AIMS: Although voltage-sensitive dye di-4-ANEPPS is a common tool for mapping cardiac electrical activity, reported effects on electrophysiological parameters are rather. The main goals of the study were to reveal effects of the dye on rabbit isolated heart and to verify, whether rabbit isolated heart stained with di-4-ANEPPS is a suitable tool for myocardial ischemia investigation. METHODS AND RESULTS: Study involved experiments on stained (n = 9) and non-stained (n = 11) Langendorff perfused rabbit isolated hearts. Electrophysiological effects of the dye were evaluated by analysis of various electrogram (EG) parameters using common paired and unpaired statistical tests. It was shown that staining the hearts with di-4-ANEPPS leads to only short-term sporadic prolongation of impulse conduction through atria and atrioventricular node. On the other hand, significant irreversible slowing of heart rate and ventricular conduction were found in stained hearts as compared to controls. In patch clamp experiments, significant inhibition of sodium current density was observed in differentiated NG108-15 cells stained by the dye. Although no significant differences in mean number of ventricular premature beats were found between the stained and the non-stained hearts in ischemia as well as in reperfusion, all abovementioned results indicate increased arrhythmogenicity. In isolated hearts during ischemia, prominent ischemic patterns appeared in the stained hearts with 3-4 min delay as compared to the non-stained ones. Moreover, the ischemic changes did not achieve the same magnitude as in controls even after 10 min of ischemia. It resulted in poor performance of ischemia detection by proposed EG parameters, as was quantified by receiver operating characteristics analysis. CONCLUSION: Our results demonstrate significant direct irreversible effect of di-4-ANEPPS on spontaneous heart rate and ventricular impulse conduction in rabbit isolated heart model. Particularly, this should be considered when di-4-ANEPPS is used in ischemia studies in rabbit. Delayed attenuated response of such hearts to ischemia might lead to misinterpretation of obtained results.

Centre of Biosciences Institute of Molecular Physiology and Genetics Slovak Academy of Sciences Bratislava Slovakia

Department of Biomedical Engineering Faculty of Electrical Engineering and Communication Brno University of Technology Brno Czechia

Department of Physiology Faculty of Medicine Masaryk University Brno Czechia

International Clinical Research Center St Anne's University Hospital Brno Brno Czechia

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Acker C. D., Yan P., Loew L. M. (2020). Recent progress in optical voltage-sensor technology and applications to cardiac research: from single cells to whole hearts. Prog. Biophys. Mol. Biol. 154 3–10. 10.1016/j.pbiomolbio.2019.07.004 PubMed DOI PMC

Baker L. C., Wolk R., Choi B. R., Watkins S., Plan P., Shah A., et al. (2004). Effects of mechanical uncouplers, diacetyl monoxime, and cytochalasin-D on the electrophysiology of perfused mouse hearts. Am. J. Physiol. Heart Circ. Physiol. 287 H1771–H1779. 10.1152/ajpheart.00234.2004 PubMed DOI

Baruscotti M., Westenbroek R., Catterall W. A., DiFrancesco D., Robinson R. B. (1997). The newborn rabbit sino-atrial node expresses a neuronal type I-like N+ channel. J. Physiol. 498 641–648. 10.1113/jphysiol.1997.sp021889 PubMed DOI PMC

Bernardo N. L., D’Angelo M., Okubo S., Joy A., Kukreja R. C. (1999). Delayed ischemic preconditioning is mediated by opening of ATP-sensitive potassium channels in the rabbit heart. Am. J. Physiol. 45 H1323–H1330. 10.1152/ajpheart.1999.276.4.H1323 PubMed DOI

Bernier M., Curtis M. J., Hearse D. J. (1989). Ischemia-induced and reperfusion-induced arrhythmias: importance of heart rate. Am. J. Physiol. 256 H21–H31. 10.1152/ajpheart.1989.256.1.H21 PubMed DOI

Bers D. M. (2002). Cardiac Na/Ca exchange function in rabbit, mouse and man: what’s the difference? J. Mol. Cell Cardiol. 34 369–373. 10.1006/jmcc.2002.1530 PubMed DOI

Brack K. E., Narang R., Winter J., Ng G. A. (2013). The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart. Exp. Physiol. 98 1009–1027. 10.1113/expphysiol.2012.069369 PubMed DOI PMC

Ceconi C., Cargnoni A., Francolini G., Parinello G., Ferrari R. (2009). Heart rate reduction with ivabradine improves energy metabolism and mechanical function of isolated ischaemic rabbit heart. Cardiovasc. Res. 84 72–82. 10.1093/cvr/cvp158 PubMed DOI

Cheng Y., Van Wagoner D. R., Mazgalev T. N., Tchou P. J., Efimov I. R. (1998). Voltage-sensitive dye RH421 increases contractility of cardiac muscle. Can. J. Physiol. Pharmacol. 76 1146–1150. 10.1139/y98-124 PubMed DOI

Dhein S., Mohr D. W., Delmar M. (2005). Practical Methods in Cardiovascular Research. Berlin Heidelberg: Springer.

