Enhancing Cardiac Ablation Efficacy with Electroporation-Assisted Intracellular Delivery of the Ribosome-Inactivating Protein Gelonin

Background: Pulsed field ablation (PFA) is a promising non-thermal approach for cardiac ablation that induces cardiomyocyte death through electroporation. In our previous studies, we demonstrated that despite high electric field strengths, a proportion of cells remain only reversibly electroporated and survive pulsed electric fields (PEF) exposure. Achieving complete ablation therefore requires higher electric fields, which increases the risk of vasospasm or collateral injury. One possible strategy to enhance ablation efficacy without raising field strength is selective pharmacological targeting of reversibly electroporated cells. Gelonin, a type I ribosome-inactivating protein, inhibits protein synthesis once inside the cytosol but is completely membrane-impermeant and therefore non-toxic to cells with intact plasma membranes. We investigated whether transient membrane permeabilization during PEF exposure enables intracellular gelonin delivery to enhance cardiomyocyte ablation efficacy.
Methods: The potential cardiotoxicity of gelonin was first evaluated by assessing long-term viability of AC16 cardiomyocytes using the IncuCyte live-cell imaging platform. Spontaneous penetration of gelonin through the intact plasma membrane was excluded with molecular dynamics simulations. The synergistic cytotoxic effect of gelonin in combination with PFA was investigated by exposing monolayers of cardiomyocytes to µsPEF (20 x 100 microsecond pulses at 1 Hz) or nsPEF (200 x 300 nanosecond pulses at 10 Hz) in the presence of 0–150 nM gelonin, using contact electrodes precisely positioned by an automated system. Cell death was quantified by propidium iodide uptake, and caspase-3/7 activity was measured to monitor the progression of apoptosis.
Results: Long-term observation of cardiomyocytes exposed to gelonin alone revealed no cytotoxic effects. Molecular dynamics simulations demonstrated that gelonin rapidly adsorbs onto the lipid bilayer without penetrating the membrane, confirming that it remains extracellular in the absence of electroporation. Under µsPEF exposure, gelonin significantly enhanced cytotoxicity by lowering the electric field strength required to kill 50% of cardiomyocytes (ED₅₀) from 0.8 kV/cm (95% CI 0.7–0.9) to 0.5 kV/cm (95% CI 0.4–0.6; p < 0.0001). In contrast, nsPEF-induced ablation was not affected by the presence of gelonin (ED₅₀ ≈ 6 kV/cm; p = 0.49). Caspase-3/7 activity was elevated in cardiomyocytes exposed to µsPEF, indicating activation of apoptotic pathways.
Conclusion: Gelonin acts as a sensitizer that selectively enhances µsPEF-mediated cardiomyocyte death without inducing toxicity in non-electroporated cells. Combining electroporation with non-permeant ribosome-inactivating protein represents a novel strategy to improve ablation efficacy and limits the reversibility of electroporation caused by pulsed field ablation.