Analytical Biochemistry 447 (2014) 39–42
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy Yu Hirano a, Mikie Kodama a, Masahiro Shibuya b, Yoshiyuki Maki b, Yasuo Komatsu a,⇑ a b
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Toyohira, Sapporo 062-8517, Japan Cosmo Bio Co., Ltd., Nishi-Ku, Sapporo, Japan
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 18 October 2013 Accepted 7 November 2013 Available online 16 November 2013 Keywords: Scanning electrochemical microscopy Cardiomyocytes Contraction behavior Oxygen consumption
a b s t r a c t The contractile behavior of cardiomyocytes can be monitored by measuring their action potentials, and the analysis is essential for screening the safety of potential drugs. However, immobilizing cardiac cells on a specific electrode is considerably complicated. In this study, we demonstrate that scanning electrochemical microscopy (SECM) can be used to analyze rapid topographic changes in beating cardiomyocytes in a standard culture dish. Various cardiomyocyte contraction parameters and oxygen consumption based on cell respiration could be determined from SECM data. We also confirmed that cellular changes induced by adding the cardiotonic agent digoxin were conveniently monitored by this SECM system. These results show that SECM can be a potentially powerful tool for use in drug development for cardiovascular diseases. Ó 2013 Elsevier Inc. All rights reserved.
Analysis of cardiomyocyte contraction is currently used to evaluate the pharmacological and toxicological properties of drugs [1–3]. Measurements of action potentials and topographic changes have been developed to characterize cardiomyocyte contraction. Action potentials can be recorded by a microelectrode array (MEA) [4–6], whole-cell patch clamping [7], or voltage-sensitive dyes [8]. MEA systems detect rhythmic changes in electrical potentials that are generated by beating cells when they are immobilized on a microelectrode array. Whole-cell patch-clamp techniques are used with human ether-a-go-go-related gene-transfected HEK293/CHO cells; however, electrical recordings are technically difficult. Although Ca2+-dependent fluorescent dyes can be used to monitor contraction behaviors in a standard culture dish, fluorescent signal measurements are not suitable for long-term monitoring because of photodamage and photobleaching effects [9]. Video microscopy can be used to determine the kinetics of cardiomyocyte topographic changes, and it provides valuable information on heart contraction [10]. However, to record cell motion, isolation of a single cardiomyocyte is required [11]. Quantifying cardiomyocyte oxygen consumption is also essential for analysis of energy metabolism in the heart. Oxygen consumption can be measured using a Clark electrode [12], by myoglobin saturation [13], or by electron paramagnetic resonance oximetry [14]. These methods measure the oxygen concentration in the bulk culture medium, which provides for quantitative
⇑ Corresponding author. Fax: +81 11 857 8954. E-mail address: [email protected] (Y. Komatsu). 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.11.008
analysis, but it is difficult to evaluate the oxygen consumption for a single cell. Scanning electrochemical microscopy (SECM) provides information on the local distributions of electrochemically active species by microelectrode scanning [15]. SECM has recently been used for analyzing various cellular functions [16–20]. SECM provides for noninvasive measurements of cellular topography by scanning in the vicinity of target cells adsorbed on a standard culture dish [16,17]. In addition, SECM measurements of oxygen concentrations around these cells directly reflect their respiratory activity and can be used to analyze energy metabolism in viable cells [20]. However, SECM has not yet been used for measuring the motions or oxygen consumption of beating cardiomyocytes. In this study, we developed an SECM system for analyzing the contraction kinetics and oxygen consumption of cultured cardiomyocytes. This system enabled us to investigate changes in cardiomyocyte function that resulted from changes in temperature and after adding a cardiotonic agent. Thus, this SECM system may become a powerful tool for studying pharmacological and toxicological effects in cardiomyocytes.