Du Y.-M., Nathan R. D. (2007). Ionic basis of ischemia-induced bradycardia in the rabbit sinoatrial node. J. Mol. Cell Cardiol. 42 315–325. 10.1016/j.yjmcc.2006.10.004 PubMed DOI

Efimov I. R., Nikolski V., Salama G. (2004). Optical imaging of the heart. Circ. Res. 94 21–33. 10.1161/01.RES.0000130529.18016.35 PubMed DOI

Feng Y., Cona M. M., Vunckx K., Li Y., Chen F., Nuyts J., et al. (2013). Detection and quantification of acute reperfused myocardial infarction in rabbits using DISA-SPECT/CT and 3.0 T cardiac MRI. Int. J. Cardiol. 168 4191–4198. 10.1016/j.ijcard.2013.07.108 PubMed DOI

Fialova K., Kolarova J., Janousek O., Ronzhina M., Provaznik I., Novakova M. (2011). Effects of voltage-sensitive dye di-4-ANEPPS on isolated rat heart electrogram. Comput. Cardiol. 38 721–724.

Fialova K., Kolarova J., Provaznik I., Novakova M. (2010). Comparison of voltage-sensitive dye di-4-ANEPPS effects in isolated hearts of rat, guinea pig, and rabbit. Comput. Cardiol. 2010 1–4.

Gintant G. A., Gallacher D. J., Pugsley M. K. (2011). The ‘overly-sensitive’ heart: sodium channel block and QRS interval prolongation. Br. J. Pharmacol. 164 254–259. 10.1111/j.1476-5381.2011.01433.x PubMed DOI PMC

Hardy M. E., Lawrence C. L., Standen N. B., Rodrigo G. C. (2006). Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes? J. Pharmacol. Toxicol. Methods 54 173–182. 10.1016/j.vascn.2006.02.013 PubMed DOI

Hardy M. E., Pollard C. E., Small B. G., Bridgland-Taylor M., Woods A. J., Valentin J. P., et al. (2009). Validation of a voltage-sensitive dye (di-4-ANEPPS)-based method for assessing drug-induced delayed repolarisation in beagle dog left ventricular midmyocardial myocytes. J. Pharmacol. Toxicol. Methods 60 94–106. 10.1016/j.vascn.2009.03.005 PubMed DOI

Hejc J., Vitek M., Ronzhina M., Novakova M., Kolarova J. (2015). A wavelet-based ECG delineation method: adaptation to an experimental electrograms with manifested global ischemia. Cardiovasc. Eng. Technol. 6 364–375. 10.1007/s13239-015-0224-z PubMed DOI

Holcomb M. R., Woods M. C., Uzelac I., Wikswo J. P., Gilligan J. M., Sidorov V. Y. (2009). The potential of dual camera systems for multimodel imaging of cardiac electrophysiology and metabolism. Exp. Biol. Med. 234 1355–1373. 10.3181/0902-RM-47 PubMed DOI PMC

Johnson P. L., Smith W., Bayhnam T. C., Knisley S. B. (1999). Errors caused by combination of di-4-ANEPPS and fluo 3/4 for simultaneous measurements of transmembrane potentials and intracellular calcium. Ann. Biomed. Eng. 27 563–571. 10.1114/1.198 PubMed DOI

Kaese S., Frommeyer G., Verheule S., van Loon G., Gehrmann J., Breithardt G., et al. (2013). The ECG in cardiovascular-relevant animal models of electrophysiology. Herzschr. Elektrophys. 2 84–91. 10.1007/s00399-013-0260-z PubMed DOI

Kang C., Brennan J. A., Kuzmiak-Glancy S., Garrott K. E., Kay M. W., Efimov I. R. (2016). Technical advances in studying cardiac electrophysiology – role of rabbit models. Prog. Biophys. Mol. Biol. 121 97–109. 10.1016/j.pbiomolbio.2016.05.006 PubMed DOI

Kappadan V., Telele S., Uzelac I., Fenton F., Parlitz U., Luther S., et al. (2020). High- resolutional optical measurement of cardiac restitution, contraction, and fibrillation dynamics in beating vs. blebbistatin-uncoupled isolated rabbit hearts. Front. Physiol 26:464. 10.3389/fphys.2020.00464 PubMed DOI PMC

Kawaguchi A., Asano H., Matsushima K., Wada T., Yoshida S., Ichida S. (2007). Enhancement of sodium current in NG108-15 cells during neural differentiation is mainly due to an increase in NaV1.7 expression. Neurochem. Res. 32 1469–1475. 10.1007/s11064-007-9334-9 PubMed DOI

