Being able to witness the precise events that form the heart's orchestral rhythm or the rat-a-tat-tat of irregular heartbeats could enable researchers to better understand the underlying causes of arrhythmias and sudden cardiac death. Indeed, a team from the University of Pittsburgh School of Medicine and Carnegie Mellon University report they have developed unique chemical dyes that have made it possible to see what the naked eye has never seen before: action potentials, or voltage changes, of cardiac cells -- including those deep inside the heart, which trigger and determine the pace of heartbeats.
The researchers describe seven of these "Pittsburgh" dyes -- PGH I to IV and VI to VIII, for short -- in the current issue of the Journal of Membrane Biology. Importantly, the PGH dyes are able to follow the electrical activity of cells several layers below the surface of the heart where the cardiac contractions are initiated and propagated.
"What exactly causes arrhythmias and sudden cardiac death remains an important question we hope to answer through our studies that make use of a combination of novel imaging approaches. Toward this end, these dyes have proved to be particularly important for recording membrane potential changes and capturing in detail, and in real time, the synchronicity or asynchronicity of the heart. Obtaining such images had long been a challenge due to confounding motions of the heart," said lead author Guy Salama, Ph.D., professor of cell biology and physiology at the University of Pittsburgh School of Medicine.
Like a light switch that's quickly flipped on and off, a heart beat begins in similar fashion. A rapid change in electrical charge -- from negative to positive and back again -- occurs within each cell, producing a current that takes about 3/10 of a second to spread in a staccato yet fluid motion across the heart. As these voltage changes occur, so do the traffic patterns of potassium, sodium and calcium, each of these ions entering and leaving cells through their designated portals called channels. Should any of these channels be blocked, or open too fast or too slow, the entire orchestral rhythm of the heart can at once become chaotic, causing irregular heartbeats. Of the different types of arrhythmias, those originating in the heart's ventricles are the most common underlying cause of sudden cardiac death.
To create a method capable of yielding images of a cell as its voltage changes, Dr. Salama teamed up with Alan Waggoner, Ph.D., and Lauren Ernst, Ph.D., of Carnegie Mellon's Molecular Biosensor and Imaging Center (MBIC). Together, they developed the long wavelength, voltage-sensitive dyes described in the paper.
The PGH dyes emit fluorescent light according to changes in voltage across cell membranes that are produced by activity of sodium and potassium channels, which open and close as the cell's voltage changes. The dyes make it possible to actually see changes in the electrical potential of a single cell, multiple cells, or even the entire heart. Importantly, the researchers found that the PGH dyes also could be used simultaneously with other probes, such as for calcium, to provide a more complete picture of the processes influencing normal and abnormal rhythms. They can now map, in real time, action potentials, or voltage changes, of cardiac cells below the surface of the heart while following calcium transients (a measure of the local force generated by each cell) during each action potential.
"A unique feature of the PGH dyes is their large Stokes shifts, the wavelength difference between excitation and fluorescence, which make them particularly advantageous for simultaneously mapping action potentials and calcium transients with less interference and therefore greater sensitivity," said Dr. Waggoner, professor of biological sciences and MBIC director, Mellon College of Science, Carnegie Mellon University.
"One of the dyes in particular exhibited excitation at wavelengths far into the red region of the spectrum, wavelengths significantly longer than the other dyes. Since longer wavelengths of light can penetrate farther into tissue, this dye can image the electrical activity of cells deeper inside the heart, noted Dr. Ernst, senior research scientist at Carnegie Mellon's MBIC.
Using animal models, the researchers have been interested in understanding what mechanisms influence long QT intervals, the time that it takes a depolarized cell to return to its repolarized state. Structural abnormalities of potassium channels have been associated with a hereditary condition called Long QT Syndrome, which affects between 5,000 and 10,000 people in the United States and can have fatal consequences, with boys and women with the syndrome at greatest risk for sudden cardiac death. Yet, for reasons not well understood, it's normal for women of pre-menopausal age to have slightly prolonged QT intervals.
Because many commonly used drugs, including the antibiotic erythromycin, decongestants containing epinephrine and anti-depressants, can block potassium channels, any woman in her 30s and 40s can be at risk for drug-induced ventricular arrhythmias, and potentially, sudden cardiac death.
"The prevailing view is that abnormal potassium channels cause long QT intervals, and therefore, must be what causes arrhythmias as well. But not everyone with a long QT interval experiences arrhythmias. We're beginning to see that the determining factor is an added problem involving the sodium-calcium exchanger and may help explain the differences in risk between men and women," said Dr. Salama.
The new voltage-sensitive dyes, together with novel optical techniques, have greatly enhanced the understanding of how the heart works. Research will continue toward the development of a high-speed, depth-resolved 3-D imaging system that makes use of what may be the fastest camera at 100x100 pixels, with the ability to capture 10,000 images per second.
In addition to Drs. Salama, Waggoner and Ernst, other authors of the paper are Bum-Rak Choi, Ph.D., Ghassan Azour and Mitra Lavasani, all of the University of Pittsburgh School of Medicine; Brian M. Salzberg, Ph.D., of the University of Pennsylvania; and Michael J. Patrick, from Carnegie Mellon University.
The research was supported by the National Heart Lung and Blood Institute, the National Institute of Neurological Disorders and Stroke and the National Cancer Institute, all of the National Institutes of Health.
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