Material and methods Cell culture Primary cardiomyocytes were prepared from 1- to 2-day-old neonatal rat ventricles (Cosmo Bio Co., Ltd., Japan) and used for SECM measurements [21]. Cardiomyocytes were seeded on
Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy / Y. Hirano et al. / Anal. Biochem. 447 (2014) 39–42
Instrumentation SECM measurements were performed using an instrument built in our laboratory as previously described [17] but with slight modifications. In brief, our SECM setup comprised a piezomotor positioning system (Physik Instrumente, Germany), an inverted microscope, a temperature-controlled plate (Tokai Hit, Japan), a data acquisition and instrument control system (National Instruments, USA), and a potentiostat (Hokuto Denko, Japan). We used a Pt microdisk electrode (5-lm radius) as the electrochemical probe for SECM measurements and a leak-free Ag/AgCl electrode (Warner Instruments, USA) as the reference and auxiliary electrode. The radius of the Pt microelectrode, including a glass sheath, was 10 lm. Measurement procedures for beating cardiomyocytes by SECM We applied a 0.5-V potential or a 0.5-V potential (vs Ag/AgCl) to the probe electrode for ferrocyanide oxidation or oxygen reduction, respectively. To detect topographical changes in a beating cardiomyocyte, we positioned the microelectrode tip 9 lm away from a cell’s surface by approach curve measurements (Supplementary Fig. S-1) [22]. We subsequently measured the responses of the oxidation currents of ferrocyanide with a time resolution of 104 points/s (Supplementary Fig. S-2). A plot of current versus time was converted to a plot of topographical change versus time using a calibration curve (Supplementary Fig. S-4). To analyze the contraction and relaxation motions of cardiomyocytes, a single average waveform was calculated from the topographical change plot using a software that we developed with LabView (National Instruments). This program identified all pulse peaks in SECM data for 30 s, which provided a peak-to-peak interval time, a peak fluctuation value, and an average waveform. In addition, the duration of contraction, the duration of relaxation, and the contraction amplitude were determined from this average waveform (Supplementary Fig. S-3). Oxygen consumption was determined from measurements of the oxygen reduction current in the vicinity of the target cells (Supplementary Fig. S-2). We also measured the contraction behaviors and oxygen consumption of the beating cardiomyocytes by SECM at various temperatures. For these experiments, we used spontaneously beating cells 4 days after they were initially seeded. We first measured the current response at 37 °C. Further, we changed the temperature to 35 °C and waited for 7 min, after which we measured the current responses at the same position. These measurements were repeated at 33 and 31 °C. For a representative pharmacological intervention, we measured the contraction behaviors of cardiomyocytes by SECM before and after adding digoxin. For these experiments, we used spontaneously beating heart cells after incubation for 10 days. After measuring the current response at 37 °C (0 lM), we added digoxin to a final concentration of 2.5 or 10 lM, waited for 20 min, and then measured current responses at the same position.
Results and discussion Contraction kinetics analysis To prevent serum proteins from being adsorbed on the electrode surface, we performed SECM measurements of the cardiomyocytes in serum-free medium that contained 1 mM potassium ferrocyanide. Cardiomyocytes exhibited continuous spontaneous beating for at least 4 h under these conditions. In addition, a leak-free reference electrode was used so as not to interfere with cardiomyocytes that were sensitive to changes in ion concentrations. The microelectrode was positioned above the central part of the contractile motion, which was identified using an optical microscope. We measured the contraction behaviors of cardiomyocytes by our SECM system at 37 °C. A time resolution of 102 points/s failed to detect any current changes, whereas a resolution of 104 points/s obtained current pulses that were synchronized with cardiomyocyte contractions (Fig. 1, black line). A plot of current versus time could be converted to a plot of topographical change versus time (Fig. 1, gray line) using a calibration curve (Supplementary Fig. S-4). From this plot, we calculated the peak-to-peak interval time and peak fluctuation value, which showed fluctuations in the beating rhythm (Supplementary Table S-1). To analyze the contraction and relaxation motions of cardiac cells, an average waveform for all pulse peaks is required, which can be derived from active potential measurements or video microscopic analysis [4,9,11]. Thus, we developed a software program that could convert a single average waveform from the plot of topographical changes, which resulted in generating an average waveform at 37 °C (Fig. 2A, black line). The following three parameters reflected the contraction behavior: contraction duration, relaxation duration, and contraction amplitude (Supplementary Fig. S-3). Both duration values indicated the rate of cardiac contraction, and the contraction amplitude reflected the cardiac contractility. These parameters could be determined from an average plot (Supplementary Table S-1). To validate our analysis method, SECM measurements were performed for the same cell at 35, 33, and 31 °C (Supplementary Fig. S-5). The average waveform was determined as that at 37 °C
microelectrode
relaxation
contraction
0.73
0.5
0.72
0.4
0.71 0.3 0.70 0.2 0.69 0.1
0.68 0.67
Topographic change (µm)
35-mm dishes that were coated with 0.01% collagen (Koken, Japan) at a concentration of 5 104 cells/cm2. These cells were then incubated in cardiomyocyte culture medium (Cosmo Bio) supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. The medium was changed every 2 days. For SECM experiments, we used spontaneously beating cells after 4–10 days in a culture with serum-free medium that contained 1 mM potassium ferrocyanide (K4[Fe(CN)6]) and 2% Knockout serum replacement (Life Technologies, USA) in MCDB107 (Cosmo Bio). All other reagents were of analytical grade. Solutions were prepared with ultrapure water from a Millipore system.
