Signaling and Waves in a Single Living Ventricular Myocyte C

16 Dec 2008 - Real-Time Observations of Intracellular Mg. 2+. Signaling and Waves in a Single Living Ventricular. Myocyte Cell. Seungah Lee,† Hee Gu...
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Anal. Chem. 2009, 81, 538–542

Real-Time Observations of Intracellular Mg2+ Signaling and Waves in a Single Living Ventricular Myocyte Cell Seungah Lee,† Hee Gu Lee,‡ and Seong Ho Kang*,† Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University, Jeonju 561-756, South Korea, and Cellomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, South Korea Despite the important regulatory role of Mg2+ in metabolic pathways, its underlying mechanism is not completely understood at the single-cell level. This study examined the propagation and dynamics of Mg2+ signaling across the cell membrane by employing the real-time visualization of intracellular Mg2+ waves in living ventricular myocytes using a combination of total internal reflection fluorescence microscopy and Nomarski differential interference contrast. Real-time Mg2+ waves and sparks in a living cell membrane were observed using a fluorescent Mg2+ indicator (mag-fluo-4-AM) in the concentration range of 5 aM-5 µM. The intracellular locations of the fluorescent Mg2+ indicator were confirmed by adding Na+ATP. The Mg2+ sparks and waves showed random temporal propagation patterns in nonhomogeneous substructures. These results show that spatiotemporal intracellular Mg2+ signaling information can be obtained for individual living cells. The magnesium ion (Mg2+) is an important mediator and regulator of intracellular Ca2+ signaling in muscle cells.1 Mg2+ regulates a variety of cellular functions and serves as a cofactor in many enzymatic pathways. In cardiac myocytes, the Na+-Mg2+exchange mechanism has been studied for a long time.2 In particular, Mg2+ efflux was found to be activated only in the presence of extracellular Na+.3,4 However, the detailed dynamics of Mg2+ are much less well understood than those of the other major cations,5,6 which is attributable to the high concentrations of free Mg2+ in cells that are several orders of magnitude greater than those of Ca2+ or H+.7 Because of this, * To whom correspondence should be addressed. Tel: +82-63-270-3421. Fax: +82-63-270-3408. E-mail: [email protected]. † Chonbuk National University. ‡ Korea Research Institute of Bioscience and Biotechnology. (1) Dawson, R. M. C.; Hauser, H. Binding of calcium to phospholipids. In Calcium and Cellular Function; Cuthbert, A. W., Ed.; St. Martins Press: New York, 1970; pp 17-41. (2) Murphy, E.; Freudenrich, C. C.; Lieberman, M. Annu. Rev. Physiol. 1991, 53, 273–287. (3) Tursun, P.; Tashiro, M.; Konishi, M. Biophys. J. 2005, 88, 1911–1924. (4) Romani, A.; Marfella, C.; Scarpa, A. Circ. Res. 1993, 72, 1139–1148. (5) Bai, Y.; Tang, A.; Wang, S.; Zhu, X. Proc. SPIE 2005, 5635, 69–76. (6) Wang, S. Q.; Wei, C.; Zhao, G.; Brochet, D. X.; Shen, J.; Song, L. S.; Wang, W.; Yang, D.; Cheng, H. Circ. Res. 2004, 94, 1011–1022. (7) Romani, A.; Scarpa, A. Arch. Biochem. Biophys. 1992, 298, 1–12.

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even large changes in intracellular Mg2+ mobilization involve only small fractions of the Mg2+ present. Developments in optical technologies, such as single-photon and two-photon excitation confocal microscopy,8,9 wide-field microscopy coupled with low-noise CCD cameras,10-14 and singlemolecule fluorescence microscopy,15,16 have made it possible to image the activities of individual ion channels. However, the realtime visualization of intracellular Mg2+ signaling and waves in single living cells is difficult, because the Mg2+ fluxes across the plasma and mitochondrial membranes have much smaller gradients than those of Ca2+.7 Recently, total internal reflection fluorescence microscopy (TIRFM) has been successfully applied in Ca2+ signal transduction studies.5,17 This technique allows low fluctuations in background noise and restricted fluorescence excitation in thin evanescent field layers (5 aM. However, the real-time propagation of these sparks could not be identified in the living single cells stained with high concentrations of mag-fluo-4-AM (i.e., >50 nM), because of the substantial Mg2+ background level throughout the cells (Figure 2A,B). In addition, no Mg2+ sparks were detected at mag-fluo-4-AM concentrations of e5 aM (Figure 2D). These findings demonstrate that 500 aM magfluo-4-AM was the optimum concentration for obtaining good TIRFM images of the intracellular spark phenomenon with reasonable spatial and temporal resolutions (Figure 2C).

Figure 4. Typical spontaneous Mg2+ spark in a living single rat ventricular myocyte. (upper panel, A-H) TIRFM images of Mg2+ spark fluorescence in a living single cell. (bottom panel) The relative fluorescence intensity versus time of 11 sparks in a single cell. The numbers in the cell indicate the order of the spherical Mg2+ sparks. The yellow bold line with an arrowhead shows the direction of wave propagation. Mag-fluo-4-AM concentration, 500 aM. The RFU intensity values were calculated after background subtraction. The TIRFM condition was the same as that shown in Figure 2.

