Figure 1: Photomicrographs of sections approximately parallel to and 0.1-0.2 mm below anterior ventricular epicardial surface of the left ventricle (panel a) and right ventricle (panel b). The white bar corresponds to 0.5 mm. Both ventricles of the heart contain fibers.

Description:

Ventricular fibrillation is lethal if it is not halted within minutes. Electrical defibrillation shocks are the only effective means to halt fibrillation. Patients with recurrent fibrillation frequently receive an implantable defibrillator that senses fibrillation and quickly applies a shock from electrodes in or near the heart. Our research is exploring the use of elongated, or line, electrodes to deliver the shock to the heart. Line electrodes are advantageous because they can be fabricated as catheters and implanted in the heart by threading the catheter through veins. This eliminates the need for traumatic open-chest surgery during implantation. We have sought ways to optimize shock delivery from line electrodes. We use optical dyes and a laser scanner to measure how the shock interacts with the fibers that make up the heart. We have found that a line electrode produces a more homogeneous response in the heart if the electrode is oriented parallel to the fibers compared with other directions. Ongoing work is exploring how we may incorporate line electrodes parallel to fibers into a therapy that takes advantage of the homogeneous response. Also we are attempting to further increase the homogeneity of the response by adjusting the distribution of current that emanates from the line electrode. We have measured the distribution of current and performed finite element model calculations to determine the distribution in detail. We plan to fabricate a modified electrode in which we will introduce resistance near the ends of the line electrode to lessen the rise in current that emanates from the ends. We will then determine with the laser scanner whether such a modified electrode increases the homogeneity of the response in the heart.

Figure 2: Distribution of current emanating from a line electrode calculated with a finite element model (top graph) and measured in discrete terminals of a segmented line electrode in 4 rabbit hearts (center graph, mean±one sd), and effect of introducing series resistors of 4 Kohm, 2 Kohm, 1 Kohm and 0.5 Kohm, respectively, in the leads for terminals at or near the left end of a line electrode (bottom graph). Distances of 0 and 10 mm correspond to electrode ends. With no resistors, elevated current occurs near electrode ends in the model and in hearts during stimulation. Introducing the series resistors near the left end of the electrode, such that the end terminal has highest resistance and consecutive terminals have decreasing resistance, eliminated the rise in current at the electrode end.

Selected publications about this project:

Baynham TC, Knisley SB, "Roles of Line Stimulation-Induced Virtual Electrodes and Action Potential Prolongation in Arrhythmic Propagation," (manuscript in press in Journal Of Cardiovascular Electrophysiology, 21 typed pages): publication in February or March 2001.Baynham TC, Knisley SB, "Effective Epicardial Resistance of Rabbit Ventricles,"Annals of Biomedical Engineering, 27;96-102:1998Knisley SB, Baynham TC, "Line stimulation parallel to myofibers enhances regional uniformity of transmembrane voltage changes in rabbit hearts," Circulation Research 81;229-241: 1997

Figure 1: Diagram of the method used to record action potentials from the epicardium of isolated perfused hearts stained with a transmembrane voltage-sensitive dye. A computer-controlled laser beam scanned a grid of 128 recording spots on the heart every ms. The fluorescent light intensity, which changes in proportion to changes in the transmembrane potential of cells at each recording spot, was detected with a photomultiplier tube. Individual transmembrane potential recordings at each spot were then obtained by demultiplexing the fluorescence signal.

Description:

This project uses optical mapping techniques to explore how defibrillation occurs. We think that the defibrillation shock produces rapid changes in the transmembrane voltages of cells during the time that the shock is turned on, and that these changes ultimately produce defibrillation. The shock lasts only about 5 milliseconds, and so our measurements need to be fast and their times precisely controlled. We use a fast laser scanner system in which the beam is steered to spots on the heart with acousto-optic deflectors. This allows us to map the transmembrane voltage changes quickly at many spots on the heart. The effects of the shocks in hearts have proven to be very complex. The effects probably involve many factors including the shock electric field and the cellular or fiber structure of the heart. Several of the factors were predicted by mathematical models before measurements such as ours were possible. Our experiments have tested some of the predictions. The most interesting discoveries have occurred when a result did not fit a model prediction, e.g., we found "virtual electrode effects" in hearts (i.e., regions where transmembrane voltage changes were negative when they were predicted by a model to be positive). That model has been rejected. We plan to test a model called the "generalized activating function." If it is correct, this model can account for potentially all of the factors that determine the transmembrane voltage changes during a shock. This model is already known to apply to electrical stimulation of nerve fibers, but it is not known whether the model applies to defibrillation shocks in hearts. We hope to determine whether or not it applies to hearts.