Current (nA)
40
0.0 0
2
4
Time (s) Fig.1. Current–time responses of a beating heart cell as determined by scanning electrochemical microscopy at 37 °C. The black line indicates cardiomyocyte current–time responses, and the gray line indicates topographical changes.
Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy / Y. Hirano et al. / Anal. Biochem. 447 (2014) 39–42
(A)
cardiomyocytes (dashed line in Fig. 2B). This decreasing oxygen concentration near cardiomyocytes was dependent on the incubation temperature (Fig. 2B). These results indicated that SECM had detected oxygen concentration decreases that were due to the beating cardiomyocytes’ respiration.
1.2
Normalized contraction
1 0.8 0.6
Analyzing the effects of digoxin on cardiomyocytes
0.4
33ഒ 31ഒ
37ഒ 0.2
35ഒ
0 -0.2 0
0.2
0.4
0.6
0.8
Time (s) 0.210
cardiac fibroblast
Oxygen concentration (mM)
0.209
Measurements of the contraction amplitude and oxygen consumption are essential for analyzing the effects of cardiotonic agents on the heart. We investigated whether SECM could be used to detect these changes after treatment with the cardiotonic agent digoxin. Adding digoxin resulted in an increase in the contraction amplitude (Fig. 3A, Supplementary Fig. S-6, and Supplementary Table S-2) and a decrease in both the beat rate and the oxygen consumption (Supplementary Table S-2 and Fig. 3B) in a digoxin concentration-dependent manner. These results were consistent with those of previous studies [5,26,27]. Thus, we believe that our SECM system is applicable for analyzing dynamic changes that are induced by a cardiotonic agent.
0.208 0.207
(A)
0.206 0.205
31ഒ
0.204
33ഒ
10 µM
0.203 0.202
35ഒ
Topographic change
(B)
41
0.201 0.200
37ഒ
0.199 0
50
100
150
200
Distance from cell surface (µm) Fig.2. (A) Comparison of average waveforms at 31, 33, 35, and 37 °C. (B) Oxygen consumption analysis among local cardiomyocytes by scanning electrochemical microscopy. Plots are oxygen concentration profiles versus tip–sample distance at 31, 33, 35, and 37 °C. Dotted line indicates oxygen concentration profiles of a cardiac fibroblast (nonbeating cell).
2.5 µM
0 µM 0.5 µm
0
0.5
1
1.5
Time (s)
Respiration activity analysis Quantifying the oxygen consumption by viable cells provides valuable information regarding metabolic mechanisms [24]. Cardiomyocytes consume considerably more oxygen than the other types of nonbeating cells [25]. Although SECM can be potentially used to evaluate oxygen consumption [20], it has not been previously used for the beating cardiomyocytes. To examine the cardiomyocyte oxygen consumption, we measured the approach curves for cardiac cells comprising both cardiomyocytes and nonbeating cardiac fibroblasts. The oxygen concentration did not change either for the cardiac fibroblasts (dotted line in Fig. 2B) or on the dish surface (data not shown), whereas the oxygen concentration clearly decreased at distances closer to the beating
(B)
0.21 0.208
Oxygen concentration (mM)
(Fig. 2A). When the cell was incubated at lower temperatures, the peak-to-peak interval time (Supplementary Table S-1) and peak broadening (Fig. 2A) increased. Because these results were consistent with those obtained in a previous study [23], our SECM system was effective for analyzing cardiomyocyte contraction.
0.206 10 µM 0.204 0.202 0.2
2.5 µM
0.198 0 µM
0.196 0
50
100
150
200
Distance (µm) Fig.3. (A) Contraction and (B) oxygen consumption responses of beating heart cells before and after adding digoxin at 2.5 and 10 lM.
