (nϭ7) or cocultures containing only 1% MSCs (nϭ4). Immunostaining, Western blot, and dye transfer revealed the presence of functional gap junctions involving MSCs. Conclusions-Our results suggest that mixtures of MSCs and NRVMs can produce an arrhythmogenic substrate. The mechanism of reentry is probably increased tissue heterogeneity resulting from electric coupling of inexcitable MSCs with myocytes.

QT interval variation is assumed to arise from variation in repolarization as evidenced from rare Na- and K-channel mutations in Mendelian QT prolongation syndromes. However, in the general population, common noncoding variants at a chromosome 1q locus are the most common genetic regulators of QT interval variation. In this study, we use multiple human genetic, molecular genetic, and cellular assays to identify a functional variant underlying trait association: a noncoding polymorphism (rs7539120) that maps within an enhancer of NOS1AP and affects cardiac function by increasing NOS1AP transcript expression. We further localized NOS1AP to cardiomyocyte intercalated discs (IDs) and demonstrate that overexpression of NOS1AP in cardiomyocytes leads to altered cellular electrophysiology. We advance the hypothesis that NOS1AP affects cardiac electrical conductance and coupling and thereby regulates the QT interval through propagation defects. As further evidence of an important role for propagation variation affecting QT interval in humans, we show that common polymorphisms mapping near a specific set of 170 genes encoding ID proteins are significantly enriched for association with the QT interval, as compared to genome-wide markers. These results suggest that focused studies of proteins within the cardiomyocyte ID are likely to provide insights into QT prolongation and its associated disorders.

Abstract-Previous studies have postulated an important role for the inwardly rectifying potassium current (I K1 ) in controlling the dynamics of electrophysiological spiral waves responsible for ventricular tachycardia and fibrillation. In this study, we developed a novel tissue model of cultured neonatal rat ventricular myocytes (NRVMs) with uniform or heterogeneous Kir2.1expression achieved by lentiviral transfer to elucidate the role of I K1 in cardiac arrhythmogenesis. Kir2.1-overexpressed NRVMs showed increased I K1 density, hyperpolarized resting membrane potential, and increased action potential upstroke velocity compared with green fluorescent protein-transduced NRVMs. Opposite results were observed in Kir2.1-suppressed NRVMs. Optical mapping of uniformly Kir2.1 gene-modified monolayers showed altered conduction velocity and action potential duration compared with nontransduced and empty vector-transduced monolayers, but functional reentrant waves could not be induced. In monolayers with an island of altered Kir2.1 expression, conduction velocity and action potential duration of the locally transduced and nontransduced regions were similar to those of the uniformly transduced and nontransduced monolayers, respectively, and functional reentrant waves could be induced. The waves were anchored to islands of Kir2.1 overexpression and remained stable but dropped in frequency and meandered away from islands of Kir2.1 suppression. In monolayers with an inverse pattern of I K1 heterogeneity, stable high frequency spiral waves were present with I K1 overexpression, whereas lower frequency, meandering spiral waves were observed with I K1 suppression. Our study provides direct evidence for the contribution of I K1 heterogeneity and level to the genesis and stability of spiral waves and highlights the potential importance of I K1 as an antiarrhythmia target. Key Words: Kir2.1 Ⅲ inwardly rectifying potassium current Ⅲ reentry Ⅲ spiral waves Ⅲ ventricular tachycardia Ⅲ ventricular fibrillation V entricular fibrillation (VF) is the leading cause of cardiac arrest and sudden cardiac death in the industrialized world. 1 Studies in the 1970s suggested that the heart could sustain electrical activity that rotated around a functional obstacle. 2,3 These reentrant waves are believed to be the unitary components of fibrillation. Several other studies that focused on understanding the mechanisms of initiation and maintenance of VF concluded that the stability of spiral waves (functional form of reentrant waves) depends on the abbreviation of action potential duration, as well as the reduction of wavefront-wavetail interactions, at fibrillation frequencies. 4 -6 In addition, ionic heterogeneity may be a key factor in the initiation of spiral waves and their transition to the irregular spatiotemporal pattern seen in VF. [7][8][9] Numerous studies pioneered mainly by Jalife and colleagues have indicated that I K1 plays an important role in determining cardiac excitability and arrhythmogenesis and that I K1 block has a significa...

Although stretch-activated currents have been extensively studied in isolated cells and intact hearts in the context of mechanoelectric feedback (MEF) in the heart, quantitative data regarding other mechanical parameters such as pressure, shear, bending, etc, are still lacking at the multicellular level. Cultured cardiac cell monolayers have been used increasingly in the past decade as an in vitro model for the studies of fundamental mechanisms that underlie normal and pathological electrophysiology at the tissue level. Optical mapping makes possible multisite recording and analysis of action potentials and wavefront propagation, suitable for monitoring the electrophysiological activity of the cardiac cell monolayer under a wide variety of controlled mechanical conditions. In this paper, we review methodologies that have been developed or could be used to mechanically perturb cell monolayers, and present some new results on the acute effects of pressure, shear stress and anisotropic strain on cultured neonatal rat ventricular myocyte (NRVM) monolayers.