Selected publications about this project:

Knisley SB, "Evidence for Roles of the Activating Function in Electric Stimulation," IEEE Transactions on Biomedical Engineering 47;1114-1119: 2000Stephen B. Knisley, Andrew E. Pollard, Vladimir G. Fast, "Effects of Electrode Myocardial Separation on Cardiac Stimulation in Conductive Solution," Journal of Cardiovascular Electrophysiology 11;1132-1143:2000.Stephen B. Knisley, Natalia Trayanova, Felipe Aguel, "Roles of Electric Field and Fiber Structure in Cardiac Electric Stimulation" Biophysical Journal 77; 1404 1417:1999

Figure 1: Pattern of an array of transparent stimulating and recording electrodes. The array was fabricated from transparent indium tin oxide sputtered onto glass and photolithographically etched. A array of 16 recording electrode terminals (circles) is in the center. A semicircular array of stimulation electrode terminals (squares) is below.

Description:

This project performs simultaneous collocated extracellular and transmembrane potential measurements to improve interpretation of cardiac electrograms. Present interpretations apply to a limited set of conditions and may not apply to all arrhythmic conditions. The transmembrane potential, which is considered the "gold standard" with which to determine membrane excitation, can be mapped optically using transmembrane voltage-sensitive fluorescent dyes. This project maps the transmembrane potential and also simultaneously the extracellular potential at the same locations. The measurements directly reveal features of the extracellular potentials that indicate membrane excitation.

Previous technology has not allowed such simultaneous collocated optical and electrical mapping. This is because the metallic electrodes that have been used for electrical mapping block the light that is needed for the optical mapping. By developing in collaboration with another institution transparent recording electrodes that do not block the light, we found that simultaneous collocated electrical and optical recordings are possible. The electrodes use thin transparent electrically-conductive film on glass, and photolithographic patterning of the film.

Figure 2: An example of the extracellular potential and the simultaneous fluorescence signal recorded from the same location with a transparent indium tin oxide electrode during a single heartbeat.

Selected publications about this project:

Stephen B. Knisley, Michael R. Neuman, "Cardiac Excitation Time Differences Determined by Simultaneous Collocal Mapping of Extracellular Voltages and Transmembrane Voltages," Circulation 102 No. 18;II-336: 2000

The possibility to use multiple dyes simultaneously to sense different physiological variables is potentially very important for understanding aspects of heart function and arrhythmias. A problem is that errors can occur in measurements of emitted fluorescence when combinations of dyes are present due to overlap of the emission spectra. A spectrofluorometer is being used to measure fluorescence emission spectra from charge-shift transmembrane voltage-sensitive dyes and from combinations of transmembrane voltage-sensitive dyes and calcium sensitive dyes during laser excitation at a single wavelength. Measurements and theory have begun to address minimization of errors by the choices of dyes and excitation or measurement wavelength bands.

Selected publications about this project:

P.L. Johnson, W. Smith, T.C. Baynham, and S.B. Knisley, "Errors caused by combination of Di-4-ANEPPS and Fluo3/4 for simultaneous measurements of transmembrane potentials and intracellular calcium," Annals of Biomedical Engineering, vol. 27, pp. 563-571, 1999.P.L. Johnson, S.B. Knisley, D. Rollins, and W.M. Smith, "Spectral overlap considerations for simultaneous measurement of intracellular calcium concentration and transmembrane potentials in cardiac tissue," Proc. 19th Annual International Conf. Institute of Electrical and Electronics Engineers, Engineering in Medicine And Biology Society, vol. 19, 1997.

Description:

Ratiometry of emitted fluorescence signals at different wavelengths is being investigated as a way to overcome the problems of photobleaching and heart motion artifacts in optical mapping. The ratiometry may allow optical measurements of slow changes in resting transmembrane potential, which are usually obscured by photobleaching of dye. Ratiometry may also allow measurements of transmembrane action potentials and intracellular calcium transients in beating hearts without requiring pharmacological or mechanical techniques to inhibit heart movement.

Selected publications about this project:

Knisley SB, Robert K. Justice, Wei Kong, Philip L. Johnson, "Ratiometry of Transmembrane Voltage-Sensitive Fluorescent Dye Emission in Hearts," American Journal of Physiology 279;1421-1432:2000.Wei Kong, Stephen B. Knisley, "Effect of diacetyl monoxime on action potential duration in rabbit hearts as revealed by fluorescence emission ratiometry," (abstract presented at BMES conference, China, 2000)Wei Kong, Philip Johnson Jr., Stephen B. Knisley, "Emission Overlap for RH237 and Fluo-4 Used Simultaneously to Record Optical Vm and Intracellular Calcium Transients in Rabbit Hearts," Annals of Biomedical Engineering 28(1);S57:2000 (abstract)Wei Kong, Philip L. Johnson, Stephen B. Knisley, "Reduction of motion artifacts and photobleaching during multiwavelength ratiometric optical recording of action potentials and intracellular calcium transients in rabbit hearts," Pacing and Clinical Electrophysiology 23 (4);608:2000 (abstract).

