Mark R. Holcomb

Polarity reversal lowers activation time during diastolic field stimulation of the rabbit ventricles: insights into mechanisms

To fully characterize the mechanisms of defibrillation, it is necessary to understand the response, within the three-dimensional (3D) volume of the ventricles, to shocks given in diastole. Studies that have examined diastolic responses conducted measurements on the epicardium or on a transmural surface of the left ventricular (LV) wall only. The goal of this study was to use optical imaging experiments and 3D bidomain simulations, including a model of optical mapping, to ascertain the shock-induced virtual electrode and activation patterns throughout the rabbit ventricles following diastolic shocks. We tested the hypothesis that the locations of shock-induced regions of hyperpolarization govern the different diastolic activation patterns for shocks of reversed polarity. In model and experiment, uniform-field monophasic shocks of reversed polarities (cathode over the right ventricle is RV-, reverse polarity is LV-) were applied to the ventricles in diastole. Experiments and simulations revealed that RV- shocks resulted in longer activation times compared with LV- shocks of the same strength. 3D simulations demonstrated that RV- shocks induced a greater volume of hyperpolarization at shock end compared with LV- shocks; most of these hyperpolarized regions were located in the LV. The results of this study indicate that ventricular geometry plays an important role in both the location and size of the shock-induced virtual anodes that determine activation delay during the shock and subsequently affect shock-induced propagation. If regions of hyperpolarization that develop during the shock are sufficiently large, activation delay may persist until shock end.

The Potential of Dual Camera Systems for Multimodal Imaging of Cardiac Electrophysiology and Metabolism

Fluorescence imaging has become a common modality in cardiac electrodynamics. A single fluorescent parameter is typically measured. Given the growing emphasis on simultaneous imaging of more than one cardiac variable, we present an analysis of the potential of dual camera imaging, using as an example our straightforward dual camera system that allows simultaneous measurement of two dynamic quantities from the same region of the heart. The advantages of our system over others include an optional software camera calibration routine that eliminates the need for precise camera alignment. The system allows for rapid setup, dichroic image separation, dual-rate imaging, and high spatial resolution, and it is generally applicable to any two-camera measurement. This type of imaging system offers the potential for recording simultaneously not only transmembrane potential and intracellular calcium, two frequently measured quantities, but also other signals more directly related to myocardial metabolism, such as [K+]e, NADH, and reactive oxygen species, leading to the possibility of correlative multimodal cardiac imaging. We provide a compilation of dye and camera information critical to the design of dual camera systems and experiments.

Ratiometric imaging of calcium during ischemia-reperfusion injury in isolated mouse hearts using Fura-2

BackgroundWe present an easily implementable method for measuring Fura-2 fluorescence from isolated mouse hearts using a commercially available switching light source and CCD camera. After calibration, it provides a good estimate of intracellular [Ca2+] with both high spatial and temporal resolutions, permitting study of changes in dispersion of diastolic [Ca2+], Ca2+ transient dynamics, and conduction velocities in mouse hearts. In a proof-of-principle study, we imaged isolated Langendorff-perfused mouse hearts with reversible regional myocardial infarctions.MethodsIsolated mouse hearts were perfused in the Landendorff-mode and loaded with Fura-2. Hearts were then paced rapidly and subjected to 15 minutes of regional ischemia by ligation of the left anterior descending coronary artery, following which the ligation was removed to allow reperfusion for 15 minutes. Fura-2 fluorescence was recorded at regular intervals using a high-speed CCD camera. The two wavelengths of excitation light were interleaved at a rate of 1 KHz with a computer controlled switching light source to illuminate the heart.ResultsFura-2 produced consistent Ca2+ transients from different hearts. Ligating the coronary artery rapidly generated a well defined region with a dramatic rise in diastolic Ca2+ without a significant change in transient amplitude; Ca2+ handling normalized during reperfusion. Conduction velocity was reduced by around 50% during ischemia, and did not recover significantly when monitored for 15 minutes following reperfusion.ConclusionsOur method of imaging Fura-2 from isolated whole hearts is capable of detecting pathological changes in intracellular Ca2+ levels in cardiac tissue. The persistent change in the conduction velocities indicates that changes to tissue connectivity rather than altered intracellular Ca2+ handling may be underlying the electrical instabilities commonly seen in patients following a myocardial infarction.