42
Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy / Y. Hirano et al. / Anal. Biochem. 447 (2014) 39–42
Conclusion We constructed a convenient system for analyzing cardiomyocyte behaviors in a standard culture dish. After improving the time resolution and programming software suited to our tasks, we could use our SECM system to analyze cardiomyocyte contractions. We demonstrated that kinetic parameters that reflected contraction behaviors could be extracted from our SECM data and that oxygen consumption associated with cell respiration could be quantified. On the basis of these capabilities, we evaluated the effects of adding a cardiotonic agent on the cardiomyocyte behavior. Taken together, our results indicate that our SECM system has significant potential for analyzing cardiomyocytes. Acknowledgments We thank Dr. Yasuhiro Mie and Dr. Masiki Ikegami for their helpful discussions. The financial support received, in part, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Grant-in-Aid for Scientific Research No. 25410162) and from the Ministry of Economy, Trade, and Industry, Japan, is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab. 2013.11.008. References [1] M.L. De Bruin, M. Pettersson, R.H.B. Meyboom, A.W. Hoes, H.G.M. Leufkens, Anti-HERG activity and the risk of drug-induced arrhythmias and sudden death, Eur. Heart J. 26 (2005) 590–597. [2] W.S. Redfern, L. Carlsson, A.S. Davis, W.G. Lynch, I. MacKenzie, S. Palethorpe, P.K.S. Siegl, I. Strang, A.T. Sullivan, R. Wallis, A.J. Camm, T.G. Hammond, Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development, Cardiovasc. Res. 58 (2003) 32–45. [3] J.P. Valentin, Reducing QT liability and proarrhythmic risk in drug discovery and development, Br. J. Pharmacol. 159 (2010) 5–11. [4] A. Stett, U. Egert, E. Guenther, F. Hofmann, T. Meyer, W. Nisch, H. Haemmerle, Biological application of microelectrode arrays in drug discovery and basic research, Anal. Bioanal. Chem. 377 (2003) 486–495. [5] L. Guo, J.Y. Qian, R. Abrams, H.M. Tang, T. Weiser, M.J. Sanders, K.L. Kolaja, The electrophysiological effects of cardiac glycosides in human iPSC-derived cardiomyocytes and in guinea pig isolated hearts, Cell. Physiol. Biochem. 27 (2011) 453–462. [6] N. Yokoo, S. Baba, S. Kaichi, A. Niwa, T. Mima, H. Doi, S. Yamanaka, T. Nakahata, T. Heike, The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells, Biochem. Biophys. Res. Commun. 387 (2009) 482–488. [7] J.C. Hancox, M.J. McPate, A. El Harchi, Y.H. Zhang, The hERG potassium channel and hERG screening for drug-induced torsades de pointes, Pharmacol. Ther. 119 (2008) 118–132.
[8] G. Ravenscroft, K.J. Nowak, C. Jackaman, S. Clement, M.A. Lyons, S. Gallagher, A.J. Bakker, N.G. Laing, Dissociated flexor digitorum brevis myofiber culture system—a more mature muscle culture system, Cell Motil. Cytoskeleton 64 (2007) 727–738. [9] G.X. Bu, H. Adams, E.J. Berbari, M. Rubart, Uniform action potential repolarization within the sarcolemma of in situ ventricular cardiomyocytes, Biophys. J. 96 (2009) 2532–2546. [10] M. Das, J.W. Rumsey, C.A. Gregory, N. Bhargava, J.F. Kang, P. Molnar, L. Riedel, X. Guo, J.J. Hickman, Embryonic motoneuron-skeletal muscle co-culture in a defined system, Neuroscience 146 (2007) 481–488. [11] J. Ren, A.M. Bode, Altered cardiac excitation–contraction coupling in ventricular myocytes from spontaneously diabetic BB rats, Am. J. Physiol. Heart Circ. Physiol. 279 (2000) H238–H244. [12] S. Kinugawa, H. Huang, Z.P. Wang, P.M. Kaminski, M.S. Wolin, T.H. Hintze, A defect of neuronal nitric oxide synthase increases xanthine oxidase-derived superoxide anion and attenuates the control of myocardial oxygen consumption by nitric oxide derived from endothelial nitric oxide synthase, Circ. Res. 96 (2005) 355–362. [13] L.S.L. Arakaki, D.H. Burns, M.J. Kushmerick, Accurate myoglobin oxygen saturation by optical spectroscopy measured in blood-perfused rat muscle, Appl. Spectrosc. 