Di-4-ANEPPS Modulates Electrical Activity and Progress of Myocardial Ischemia in Rabbit Isolated Heart

Abstract

AimsAlthough 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 ResultsStudy 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.ConclusionOur 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.

Optical mapping and optogenetics in cardiac electrophysiology research and therapy: a state-of-the-art review

“…For example, voltage-sensitive dye di-4-ANEPPS has demonstrated reduced spontaneous heart rate, sodium current, T-wave amplitude, and AV-node conduction in ex vivo and in vitro preparations. 12 , 25 …”

“…Cardiac optical mapping uses voltage and calcium (Ca 2+ )-sensitive dye to image multi-cellular preparations at high spatiotemporal resolutions. 1 Cultured cardiomyocytes, 7 induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), 8 engineered heart tissue (EHT), 9 myocardial slices, 10 isolated atria, 11 and whole hearts 12 have all been optically mapped. It has been crucial in deciphering the role of rotors in atrial fibrillation, 13 the virtual electrode phenomena, 14 and autonomic regulation of cardiac arrhythmias.…”

Experimental models of myocardial ischemia: classical approaches and innovations (review)

“…2. Principle scheme of "Heart-On-Chip" [13] 2024;39(1): [18][19][20][21][22][23][24][25][26][27] Для изучения ишемии / реперфузии миокарда разработаны модели «сердца-на-чипе», в которых осуществляется регуляция подачи кислорода [15]. Создание модели, имитирующей постинфарктные изменения, возможно путем воздействия повреждающих факторов (например, лазера) или регуляцией количества фибробластов и коллагена [16].…”

Optical mapping of contracting hearts

“…Although multiple voltage‐sensitive dyes with different values of ∆ F / F are already available in the market, it is important to note the adverse effects of these dyes in addition to their benefits. For example, the most commonly used ratiometric potentiometric dye, di‐4‐ANEPPS, was observed to have effects on heart rate (Fialova et al., 2010 ; Janoušek et al., 2015 ) in rat, guinea pig and rabbit, conduction velocity (Larsen et al., 2012 ) in guinea pig, and ischaemia (Ronzhina et al., 2021 ) in rabbit. However, intracoronary injection of the dye di‐4‐ANBDQBS (JPW‐6033), designed for blood‐perfusion optical mapping (Matiukas et al., 2007 ), showed no signs of cardiac toxicity during continuous monitoring of ECG from baseline to 30 min after dye/solvent injection in pig models.…”