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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$a Ronzhina, Marina $u Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, Czechia

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$a Di-4-ANEPPS Modulates Electrical Activity and Progress of Myocardial Ischemia in Rabbit Isolated Heart / $c M. Ronzhina, T. Stracina, L. Lacinova, K. Ondacova, M. Pavlovicova, L. Marsanova, R. Smisek, O. Janousek, K. Fialova, J. Kolarova, M. Novakova, I. Provaznik

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$a 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.

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$a Stracina, Tibor $u Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czechia

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$a Lacinova, Lubica $u Centre of Biosciences, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia

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$a Ondacova, Katarina $u Centre of Biosciences, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia

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$a Pavlovicova, Michaela $u Centre of Biosciences, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia

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$a Marsanova, Lucie $u Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, Czechia

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$a Smisek, Radovan $u Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, Czechia

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$a Janousek, Oto $u Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, Czechia

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$a Fialova, Katerina $u Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czechia

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$a Kolarova, Jana $u Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, Czechia

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$a Novakova, Marie $u Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czechia $u International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia

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$a Provaznik, Ivo $u Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, Czechia

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