61 (2007) 978–985. [14] G. Ilangovan, T. Liebgott, V.K. Kutala, S. Petryakov, J.L. Zweier, P. Kuppusamy, EPR oximetry in the beating heart: myocardial oxygen consumption rate as an index of postischemic recovery, Magn. Reson. Med. 51 (2004) 835–842. [15] A.J. Bard, M.V. Mirkin, Scanning Electrochemical Microscopy, Dekker, New York, 2001. [16] C.X. Cai, B. Liu, M.V. Mirkin, H.A. Frank, J.F. Rusling, Scanning electrochemical microscopy of living cells. 3. Rhodobacter sphaeroides, Anal. Chem. 74 (2002) 114–119. [17] Y. Hirano, Y. Nishimiya, K. Kowata, F. Mizutani, S. Tsuda, Y. Komatsu, Construction of time-lapse scanning electrochemical microscopy with temperature, control and its application to evaluate the preservation effects of antifreeze proteins on living cells, Anal. Chem. 80 (2008) 9349–9354. [18] J.D. Guo, S. Amemiya, Permeability of the nuclear envelope at isolated Xenopus oocyte nuclei studied by scanning electrochemical microscopy, Anal. Chem. 77 (2005) 2147–2156. [19] T. Yasukawa, T. Kaya, T. Matsue, Dual imaging of topography and photosynthetic activity of a single protoplast by scanning electrochemical microscopy, Anal. Chem. 71 (1999) 4637–4641. [20] H. Shiku, T. Shiraishi, H. Ohya, T. Matsue, H. Abe, H. Hoshi, M. Kobayashi, Oxygen consumption of single bovine embryos probed by scanning electrochemical microscopy, Anal. Chem. 73 (2001) 3751–3758. [21] H. Ono, H. Nakamura, M. Matsuzaki, A NADH dehydrogenase ubiquinone flavoprotein is decreased in patients with dilated cardiomyopathy, Intern. Med. 49 (2012) 2039–2042. [22] Y.H. Shao, M.V. Mirkin, Probing ion transfer at the liquid/liquid interface by scanning electrochemical microscopy (SECM), J. Phys. Chem. B 102 (1998) 9915–9921. [23] Y.S. Han, T. Tveita, Y.S. Prakash, G.C. Sieck, Mechanisms underlying hypothermia-induced cardiac contractile dysfunction, Am. J. Physiol. Heart Circ. Physiol. 298 (2010) H890–H897. [24] S.K. Jung, L.M. Kauri, W.J. Qian, R.T. Kennedy, Correlated oscillations in glucose consumption, oxygen consumption, and intracellular free Ca2+ in single islets of Langerhans, J. Biol. Chem. 275 (2000) 6642–6650. [25] T. Yamada, J.J. Yang, N.V. Ricchiuti, M.W. Seraydarian, Oxygen-consumption of mammalian myocardial-cells in culture—measurements in beating cells attached to the substrate of the culture dish, Anal. Biochem. 145 (1985) 302–307. [26] E.J. Eichhorn, M. Gheorghiade, Digoxin, Prog. Cardiovasc. Dis. 44 (2002) 251– 266. [27] E. Braunwald, Effects of digitalis on the normal and the failing heart, J. Am. Coll. Cardiol. 5 (1985) A51–A59.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy Yu Hirano a, Mikie Kodama a, Masahiro Shibuya b, Yoshiyuki Maki b, Yasuo Komatsu a,⇑ a b
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Toyohira, Sapporo 062-8517, Japan Cosmo Bio Co., Ltd., Nishi-Ku, Sapporo, Japan
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 18 October 2013 Accepted 7 November 2013 Available online 16 November 2013 Keywords: Scanning electrochemical microscopy Cardiomyocytes Contraction behavior Oxygen consumption
a b s t r a c t The contractile behavior of cardiomyocytes can be monitored by measuring their action potentials, and the analysis is essential for screening the safety of potential drugs. However, immobilizing cardiac cells on a specific electrode is considerably complicated. In this study, we demonstrate that scanning electrochemical microscopy (SECM) can be used to analyze rapid topographic changes in beating cardiomyocytes in a standard culture dish. Various cardiomyocyte contraction parameters and oxygen consumption based on cell respiration could be determined from SECM data. We also confirmed that cellular changes induced by adding the cardiotonic agent digoxin were conveniently monitored by this SECM system. These results show that SECM can be a potentially powerful tool for use in drug development for cardiovascular diseases. Ó 2013 Elsevier Inc. All rights reserved.
