**6. Conclusions**

In the discussion sections of many published *in vivo* studies, the results obtained are compared with previously published findings. Although changes in ECG parameters are often described, the type of anesthesia used in the experiments is not taken into account. Moreover, in acute *in vivo* experiments, the time of day the experiments are performed, and the adaptation of the animals to the LD cycle, and/or sex, are not taken into account whatsoever. This approach is self-evident and logical because the experiments are mostly performed only on males and during the workday, often without regard for chronobiological principles.

However, if changes in ECG parameters are considered to be important indicators of arrhythmogenesis, such comparisons may be misleading and must not be immediately regarded to indicate a difference in myocardial electrical stability. We should be more careful in interpreting results and, in discussing the mechanisms underlying a given type of arrhythmia, acknowledge that initial ECG parameters may already be affected to some extent by the anesthesia used and by regular daytime experimentation. The data presented in the tables clearly demonstrate the differences in baseline or control values with different types of anesthesia and whether the baseline or control value is "normal" or already altered by anesthesia should be taken into account. For example, a change in the evaluated ECG parameter after an intervention may not necessarily indicate a possible electrophysiological substrate for the development of an arrhythmia, it can only be "adjusted to a normal value" because we do not know the reference value.

Similarly, sex and time of day the experiments are performed can be a problem because it is not possible to determine sex differences as well as changes during the active and nonactive period of rat regimen day because there are no studies that have directly addressed this aspect. Telemetry studies that would reveal changes in ECG parameters in circadian dependence, to describe reference values and, possibly, sex differences, could help to facilitate interpretation of the results obtained. However, it is highly speculative to consider the values from the cited telemetry studies as reference values (although the ECG is measured from nonanesthetized rats) because the methodologies do not report whether the indicated baseline value is the 24 h average (mesor) or is the current value measured immediately before the intervention. Most likely, they are baseline values before the experimental intervention and this only applies to male rats, whereas the lighted (light or dark) period when the experiment is performed is not reported, although an adaptation of animals to the LD cycle is described.

Thus, the question "Which anesthetic is the most suitable anesthetic in *in vivo* rat cardiological experiments so that the initial electrophysiology of the heart is not significantly affected" is relatively difficult to address for several reasons. First, we do not currently have specified sex-related reference values for rats. Second, because there are circadian variations in the measurable parameters of the cardiovascular system, there are also changes in individual ECG parameters, depending on the light cycle (inactive period) and dark (active period). Finally, the effects of anesthetics at the level of ion channels are not described in detail because the entire electrophysiology of the myocardium depends on ionic currents and the overall metabolism of minerals.

As such, when evaluating changes in ECG parameters in rats, these possible variations should also be taken into account. The correct assessment of changes, in turn, depends on knowledge of the reference values according to sex and on the time of day the experiments or measurements are performed. Although rat ECG parameters are only analyzed in this study, these can be of basis to further researches and studies that may involve humans in the future.