View 1: The first demonstration of electric stimulation-induced transmembrane voltage changes at the ends of an excitable cell. Recordings and photographs of a single isolated rabbit cardiac cell (approximately 15x70 micrometers) aligned with the stimulation electric field and illuminated with a laser spot (wavelength 488 nm) at the cell end facing the S2 anode (left) or cathode (right). Cell was stained with transmembrane voltage-sensitive fluorescent dye, di-4-ANEPPS. Before experimental trials, the laser beam was blocked by an acousto-optic modulator. In each trial, laser illumination at a cell end began 50 ms before the S1 stimulation pulse (You can see the inverted fluorescence signal change upon beginning laser illumination, which produced the large downward deflection near the beginning of fluorescence recording). Laser illumination was then terminated 500 ms later.

The pacing stimulation pulse (S1 in upper recordings) produced a transition from the diastolic transmembrane voltage to the depolarized transmembrane voltage or action potential plateau (i.e., phase zero depolarization), as evident from decrease in emitted fluorescence at both cell ends during S1. Then a strong shock (S2, 20 V/cm) caused a further change in transmembrane voltage in the upward direction at the cell end facing the cathode (right), but a change in the downward direction at the cell end facing the anode (left). Thus, transmembrane voltages at the two ends changed in opposite directions during the pulse. Magnitudes estimated from the heights of the phase zero depolarizations fit magnitudes predicted from electric field strength multiplied by cell length.

References:

Knisley S, Blitchington T, Hill B, Grant A, Smith W, Pilkington T, Ideker R, "Optical Measurements of Transmembrane Potential Changes During Electric Field Stimulation of Ventricular Cells," Circulation Research 72;255-270:1993.Knisley S, Grant A, "Asymmetrical Electrically-Induced Injury of Rabbit Ventricular Myocytes," Journal of Molecular and Cellular Cardiology 27; 1111-1122: 1995

View 2: Fiber structure of heart as revealed by transillumination. We reported the first transmembrane voltage measurements indicating that the cardiac fiber orientation can determine the signs of transmembrane voltage changes during a shock, an effect sometimes called "The Virtual Electrode Effect." Since the fiber direction can have a strong effect, we wanted to understand the fiber structure that exists in hearts. We have studied fiber structure by various methods including the measurement of the fast axis of action potential propagation, examination of stained thin sections cut from hearts, and, as shown here, staining and transillumination of the ventricular wall in whole hearts. The picture shows an anterior view of a rabbit heart with epicardial fiber directions highlighted by brief application of concentrated stain and transillumination with an optical fiber placed inside the left ventricle. Average fiber orientation was from upper left to lower right in the picture, however not all fibers were parallel. You can see that the fibers became more horizontal in epicardium toward the apex (bottom of picture), and more vertical in left lateral epicardium (right side of picture).

Reference:

Knisley S, Hill B, Ideker R, "Virtual Electrode Effects in Myocardial Fibers," Biophysical Journal 66;719-728: 1994.Knisley S, Natalia Trayanova, Felipe Aguel, "Roles of Electric Field and Fiber Structure in Cardiac Electric Stimulation" Biophysical Journal 77; 1404 1417:1999

View 3: Fiber structure on endocardium of heart. Defibrillation shocks are often applied from electrodes placed inside the heart. Thus we want to understand fiber orientation inside the heart. This photograph of right septal endocardium with right freewall lifted shows fiber orientation in right ventricular cavity, which is where defibrillation shocks are applied with implantable defibrillators. Heart was formalin-fixed, septal endothelial tissue was removed, and septum was stained with concentrated Evan’s blue dye to highlight structure. Heart was transilluminated with optical fiber in left ventricular cavity. You can see that fibers have average orientation from upper left to lower right.

Reference:

Knisley S, Andrew E. Pollard, Vladimir G. Fast, "Effects of Electrode Myocardial Separation on Cardiac Stimulation in Conductive Solution," Journal of Cardiovascular Electrophysiology 11;1132-1143:2000.

An addition, a project conducted in the laser laboratory at UNC is currently testing the predictions of the models. A femtosecond pulsed near-infrared laser and a scanning system are used to produce two-photon fluorescence in biological tissue. Fluorescence is measured with spectrophotometer and photomultiplier systems to image rapid electrophysiological events such as cardiac action potentials and calcium ion transients.