Analysis of cardiomyocyte contraction is currently used to evaluate the pharmacological and toxicological properties of drugs [1–3]. Measurements of action potentials and topographic changes have been developed to characterize cardiomyocyte contraction. Action potentials can be recorded by a microelectrode array (MEA) [4–6], whole-cell patch clamping [7], or voltage-sensitive dyes [8]. MEA systems detect rhythmic changes in electrical potentials that are generated by beating cells when they are immobilized on a microelectrode array. Whole-cell patch-clamp techniques are used with human ether-a-go-go-related gene-transfected HEK293/CHO cells; however, electrical recordings are technically difficult. Although Ca2+-dependent fluorescent dyes can be used to monitor contraction behaviors in a standard culture dish, fluorescent signal measurements are not suitable for long-term monitoring because of photodamage and photobleaching effects [9]. Video microscopy can be used to determine the kinetics of cardiomyocyte topographic changes, and it provides valuable information on heart contraction [10]. However, to record cell motion, isolation of a single cardiomyocyte is required [11]. Quantifying cardiomyocyte oxygen consumption is also essential for analysis of energy metabolism in the heart. Oxygen consumption can be measured using a Clark electrode [12], by myoglobin saturation [13], or by electron paramagnetic resonance oximetry [14]. These methods measure the oxygen concentration in the bulk culture medium, which provides for quantitative
⇑ Corresponding author. Fax: +81 11 857 8954. E-mail address: [email protected] (Y. Komatsu). 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.11.008
analysis, but it is difficult to evaluate the oxygen consumption for a single cell. Scanning electrochemical microscopy (SECM) provides information on the local distributions of electrochemically active species by microelectrode scanning [15]. SECM has recently been used for analyzing various cellular functions [16–20]. SECM provides for noninvasive measurements of cellular topography by scanning in the vicinity of target cells adsorbed on a standard culture dish [16,17]. In addition, SECM measurements of oxygen concentrations around these cells directly reflect their respiratory activity and can be used to analyze energy metabolism in viable cells [20]. However, SECM has not yet been used for measuring the motions or oxygen consumption of beating cardiomyocytes. In this study, we developed an SECM system for analyzing the contraction kinetics and oxygen consumption of cultured cardiomyocytes. This system enabled us to investigate changes in cardiomyocyte function that resulted from changes in temperature and after adding a cardiotonic agent. Thus, this SECM system may become a powerful tool for studying pharmacological and toxicological effects in cardiomyocytes.
Material and methods Cell culture Primary cardiomyocytes were prepared from 1- to 2-day-old neonatal rat ventricles (Cosmo Bio Co., Ltd., Japan) and used for SECM measurements [21]. Cardiomyocytes were seeded on
Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy / Y. Hirano et al. / Anal. Biochem. 447 (2014) 39–42
Instrumentation SECM measurements were performed using an instrument built in our laboratory as previously described [17] but with slight modifications. In brief, our SECM setup comprised a piezomotor positioning system (Physik Instrumente, Germany), an inverted microscope, a temperature-controlled plate (Tokai Hit, Japan), a data acquisition and instrument control system (National Instruments, USA), and a potentiostat (Hokuto Denko, Japan). We used a Pt microdisk electrode (5-lm radius) as the electrochemical probe for SECM measurements and a leak-free Ag/AgCl electrode (Warner Instruments, USA) as the reference and auxiliary electrode. The radius of the Pt microelectrode, including a glass sheath, was 10 lm. Measurement procedures for beating cardiomyocytes by SECM We applied a 0.5-V potential or a 0.5-V potential (vs Ag/AgCl) to the probe electrode for ferrocyanide oxidation or oxygen reduction, respectively. To detect topographical changes in a beating cardiomyocyte, we positioned the microelectrode tip 9 lm away from a cell’s surface by approach curve measurements (Supplementary Fig. S-1) [22]. We subsequently measured the responses of the oxidation currents of ferrocyanide with a time resolution of 104 points/s (Supplementary Fig. S-2). A plot of current versus time was converted to a plot of topographical change versus time using a calibration curve (Supplementary Fig. S-4). To analyze the contraction and relaxation motions of cardiomyocytes, a single average waveform was calculated from the topographical change plot using a software that we developed with LabView (National Instruments). This program identified all pulse peaks in SECM data for 30 s, which provided a peak-to-peak interval time, a peak fluctuation value, and an average waveform. In addition, the duration of contraction, the duration of relaxation, and the contraction amplitude were determined from this average waveform (Supplementary Fig. S-3). Oxygen consumption was determined from measurements of the oxygen reduction current in the vicinity of the target cells (Supplementary Fig. S-2). We also measured the contraction behaviors and oxygen consumption of the beating cardiomyocytes by SECM at various temperatures. For these experiments, we used spontaneously beating cells 4 days after they were initially seeded. We first measured the current response at 37 °C. Further, we changed the temperature to 35 °C and waited for 7 min, after which we measured the current responses at the same position. These measurements were repeated at 33 and 31 °C. For a representative pharmacological intervention, we measured the contraction behaviors of cardiomyocytes by SECM before and after adding digoxin. For these experiments, we used spontaneously beating heart cells after incubation for 10 days. After measuring the current response at 37 °C (0 lM), we added digoxin to a final concentration of 2.5 or 10 lM, waited for 20 min, and then measured current responses at the same position.
