Properties of blebbistatin for cardiac optical mapping and other imaging applications.

Blebbistatin is a recently discovered myosin II inhibitor. It is rapidly becoming a compound of choice to reduce motion artifacts during cardiac optical mapping, as well as to study cell motility and cell invasion. Although blebbistatin has a number of advantages over other electromechanical uncouplers, many of its properties have yet to be addressed. Here we describe several methodological issues associated with the use of blebbistatin, including its spectral properties, reversibility, and its effect on tissue metabolic state. We show that if precautions are not taken, perfusion with blebbistatin may result in blebbistatin precipitate that accumulates in the vasculature. Although such precipitate is fluorescent, it is not detectable within wavelength bands that are typically used for transmembrane voltage fluorescence imaging (i.e., emission wavelengths >600 nm). Therefore, blockage of the microcirculation by blebbistatin may cause data misinterpretation in studies that use voltage-sensitive dyes. Blebbistatin may also impact imaging of green fluorophores due to the spectral shift it causes in endogenous tissue fluorescence. 3D excitation-emission matrices of blebbistatin in precipitate form and in various solutions (DMSO, water, and 1 % aqueous albumin) revealed significant changes in the fluorescence of this molecule in different environments. Finally, we examined the reversibility of blebbistatin's uncoupling effect on cardiac contraction. Our findings provide important new information about the properties of this myosin II inhibitor, which will aid in the proper design and interpretation of studies that use this compound.

Locations of ectopic beats coincide with spatial gradients of NADH in a regional model of low-flow reperfusion.

We studied the origins of ectopic beats during low-flow reperfusion after acute regional ischemia in excised rat hearts. The left anterior descending coronary artery was cannulated. Perfusate was delivered to the cannula using an high-performance liquid chromatography pump. This provided not only precise control of flow rate but also avoided mechanical artifacts associated with vessel occlusion and deocclusion. Optical mapping of epicardial transmembrane potential served to identify activation wavefronts. Imaging of NADH fluorescence was used to quantify local ischemia. Our experiments suggest that low-flow reperfusion of ischemic myocardium leads to a highly heterogeneous ischemic substrate and that the degree of ischemia between adjacent patches of tissue changes in time. In contrast to transient ectopic activity observed during full-flow reperfusion, persistent ectopic arrhythmias were observed during low-flow reperfusion. The origins of ectopic beats were traceable to areas of high spatial gradients of changes in NADH fluorescence caused by low-flow reperfusion.

Controlled regional hypoperfusion in Langendorff heart preparations.

We describe a new approach that combines several techniques to allow abnormal electrical and calcium activity to be visualized within hypoperfused myocardial tissue. A flexible microcannula was inserted into the left anterior descending artery of Langendorff perfused rat hearts, an air-tight seal between the coronary artery and the cannula was created, and an HPLC pump was used to deliver a specified flowrate through the microcannula. High resolution optical mapping of NADH/calcium, NADH/voltage or calcium/voltage was then conducted using a dual camera system. The ECG was acquired using surface electrodes. This perfusion technique is superior to occluding a vessel by either a tie or a clamp because it allows precise control of the composition and amount of flow to a defined ischemic bed. Another advantage is that flow can be stopped and resumed remotely, without touching the heart. This allows ectopic beats, or other arrhythmogenic activity, such as alternans, to be recorded immediately after changes in flow are imposed. Altogether, the described method provides a powerful new tool to assess how coronary flow rate affects the degree of local ischemia by the ability to record abnormal patterns of electrical activity and associated intracellular calcium transients with high spatiotemporal resolution from epicardial areas as small as 100 x 100 microm.

Generation and escape of local waves from the boundary of uncoupled cardiac tissue.

We aim to understand the formation of abnormal waves of activity from myocardial regions with diminished cell-to-cell coupling. En route to this goal, we studied the behavior of a heterogeneous myocyte network in which a sharp coupling gradient was placed under conditions of increasing network automaticity. Experiments were conducted in monolayers of neonatal rat cardiomyocytes using heptanol and isoproterenol as means of altering cell-to-cell coupling and automaticity, respectively. Experimental findings were explained and expanded using a modified Beeler-Reuter numerical model. The data suggest that the combination of a heterogeneous substrate, a gradient of coupling, and an increase in oscillatory activity of individual cells creates a rich set of behaviors associated with self-generated spiral waves and ectopic sources. Spiral waves feature a flattened shape and a pin-unpin drift type of tip motion. These intercellular waves are action-potential based and can be visualized with either voltage or calcium transient measurements. A source/load mismatch on the interface between the boundary and well-coupled layers can lock wavefronts emanating from both ectopic sources and rotating waves within the inner layers of the coupling gradient. A numerical approach allowed us to explore how 1), the spatial distribution of cells, 2), the amplitude and dispersion of cell automaticity, and 3), the speed at which the coupling gradient moves in space affect wave behavior, including its escape into well-coupled tissue.

