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RESEARCH PROFILES When good cells go bad: detecting the “use by” date of cardiac myocytes
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myocyte is important in propagating an Heart disease is the number one cause of scale of cardiac myocyte contraction electrical signal from the membrane into might be too fast to be detected by Radeath in both the U.S. and the U.K. man, but initial experiments proved oth- the sarcoplasm; degradation of this netBoth lifestyle and genetic factors can work severely impairs the functioning of erwise. The team found that a Raman contribute to this problem, and a lot of spectrum of a cardiac myocyte contains a a remodeled cell. One way to detect reresources are invested each year in carmodeling is to measure the density diac research. A common way to (a) of T-tubules with di-8-ANEPPS, a study the physiology and pathology membrane-bound fluorescent dye. of the heart muscle is to isolate and This method works well, but once analyze individual cardiac myocytes, they are stained, the cells are no but an inconvenient thing happens longer viable for further studies. to these cells when you try to culIn the current set of experiture them outside the heart organ: ments, “we use established fluoresafter several hours they begin to stop cence methods and try to correlate behaving like cardiac myocytes, in a the changes which happen within process known as remodeling. For the T-tubule networks against a researchers, it’s as if these cells have less invasive [confocal Raman] a “use by” date after which they are process so that the cell doesn’t less faithful as a model system. have to be stained,” says Cooper. To complicate matters, conven10 µm The researchers found a number tional methods are not reliable for of changes in the Raman spectrum determining whether such cells (b) over the course of 3 days. Particuhave reached this point; thus, most larly striking are a phenylalanine cardiac researchers prefer to perform –1 band at 1003 cm and the amide I experiments on fresh preparations of band, both of which almost double cells, a time-consuming and wasteful 1000 1400 1600 1200 in intensity—after 2 and 3 days, reprocess. In the June 15 issue of Analytical Chemistry (pp 4581–4587), spectively. Other changes include (a) A photomicrograph of an isolated cardiac myocyte. Jon Cooper, Godfrey Smith, and shifts in tryptophan bands and an The circle indicates a typical region from which spectra co-workers at the University of increase in peaks corresponding to were acquired. (b) A baseline-subtracted Raman specGlasgow (U.K.) report a noninvatyrosine and phenylalanine. The scitrum averaged from seven myocytes. sive confocal Raman method that entists attribute these changes to a can not only detect remodeling loss in cellular structure and to wealth of spectral information, including changes in the respective position of the but also will be useful for studying mypeaks corresponding to various amino ocytes during the brief period before amino acid side chains. Over the same acid side chains as well as main-chain they “expire”. time course, the percentage of the cell stretches and bends. One band in parThe scientists first wanted to use stained with di-8-ANEPPS decreased ticular, the amide I peak at 1658 cm–1, from 20 to 5%, which confirms that celconfocal Raman to obtain information decreased in area upon electrical stimula- lular remodeling of the cardiac myocyte about cell contraction that occurs upon tion and cell contraction; changes in this has occurred. electrical stimulation. A process called Cooper hopes that confocal Raman band could be detected in as little as excitation–contraction coupling links the electrical signal to depolarization spectroscopy will eventually be used rou300 ms. The investigators attribute this and contraction of the cardiac myocyte. tinely by researchers as a quick method decrease to protein rearrangements in “We’re interested in looking at the to reassure themselves that their cardiac the cell that reduce the degree of disorstructural and functional changes which myocytes have not started remodeling. der among cellular proteins. occur [upon contraction], particularly “This provides a new technique that The researchers also were interested in whether confocal Raman could detect with a longer-term view to looking at most importantly, I think, is noninvathe longer-term process of cellular resome disease models,” Cooper says. sive,” he says. “It can give you informamodeling. One distinct marker of reRaman spectroscopy has a relatively tion over different timescales, both the modeling is a detachment of membrane low S/N, and thus time is needed to short- and long-term changes, and it’s relatively simple to do as well.” a invaginations called T-tubules. The netcollect a good spectrum. At first, the rework of T-tubules inside a fresh cardiac searchers were concerned that the time— Jennifer Griffiths