Results and discussion Contraction kinetics analysis To prevent serum proteins from being adsorbed on the electrode surface, we performed SECM measurements of the cardiomyocytes in serum-free medium that contained 1 mM potassium ferrocyanide. Cardiomyocytes exhibited continuous spontaneous beating for at least 4 h under these conditions. In addition, a leak-free reference electrode was used so as not to interfere with cardiomyocytes that were sensitive to changes in ion concentrations. The microelectrode was positioned above the central part of the contractile motion, which was identified using an optical microscope. We measured the contraction behaviors of cardiomyocytes by our SECM system at 37 °C. A time resolution of 102 points/s failed to detect any current changes, whereas a resolution of 104 points/s obtained current pulses that were synchronized with cardiomyocyte contractions (Fig. 1, black line). A plot of current versus time could be converted to a plot of topographical change versus time (Fig. 1, gray line) using a calibration curve (Supplementary Fig. S-4). From this plot, we calculated the peak-to-peak interval time and peak fluctuation value, which showed fluctuations in the beating rhythm (Supplementary Table S-1). To analyze the contraction and relaxation motions of cardiac cells, an average waveform for all pulse peaks is required, which can be derived from active potential measurements or video microscopic analysis [4,9,11]. Thus, we developed a software program that could convert a single average waveform from the plot of topographical changes, which resulted in generating an average waveform at 37 °C (Fig. 2A, black line). The following three parameters reflected the contraction behavior: contraction duration, relaxation duration, and contraction amplitude (Supplementary Fig. S-3). Both duration values indicated the rate of cardiac contraction, and the contraction amplitude reflected the cardiac contractility. These parameters could be determined from an average plot (Supplementary Table S-1). To validate our analysis method, SECM measurements were performed for the same cell at 35, 33, and 31 °C (Supplementary Fig. S-5). The average waveform was determined as that at 37 °C
microelectrode
relaxation
contraction
0.73
0.5
0.72
0.4
0.71 0.3 0.70 0.2 0.69 0.1
0.68 0.67
Topographic change (µm)
35-mm dishes that were coated with 0.01% collagen (Koken, Japan) at a concentration of 5 104 cells/cm2. These cells were then incubated in cardiomyocyte culture medium (Cosmo Bio) supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. The medium was changed every 2 days. For SECM experiments, we used spontaneously beating cells after 4–10 days in a culture with serum-free medium that contained 1 mM potassium ferrocyanide (K4[Fe(CN)6]) and 2% Knockout serum replacement (Life Technologies, USA) in MCDB107 (Cosmo Bio). All other reagents were of analytical grade. Solutions were prepared with ultrapure water from a Millipore system.
Current (nA)
40
0.0 0
2
4
Time (s) Fig.1. Current–time responses of a beating heart cell as determined by scanning electrochemical microscopy at 37 °C. The black line indicates cardiomyocyte current–time responses, and the gray line indicates topographical changes.
Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy / Y. Hirano et al. / Anal. Biochem. 447 (2014) 39–42
(A)
cardiomyocytes (dashed line in Fig. 2B). This decreasing oxygen concentration near cardiomyocytes was dependent on the incubation temperature (Fig. 2B). These results indicated that SECM had detected oxygen concentration decreases that were due to the beating cardiomyocytes’ respiration.
1.2
Normalized contraction
1 0.8 0.6
Analyzing the effects of digoxin on cardiomyocytes
0.4
33ഒ 31ഒ
37ഒ 0.2
35ഒ
0 -0.2 0
0.2
0.4
0.6
0.8
Time (s) 0.210
cardiac fibroblast
Oxygen concentration (mM)
0.209
Measurements of the contraction amplitude and oxygen consumption are essential for analyzing the effects of cardiotonic agents on the heart. We investigated whether SECM could be used to detect these changes after treatment with the cardiotonic agent digoxin. Adding digoxin resulted in an increase in the contraction amplitude (Fig. 3A, Supplementary Fig. S-6, and Supplementary Table S-2) and a decrease in both the beat rate and the oxygen consumption (Supplementary Table S-2 and Fig. 3B) in a digoxin concentration-dependent manner. These results were consistent with those of previous studies [5,26,27]. Thus, we believe that our SECM system is applicable for analyzing dynamic changes that are induced by a cardiotonic agent.
