Significant hemolysis is present during irreversible electroporation of cardiomyocytes in vitro

Abstract

BACKGROUND: Pulsed-field ablation (PFA) of atrial fibrillation (AF) is a new method in clinical practice. Despite a favorable safety profile of PFA in AF ablation, rare cases of renal failure, probably due to hemolysis, have been recently reported. OBJECTIVE: The aim of this study was to determine the rate of hemolysis and cardiac cell death during in vitro PFA with different electric field intensities. METHODS: Blood samples from healthy volunteers and mouse HL-1 cardiomyocyte cell lines were subjected to in vitro irreversible electroporation (IRE) using 216 bipolar pulses, each lasting 2 μs with 5 μs intervals, repeated 20 times at a frequency of 1 Hz. These pulses varied in from 500 to 1500 V. Cell-free hemoglobin levels were assessed spectrophotometrically, and red blood cell microparticles (RBCμ) were evaluated using flow cytometry. Cardiomyocyte death was quantified using propidium iodide. RESULTS: PF energy (1000 V/cm, 1250 V/cm, and 1500 V/cm) was associated with a significant increase in cell-free hemoglobin (0.31 +- 0.16 g/l, 2.33 +- 0.90 g/l, and 5.7 +- 0.20 g/l, p< 0.05), and similar increase in the concentration of RBCμ. Significant rates of cardiomyocyte death were observed at electric field strengths of 750 V/cm, 1000 V/cm, 1250 V/cm and 1500 V/cm (26.5 +- 5.9%, 44.3 +- 6.2%, 55.5 +- 6.9% and 74.5 +- 17.8% of cardiomyocytes, p < 0.05). CONCLUSION: The most effective induction of cell death in vitro was observed at 1500 V/cm. This intensity was also associated with a significant degree of hemolysis.