Kolarova J., Fialova K., Janousek O., Novakova M., Provaznik I. (2010). Experimental methods for simultaneous measurement of action potentials and electrograms in isolated heart. Physiol. Res. 59 71–80. 10.33549/physiolres.932010 PubMed DOI

Larsen A. P., Olesen S.-P., Grunnet M., Poelzing S. (2010). Pharmacological activation of IKr impairs conduction in guinea pig hearts. J. Cardiovasc. Electrophysiol. 21 923–929. 10.1111/j.1540-8167.2010.01733.x PubMed DOI

Larsen A. P., Sciuto K. J., Moreno A. P., Poelzing S. (2012). The voltage-sensitive dye di-4-ANEPPS slows conduction velocity in isolated guinea pig hearts. Heart Rhythm. 9 1493–1500. 10.1016/j.hrthm.2012.04.034 PubMed DOI PMC

Liang Q., Sohn K., Punske B. B. (2006). Propagation and electrical impedance changes due to ischemia, hypoxia and reperfusion in mouse hearts. Conf. Proc. IEEE Eng. Med. Biol. Soc. 1 1560–1563. 10.1109/IEMBS.2006.260296 PubMed DOI

Liu J., Tu H., Zhang D., Zheng H., Li Y.-L. (2012). Voltage-gated sodium channel expression and action potential generation in differentiated NG108-15 cells. BMC Neurosci. 13:129. 10.1186/1471-2202-13-129 PubMed DOI PMC

Lopez-Izquierdo A., Warren M., Riedel M., Cho S., Lai S., Lux R. L., et al. (2014). A near-infrared fluorescent voltage-sensitive dye allows for moderate-throughput electrophysiological analyses of human induced pluripotent stem cell-derived cardiomyocytes. Am. J. Physiol. Heart Circ. Physiol. 307 H1370–H1377. 10.1152/ajpheart.00344.2014 PubMed DOI PMC

Mandapati R., Asano Y., Baxter W. T., Gray R., Davidenko J., Jalife J. (1998). Quantification of effects of global ischemia on dynamics of ventricular fibrillation in isolated rabbit heart. Circulation 98 1688–1696. 10.1161/01.CIR.98.16.1688 PubMed DOI

Marionneau C., Couette B., Liu J., Li H., Mangoni M. E., Nargeot J., et al. (2005). Specific pattern of ionic channel gene expression associated with pacemaker activity in mouse heart. J. Physiol. 562 223–234. 10.1113/jphysiol.2004.074047 PubMed DOI PMC

Martišiene I., Jurevièius J., Vosyliute R., Navalinskas A., Treinys R., Maèianskiene R., et al. (2015). Evolution of action potential alternans in rabbit heart during acute regional ischemia. Biomed. Res. Int. 2015:951704. 10.1155/2015/951704 PubMed DOI PMC

Matiukas A., Pertsov A. M., Cram K. P., Tolkacheva E. G. (2009). Optical mapping of electrical heterogeneities in the heart during global ischemia. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2009 6321–6324. 10.1109/IEMBS.2009.5333176 PubMed DOI PMC

Moreau A., Mercier A., Thériault O., Boutjdir M., Burger B., Keller D. I., et al. (2017). Biophysical, molecular, and pharmacological characterization of voltage-dependent sodium channels from induced pluripotent stem cell-derived cardiomyocytes. Can. J. Cardiol. 33 269–278. 10.1016/j.cjca.2016.10.001 PubMed DOI

Ng F. S., Shadi I. T., Peters N. S., Lyon A. R. (2013). Selective heart rate reduction with ivabradine slows ischaemia-induced electrophysiological changes and reduces ischaemia-reperfusion-induced ventricular arrhythmias. J. Mol. Cell Cardiol. 59 67–75. 10.1016/j.yjmcc.2013.02.001 PubMed DOI PMC

Novakova M., Bardonova J., Provaznik I., Taborska E., Bochorakova H., Paulova H., et al. (2008). Effects of voltage sensitive dye di-4-ANEPPS on guinea pig and rabbit myocardium. Gen. Physiol. Biophys. 27 45–54. PubMed

Nygren A., Clark R. B., Belke D. D., Kondo C., Giles W. R., Witkowski F. X. (2000). Voltage-sensitive dye mapping of activation and conduction in adult mouse hearts. Ann. Biomed. Eng. 28 958–967. 10.1114/1.1308501 PubMed DOI

Nygren A., Kondo C., Clark B. B., Giles W. R. (2003). Voltage-sensitive dye mapping in Langendorff-perfused rat hearts. Am. J. Physiol. Heart Circ. Physiol. 284 H892–H902. 10.1152/ajpheart.00648.2002 PubMed DOI