0.208 0.207
(A)
0.206 0.205
31ഒ
0.204
33ഒ
10 µM
0.203 0.202
35ഒ
Topographic change
(B)
41
0.201 0.200
37ഒ
0.199 0
50
100
150
200
Distance from cell surface (µm) Fig.2. (A) Comparison of average waveforms at 31, 33, 35, and 37 °C. (B) Oxygen consumption analysis among local cardiomyocytes by scanning electrochemical microscopy. Plots are oxygen concentration profiles versus tip–sample distance at 31, 33, 35, and 37 °C. Dotted line indicates oxygen concentration profiles of a cardiac fibroblast (nonbeating cell).
2.5 µM
0 µM 0.5 µm
0
0.5
1
1.5
Time (s)
Respiration activity analysis Quantifying the oxygen consumption by viable cells provides valuable information regarding metabolic mechanisms [24]. Cardiomyocytes consume considerably more oxygen than the other types of nonbeating cells [25]. Although SECM can be potentially used to evaluate oxygen consumption [20], it has not been previously used for the beating cardiomyocytes. To examine the cardiomyocyte oxygen consumption, we measured the approach curves for cardiac cells comprising both cardiomyocytes and nonbeating cardiac fibroblasts. The oxygen concentration did not change either for the cardiac fibroblasts (dotted line in Fig. 2B) or on the dish surface (data not shown), whereas the oxygen concentration clearly decreased at distances closer to the beating
(B)
0.21 0.208
Oxygen concentration (mM)
(Fig. 2A). When the cell was incubated at lower temperatures, the peak-to-peak interval time (Supplementary Table S-1) and peak broadening (Fig. 2A) increased. Because these results were consistent with those obtained in a previous study [23], our SECM system was effective for analyzing cardiomyocyte contraction.
0.206 10 µM 0.204 0.202 0.2
2.5 µM
0.198 0 µM
0.196 0
50
100
150
200
Distance (µm) Fig.3. (A) Contraction and (B) oxygen consumption responses of beating heart cells before and after adding digoxin at 2.5 and 10 lM.
42
Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy / Y. Hirano et al. / Anal. Biochem. 447 (2014) 39–42
Conclusion We constructed a convenient system for analyzing cardiomyocyte behaviors in a standard culture dish. After improving the time resolution and programming software suited to our tasks, we could use our SECM system to analyze cardiomyocyte contractions. We demonstrated that kinetic parameters that reflected contraction behaviors could be extracted from our SECM data and that oxygen consumption associated with cell respiration could be quantified. On the basis of these capabilities, we evaluated the effects of adding a cardiotonic agent on the cardiomyocyte behavior. Taken together, our results indicate that our SECM system has significant potential for analyzing cardiomyocytes. Acknowledgments We thank Dr. Yasuhiro Mie and Dr. Masiki Ikegami for their helpful discussions. The financial support received, in part, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Grant-in-Aid for Scientific Research No. 25410162) and from the Ministry of Economy, Trade, and Industry, Japan, is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab. 2013.11.008. References [1] M.L. De Bruin, M. Pettersson, R.H.B. Meyboom, A.W. Hoes, H.G.M. Leufkens, Anti-HERG activity and the risk of drug-induced arrhythmias and sudden death, Eur. Heart J. 26 (2005) 590–597. [2] W.S. Redfern, L. Carlsson, A.S. Davis, W.G. Lynch, I. MacKenzie, S. Palethorpe, P.K.S. Siegl, I. Strang, A.T. Sullivan, R. Wallis, A.J. Camm, T.G. Hammond, Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development, Cardiovasc. Res. 58 (2003) 32–45. [3] J.P. Valentin, Reducing QT liability and proarrhythmic risk in drug discovery and development, Br. J. Pharmacol. 159 (2010) 5–11. [4] A. Stett, U. Egert, E. Guenther, F. Hofmann, T. Meyer, W. Nisch, H. Haemmerle, Biological application of microelectrode arrays in drug discovery and basic research, Anal. Bioanal. Chem. 377 (2003) 486–495. [5] L. Guo, J.Y. Qian, R. Abrams, H.M. Tang, T. Weiser, M.J. Sanders, K.L. Kolaja, The electrophysiological effects of cardiac glycosides in human iPSC-derived cardiomyocytes and in guinea pig isolated hearts, Cell. Physiol. Biochem. 27 (2011) 453–462. [6] N. Yokoo, S. Baba, S. Kaichi, A. Niwa, T. Mima, H. Doi, S. Yamanaka, T. Nakahata, T. Heike, The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells, Biochem. Biophys. Res. Commun. 387 (2009) 482–488. [7] J.C. Hancox, M.J. McPate, A. El Harchi, Y.H. Zhang, The hERG potassium channel and hERG screening for drug-induced torsades de pointes, Pharmacol. Ther. 119 (2008) 118–132.
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