Based on our previous study showing accelerated conversion of ventricular activation sequence during hypoxic incubation correlating with increased apoptosis in the atrioventricular canal, we hypothesized that this slight increase in apoptosis could lead to an accelerated pruning of myocardial atrioventricular connections, which would manifest as earlier appearance of mature apex-to-base activation patterns. To test this hypothesis, we blocked apoptosis using an established caspase inhibitor in chick embryonic heart and analyzed the effects of this treatment on cell death and ventricular activation patterns. ED4 chick embryonic hearts were treated with the peptide Caspase inhibitors zVAD-fmk by intrapericardial injection in ovo. Spontaneously beating embryonic hearts isolated at ED8 were stained with voltage-sensitive dye Di-4-ANEPPS and imaged at 37 °C with Ultima L high-speed camera at 1 kHz. Amount of apoptotic cells was analyzed at ED 7 by whole mount LysoTracker Red staining and confocal microscopy. Hearts of embryos treated with zVAD showed a significantly increased proportion of immature (base to apex) activation patterns at ED8, including ventricular activation originating from right atrioventricular junction, a pattern never observed in control hearts. The number of apoptotic cells in atrioventricular canal myocardium was decreased at ED7 in the treated hearts. Apoptosis in the atrioventricular canal myocardium is thus an important contributor to ventricular activation pattern. Its inhibition at critical stages can lead to persistence of accessory atrioventricular connections, which form a morphological substrate for ventricular pre-excitation. Supported by project of MSMT VZ 0021620806, LC 06061, and by grant of AS CR AV0Z50450515.