Spatial Profiles of Mg2+ Sparks. The upper panel and bottom panel images in Figure 3A show the spatial profile images of three-dimensional (3D) and two-dimensional (2D) Mg2+ sparks at a mag-fluo-4-AM concentration of 500 aM by TIRFM, respectively. The DIC image provides precise information concerning the localization and path followed by the Mg2+ signals at the membrane sites of a living ventricular myocyte (Figure 3B). Although an inverted reflected light-type microscopic system was used in this study, a transmitted light-type DIC slider was used instead of a reflected light microscopy slider, in order to obtain better images (Figure 3B). The graph in Figure 3C shows the decay of the integrated fluorescence intensities of the Mg2+ signals throughout a living ventricular myocyte after injecting Na+ATP solution. This decay pattern of the fluorescence was in

accord with the findings of a previous study that used an ensemble average.3 Here, the Mg2+ signals in the single cells were observed within 10 s after the injection of the Na+ATP solution. All of the signals from the Mg2+ sparks in the cells disappeared after 300 s (Figure 3C). The Mg2+ signals, which were observed to cross the plasma membrane, were due to the fact that ATP strongly binds to Mg after injecting the Na+ATP solution. These observations are also consistent with previous results. When ATP decomposes to ADP with the consumption of energy, the release of free Mg from the Mg-ATP complex occurs in the living cells.26 Propagation of Nonhomogeneous Mg2+ Sparks. Three types of Ca2+ waves, viz. spherical, planar, and spiral, have been (26) Kubota, T.; Shindo, Y.; Tokuno, K.; Komatsu, H.; Ogawa, H.; Kitamura, Y.; Suzuki, K.; Oka, K. J. Am. Coll. Nutr. 2004, 23, 742S–744S.

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generally identified.27 The Mg2+ discharges in the living ventricular myocyte cells showed different types of motion compared with the Ca2+ signal, which occurs through a mechanism known as Ca2+-induced Ca2+ release.5,6 Although the mechanisms of Mg2+ movement in cardiac and liver cells have already been suggested, it is unclear if Mg2+ movement across the plasma membrane occurs via a similar mechanism, i.e., operating alternately in opposite directions, or whether the influx and efflux processes involve different pathways.7,28-30 Figure 4 shows the development of a spiral Mg2+ wave, which was the result of the random propagation of a nonhomogeneous spark (Supporting Information, movie M1). In the first frame, a spherical Mg2+ wave was generated and showed arbitrary spread with some uncertainty (Figure 4A), and its shape changed from round to elliptical (Figures 4B-D). In the 37.1-s frame (Figure 4E), the propagation of the Mg2+ sparks was random. The yellow bold line marked with an arrowhead indicates the sparks trace and the direction of wave propagation in the living cell (Figure 4H). The 3D graph, which displays the relative TIRF intensities, detection times, and numbers of Mg2+ sparks, shows that the relative fluorescence intensities of the 11 sparks (i.e., the numbers in Figure 4A-G) were similar throughout the duration of the detection time from temporal increase to decay throughout the cell (Figure 4 bottom panel). CONCLUSION The real-time signaling pathway of Mg2+ activated by Na+ATP in a living cell was visualized for the first time using a combined DIC-TIRFM system. The signaling of the Mg2+ motion across the plasma membranes in a living ventricular myocyte was observed (27) Ishida, H.; Genka, C.; Hirota, Y.; Nakazawa, H.; Barry, W. H. Biophys. J. 1999, 77, 2114–2122. (28) Schweigel, M.; Kolisek, M.; Nikolic, Z.; Kuzinski, J. Magnesium Res. 2008, 21, 118–123. (29) Sontia, B.; Touyz, R. M. Pathophysiology 2007, 14, 205–211. (30) Wabakken, T.; Rian, E.; Kveine, M.; Aasheim, H. C. Biochem. Biophys. Res. Commun. 2003, 306, 718–724.

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clearly using 50-500 aM mag-fluo-4-AM at the intracellular Mg2+ binding sites. The Mg2+ sparks were observed directly due to the high signal-to-noise ratio of TIRFM. The mechanisms of Mg2+ movement were confirmed to involve the Na+/Mg2+ exchanger, Mg-ATP complex, or other extrusion mechanisms located within the plasma membrane. In the living ventricular myocyte cells, the propagation of the Mg2+ sparks showed a nonhomogeneous nature at random positions in the intracellular space. Furthermore, the propagation pattern of the spiral Mg2+ waves was significantly different from that of the Ca2+ waves.27,31,32 The Mg2+ waves comprised the random propagation of nonhomogeneous Mg2+ discharges. Overall, this study describes intracellular Mg2+ signaling in a living single cell. It is hoped that the method utilized herein will be of assistance in studies on the regulation of various metabolic pathways in living single cells. ACKNOWLEDGMENT The authors thank Dr. Y.-G. Kwak at the Chonbuk National University Medical School for supplying the ventricular living cells. This work was supported by a grant from the Korea Ministry of Science and Technology (M1053608003-05N3608-00310) and the Korean Science & Engineering Foundation (R01-2007-000-202380). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review June 30, 2008. Accepted October 30, 2008. AC8013324 (31) Engel, J.; Fechner, M.; Sowerby, A. J.; Finch, S. A.; Stier, A. Biophys. J. 1994, 66, 1756–1762. (32) Lipp, P.; Niggli, E. Biophys. J. 1993, 65, 2272–2276.