Genesis of ectopic waves: role of coupling, automaticity, and heterogeneity.

Many arrhythmias are believed to be triggered by ectopic sources arising from the border of the ischemic tissue. However, the development of ectopic activity from individual sources to a larger mass of cardiac tissue remains poorly understood. To address this critical issue, we used monolayers of neonatal rat cardiomyocytes to create conditions that promoted progression of ectopic activity from single cells to the network that consisted of hundreds of cells. To explain complex spatiotemporal patterns observed in these experiments we introduced a new theoretical framework. The framework's main feature is a parameter space diagram, which uses cell automaticity and coupling as two coordinates. The diagram allows one to depict network behavior, quantitatively address the heterogeneity factor, and evaluate transitions between different regimes. The well-organized wave trains were observed at moderate and high cell coupling values and network heterogeneity was found to be qualitatively unimportant for these regimes. In contrast, at lower values of coupling, spontaneous ectopic activity led to the appearance of fragmented ectopic waves. For these regimes, network heterogeneity played an essential role. The ectopic waves occasionally gave rise to spiral activity in two different regions within the parameter space via two distinct mechanisms. Together, our results suggest that localized ectopic waves represent an essential step in the progression of ectopic activity. These studies add to the understanding of initiation and progression of arrhythmias and can be applied to other phenomena that deal with assemblies of coupled oscillators.

Multiple injury approach and its use for toxicity studies.

We present an experimental approach that allows exposure of cells plated on a single coverslip to multiple distinct environments. The original chamber design created a small region of injury using geometrically defined flows of the control and ischemic solutions. Modifications of the original chamber design presented in this article produce a range of flow patterns that can be advantageous for a variety of imaging applications. These applications include: experiments that address effects of different treatments applied to a cell network, parallel testing of negative and positive controls using a single coverslip, border effect studies, evaluation of the treatment's reversibility, and simultaneous monitoring of a cell layer loaded with different fluorescent indicators. The method also can be used to reveal both micro- and macroscopic features of propagation, conduction, and cell coupling in a normal or altered cardiac cell network. These possibilities are illustrated in cultures of neonatal rat cardiomyocytes using oxidant- and calcium-sensitive fluorescent indicators.

Behavior of ectopic surface: effects of beta-adrenergic stimulation and uncoupling.

By using both experimental and theoretical means, we have addressed the progression of ectopic activity from individual cardiac cells to a multicellular two-dimensional network. Experimental conditions that favor ectopic activity have been created by local perfusion of a small area of cardiomyocyte network (I-zone) with an isoproterenol-heptanol containing solution. The application of this solution initially slowed down and then fully blocked wave propagation inside the I-zone. After a brief lag period, ectopically active cells appeared in the I-zone, followed by evolution of the ectopic clusters into slowly propagating waves. The changing pattern of colliding and expanding ectopic waves confined to the I-zone persisted for as long as the isoproterenol-heptanol environment was present. On restoration of the control environment, the ectopic waves from the I-zone broke out into the surrounding network causing arrhythmias. The observed sequence of events was also modeled by FitzHugh-Nagumo equations and included a cell's arrangement of two adjacent square regions of 20 x 20 cells. The control zone consisted of well-connected, excitable cells, and the I-zone was made of weakly coupled cells (heptanol effect), which became spontaneously active as time evolved (isoproterenol effect). The dynamic events in the system have been studied numerically with the use of a finite difference method. Together, our experimental and computational data have revealed that the combination of low coupling, increased excitability, and spatial heterogeneity can lead to the development of ectopic waves confined to the injured network. This transient condition appears to serve as an essential step for the ectopic activity to "mature" before escaping into the surrounding control network.

Initiation and propagation of ectopic waves: insights from an in vitro model of ischemia-reperfusion injury.

The objective of the present study was to directly visualize ectopic activity associated with ischemia-reperfusion and its progression to arrhythmia. To accomplish this goal, we employed a two-dimensional network of neonatal rat cardiomyocytes and a recently developed model of localized ischemia-reperfusion. Washout of the ischemia-like solution resulted in tachyarrhythmic episodes lasting 15-200 s. These episodes were preceded by the appearance of multiple ectopic sources and propagation of ectopic activity along the border of the former ischemic zone. The ectopic sources exhibited a slow rise in diastolic calcium, which disappeared upon return to the original pacing pattern. Border zone propagation of ectopic activity was followed by its escape into the surrounding control network, generating arrhythmias. Together, these observations suggest that upon reperfusion, a distinct layer, which consists of ectopically active, poorly coupled cells, is formed transiently over an injured area. Despite being neighbored by a conductive and excitable tissue, this transient functional layer is capable of sustaining autonomous waves and serving as a special conductive medium through which ectopic activity can propagate before spreading into the surrounding healthy tissue.