O’Shea C., Kabir S. N., Holmes A. P., Lei M., Fabritz L., Rajpoot K., et al. (2020). Cardiac optical mapping – State-of-the-art and future challenges. Int. J. Biochem. Cell Biol. 126:105804. 10.1016/j.biocel.2020.105804 PubMed DOI PMC

Qian Y. W., Sung R. J., Lin S. F., Province R., Clusin W. T. (2003). Spatial heterogeneity of action potential alternans during global ischemia in the rabbit heart. Am. J. Physiol. Heart Circ. Physiol. 285 H2722–H2733. 10.1152/ajpheart.00369.2003 PubMed DOI

Richardson E. S., Xiao Y. (2010). “Electrophysiology of single cardiomyocytes: patch clamp and other recording methods,” in Cardiac Electrophysiology Methods and Models, 1st Edn, eds Sigg D. S., Iaizzo P. A., Xiao Y.-F., He B. (New York, NY: Springer; ), 329–348.

Ronzhina M. (2017). Study of Electrophysiological Function of The Heart in Experimental Cardiology. [dissertation]. Brno University of Technology.

Ronzhina M., Cmiel V., Janousek O., Kolarova J., Novakova M., Babula P., et al. (2013a). Application of the optical method in experimental cardiology: action potential and intracellular calcium concentration measurement. Physiol. Res. 62 125–137. 10.33549/physiolres.932369 PubMed DOI

Ronzhina M., Olejnickova V., Janousek O., Kolarova J., Novakova M., Provaznik I. (2013b). Effects of heart orientation on isolated hearts electrograms. Comput. Cardiol. 2013 543–546.

Ronzhina M., Olejnickova V., Stracina T., Novakova M., Janousek O., Hejc J., et al. (2017). Effect of increased left ventricle mass on ischemia assessment in electrocardiographic signals: rabbit isolated heart study. BMC Cardiovasc. Disord 17:216. 10.1186/s12872-017-0652-9 PubMed DOI PMC

Salama G. (2001). “Optical mapping: background and historical perspective,” in Optical Mapping of Cardiac Excitation and Arrhythmias, eds Rosenbaum D. S., Jalife J. (New York, NY: Futura Publishing Company; ), 9–33.

Salerno S., Garten K., Smith G. L., Stolen T., Kelly A. (2020). Two-photon excitation of FluoVolt allows improved interrogation of transmular electrophysiological function in the intact mouse heart. Prog. Biophys. Mol. Biol. 154 11–20. 10.1016/j.pbiomolbio.2019.08.007 PubMed DOI PMC

Shattock M. J., Rosen M. R. (2006). The control of heart rate: the physiology of the sinoatrial node and the role of the If current. Dialogues Cardiovasc. Med. 11 5–17. 10.1016/j.pbiomolbio.2019.08.007 PubMed DOI PMC

Surawicz B., Tavel M. (2008). “Stress-test,” in Chou’s Electrocardiography in Clinical Practice, eds Surawicz B., Knilans T. K. (Philadelphia, PA: Saunders Elsevier; ), 221–255.

Swift L. M., Asfour H., Posnack N. G., Arutunyan A., Kay M. W., Sarvazyan N. (2012). Properties of blebbistatin for cardiac optical mapping and other imaging applications. Pflugers. Arch. 464 503–512. 10.1007/s00424-012-1147-2 PubMed DOI PMC

Takaki H., Tahara N., Miyazaki S., Sugimachi M., Sunagawa K. (1999). Exercise-induced QRS prolongation in patients with mild coronary artery disease: computer analysis of the digitized multilead ECGs. J. Electrocardiol. 32 206–211. 10.1016/s0022-0736(99)90082-1 PubMed DOI

Varró A., Lathrop D. A., Hester S. B., Nánási P. P., Papp J. G. Y. (1993). Ionic currents and action potentials in rabbit, rat, and guinea pig ventricular myocytes. Basic Res. Cardiol. 88 93–102. 10.1007/BF00798257 PubMed DOI

Vesely P., Ronzhina M., Fialova K., Kolarova J., Halamek J., Novakova M. (2015). The effect of voltage sensitive dye di-4-ANEPPS on the RT/RR coupling in rabbit isolated heart. Comput. Cardiol. 2015 1137–1140.

Wagner G. S., Macfarlane P., Wellens H., Josephson M., Gorgels A., Mirvis D. M., et al. (2009). AHA/ACCF/HRS recommendations for the standardization and interpretation of the electrocardiogram. Part VI: acute ischemia/infarction: a scientific statement from the American Heart Association Electrocardiography and Arrhythmias Committee, Council on Clinical Cardiology; the American College of Cardiology Foundation; and the Heart Rhythm Society: Endorsed by the International Society for Computerized Electrocardiology. Circulation 119 e262–e270. 10.1016/j.jacc.2008.12.016 PubMed DOI

Golden Standard or Obsolete Method? Review of ECG Applications in Clinical and Experimental Context