Recombinant lentiviral vectors (LVs) are capable of transducing neonatal rat ventricular myocytes (NRVMs) and providing stable, long-term transgene expression. The goal of the present study was to comprehensively test whether transduction of NRVMs by LVs results in cytotoxicity and to examine the electrophysiological consequences of gene modification of NRVM monolayers by two vectors: one encoding a putatively inert enhanced green fluorescent protein (eGFP) and the other a major ion channel protein, inward rectifier K(+) channel (Kir) 2.1. Freshly isolated NRVMs were transduced and cultured in monolayers. Immunohistochemistry, Trypan blue exclusion, annexin V binding followed by flow cytometry (FCM), and terminal transferase dUTP nick-end labeling assays were performed to assess for cytotoxicity. Optical mapping studies of action potential propagation in NRVM monolayers were performed to characterize the electrophysiological alterations following transduction. The cytotoxicity assays revealed that transduction had no adverse effects on NRVM cultures. However, eGFP-transduced monolayers exhibited a decrease in conduction velocity (CV) and action potential duration (APD) compared with monolayers transduced with LVs encoding LacZ or devoid of a transgene. In addition, small interfering RNA-mediated knockdown of eGFP expression corrected this phenotype. In contrast, Kir2.1 gene-modified monolayers showed an increase in CV and a predictable decrease in APD. This study demonstrates that LVs transduce NRVMs without cytotoxic effects. However, eGFP has a significant effect on APD and CV in this experimental system and calls into question the widely held belief that GFP is physiologically inert. In addition, LV-mediated overexpression of Kir2.1 opens up the prospect of studying the functional role of inward rectifier K(+) current in cardiac arrhythmias.

Abstract-Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT G ap junctions are channels that permit intercellular communication. In mammalian tissues these channels are ubiquitously expressed and serve diverse biological functions. 1 Within the heart, gap junctions mediate electrical impulse transmission which underlies its coordinated mechanical activity. 2 Acquired heart disease and the ensuing gap junction remodeling can alter electrical conduction in a manner which begets arrhythmias. For example, this was found to be true of ventricular tachycardia (VT) occurring after chronic myocardial infarction (MI) where focal conduction slowing forms an essential part of the arrhythmogenic substrate. 2 The clinical burden of this pathophysiology is evidenced by the fact that post-MI VT is a substantial cause of sudden cardiac death (SCD). 3 Contemporary therapies for VT/SCD include antiarrhythmic drugs, implantable defibrillators, and catheter-based radiofrequency ablation. The first of these has met with limited efficacy in clinical trials largely because of increased proarrhythmic events. 3 Defibrillators, although efficacious, are associated with a considerable cost and do not directly impact the underlying pathophysiology. Radiofrequency ablation is associated with modest success rates even when performed by highly-experienced operators and, furthermore, many patients are not amenable to this approach. 3 Studies exploring new treatment approaches, such as gene therapy, are emerging with a recent example reporting inhibition of post-MI VT in a porcine model by targeting repolarization. 4 With these issues in mind, a novel alternative approach based on gene transfer technology targeting gap junctional intercellular communication (GJIC) was pursued. This approach has several potential advantages including targeting of the mechanism causing the conduction disturbance, creation of a nondestructive therapy, and the potential to confer a permanent effect depending on the vector employed.Here we explore the potential of a genetic inhibitor or loss-of-function approach directed at GJIC. Specifically, overexpression of internal loop mutants of the Cx43 gene are employed to uncouple cells connected by gap junctions. These mutants result in gap junctions that lack electrical communication when heterologously expressed in cell pairs. 5

Patterns of cellular organization in diverse tissues frequently display a complex geometry and topology tightly related to the tissue function. Progressive disorganization of tissue morphology can lead to pathologic remodeling, necessitating the development of experimental and theoretical methods of analysis of the tolerance of normal tissue function to structural alterations. A systematic way to investigate the relationship of diverse cell organization to tissue function is to engineer two-dimensional cell monolayers replicating key aspects of the in vivo tissue architecture. However, it is still not clear how this can be accomplished on a tissue level scale in a parameterized fashion, allowing for a mathematically precise definition of the model tissue organization and properties down to a cellular scale with a parameter dependent gradual change in model tissue organization. Here, we describe and use a method of designing precisely parameterized, geometrically complex patterns that are then used to control cell alignment and communication of model tissues. We demonstrate direct application of this method to guiding the growth of cardiac cell cultures and developing mathematical models of cell function that correspond to the underlying experimental patterns. Several anisotropic patterned cultures spanning a broad range of multicellular organization, mimicking the cardiac tissue organization of different regions of the heart, were found to be similar to each other and to isotropic cell monolayers in terms of local cell–cell interactions, reflected in similar confluency, morphology and connexin-43 expression. However, in agreement with the model predictions, different anisotropic patterns of cell organization, paralleling in vivo alterations of cardiac tissue morphology, resulted in variable and novel functional responses with important implications for the initiation and maintenance of cardiac arrhythmias. We conclude that variations of tissue geometry and topology can dramatically affect cardiac tissue function even if the constituent cells are themselves similar, and that the proposed method can provide a general strategy to experimentally and computationally investigate when such variation can lead to impaired tissue function.