High-Resolution High-Speed Panoramic Cardiac Imaging System

A panoramic cardiac imaging system consisting of three high-speed CCD cameras has been developed to image the surface electrophysiology of a rabbit heart via fluorescence imaging using a voltage-sensitive fluorescent dye. A robust, unique mechanical system was designed to accommodate the three cameras and to adapt to the requirements of future experiments. A unified computer interface was created for this application-a single workstation controls all three CCD cameras, illumination, stimulation, and a stepping motor that rotates the heart. The geometric reconstruction algorithms were adapted from a previous cardiac imaging system. We demonstrate the system by imaging a polymorphic cardiac tachycardia.

Continuous-waveform constant-current isolated physiological stimulator

We have developed an isolated continuous-waveform constant-current physiological stimulator that is powered and controlled by universal serial bus (USB) interface. The stimulator is composed of a custom printed circuit board (PCB), 16-MHz MSP430F2618 microcontroller with two integrated 12-bit digital to analog converters (DAC0, DAC1), high-speed H-Bridge, voltage-controlled current source (VCCS), isolated USB communication and power circuitry, two isolated transistor-transistor logic (TTL) inputs, and a serial 16 × 2 character liquid crystal display. The stimulators are designed to produce current stimuli in the range of ±15 mA indefinitely using a 20V source and to be used in ex vivo cardiac experiments, but they are suitable for use in a wide variety of research or student experiments that require precision control of continuous waveforms or synchronization with external events. The device was designed with customization in mind and has features that allow it to be integrated into current and future experimental setups. Dual TTL inputs allow replacement by two or more traditional stimulators in common experimental configurations. The MSP430 software is written in C++ and compiled with IAR Embedded Workbench 5.20.2. A control program written in C++ runs on a Windows personal computer and has a graphical user interface that allows the user to control all aspects of the device.

A high-voltage cardiac stimulator for field shocks of a whole heart in a bath

Defibrillators are a critical tool for treating heart disease; however, the mechanisms by which they halt fibrillation are still not fully understood and are the subject of ongoing research. Clinical defibrillators do not provide the precise control of shock timing, duration, and voltage or other features needed for detailed scientific inquiry, and there are few, if any, commercially available units designed for research applications. For this reason, we have developed a high-voltage, programmable, capacitive-discharge stimulator optimized to deliver defibrillation shocks with precise timing and voltage control to an isolated animal heart, either in air or in a bath. This stimulator is capable of delivering voltages of up to 500 V and energies of nearly 100 J with timing accuracy of a few microseconds and with rise and fall times of 5 micros or less and is controlled only by two external timing pulses and a control computer that sets the stimulation parameters via a LABVIEW interface. Most importantly, the stimulator has circuits to protect the high-voltage circuitry and the operator from programming and input-output errors. This device has been tested and used successfully in field shock experiments on rabbit hearts as well as other protocols requiring high voltage.

Universal serial bus powered and controlled isolated constant-current physiological stimulator

We have developed a compact, isolated, physiological, constant-current stimulator that is powered and controlled by a universal serial bus (USB) interface. The stimulator is designed to be used in ex vivo cardiac experiments but is suitable for a wide variety of settings. The cost and features compare very favorably with commercial stimulators usually used in research and student laboratories. In addition to being USB powered, other novel aspects of our stimulator include the ability to produce large currents, up to 100 mA through a typical 1 kOmega load, by means of a single high-voltage dc-to-dc converter; user-specified variable period, magnitude, and duration of complex monophasic or biphasic sequences; and easy integration via hardware or software into existing experimental setups.

Diastolic Field Stimulation: the Role of Shock Duration in Epicardial Activation and Propagation

Detailed knowledge of tissue response to both systolic and diastolic shock is critical for understanding defibrillation. Diastolic field stimulation has been much less studied than systolic stimulation, particularly regarding transient virtual anodes. Here we investigated high-voltage-induced polarization and activation patterns in response to strong diastolic shocks of various durations and of both polarities, and tested the hypothesis that the activation versus shock duration curve contains a local minimum for moderate shock durations, and it grows for short and long durations. We found that 0.1-0.2-ms shocks produced slow and heterogeneous activation. During 0.8-1 ms shocks, the activation was very fast and homogeneous. Further shock extension to 8 ms delayed activation from 1.55 ± 0.27 ms and 1.63 ± 0.21 ms at 0.8 ms shock to 2.32 ± 0.41 ms and 2.37 ± 0.3 ms (N = 7) for normal and opposite polarities, respectively. The traces from hyperpolarized regions during 3-8 ms shocks exhibited four different phases: beginning negative polarization, fast depolarization, slow depolarization, and after-shock increase in upstroke velocity. Thus, the shocks of >3 ms in duration created strong hyperpolarization associated with significant delay (P < 0.05) in activation compared with moderate shocks of 0.8 and 1 ms. This effect appears as a dip in the activation-versus-shock-duration curve.