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.
Highlights
Animal models are widespread tool for studying cardiac electric activity under various experimental conditions
Morphological Parameters of Electrogram To reveal potential direct effects of di-4-ANEPPS on electric activity of the heart various EG parameters were evaluated before the ischemia onset
In accordance with the findings presented in this study, decreased spontaneous heart rate and slowed impulse conduction through the ventricles have been reported in isolated hearts stained by di-4-ANEPPS in almost all commonly used laboratory animal species (Nygren et al, 2003; Novakova et al, 2008; Larsen et al, 2012)
Summary
Animal models are widespread tool for studying cardiac electric activity under various experimental conditions. AP can be recorded directly by microelectrodes or using optical methods. The conventional methods using microelectrodes are the gold standards for measuring electrical signals on the cellular level (Richardson and Xiao, 2010). These methods require an electrode placement in tight contact to the cell surface, which is difficult to ensure in long-term experiments. Optical methods use fluorescence properties of special chemical compounds, so called voltagesensitive dyes (VSD). Di4-ANEPPS allows reaching a time resolution better than 1 ms (Dhein et al, 2005) and exhibits changes in fluorescence of up to 10% per 100 mV (Johnson et al, 1999). VSD based approach allows non-invasive record of AP from a larger area of heart surface with high spatial resolution
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