The Role of Protein Loss and Denaturation in Determining Outcomes of Heating, Cryotherapy, and Irreversible Electroporation on Cardiomyocytes

Abstract

Atrial fibrillation affects millions of people in the US. Focal therapy is an attractive treatment for atrial fibrillation that avoids the debilitating effects of drugs for disease control. Perhaps the most widely used focal therapy for atrial fibrillation (AF) is heat-based radiofrequency (heating), although cryotherapy (cryo) is rapidly replacing it due to a reduction in side effects and positive clinical outcomes. A third focal therapy, irreversible electroporation (IRE), is also being considered. This study was designed to help guide treatment thresholds and compare mechanism of action across heating, cryo, and IRE. Testing was undertaken on HL-1 cells, a well-established cardiomyocyte cell line, to assess injury thresholds for each treatment method. Cell viability, as assessed by Hoechst and PI staining, was found to be minimal after exposure to temperatures =-40 °C (cryo), =60 °C (heating), and when field strengths =1500 V/cm (IRE) were used. Viability was then correlated to protein denaturation fraction (PDF) as assessed by Fourier Transform Infrared (FTIR) spectroscopy, and protein loss fraction (PLF) as assessed by Bicinchoninic Acid (BCA) assay after the three treatments. These protein changes were assessed both in the supernatant and the pellet of cell suspensions post treatment. We found that dramatic viability loss (=50%) correlated strongly with =12% protein change (PLF, PDF or a combination of the two) in every focal treatment. These studies help in defining both cellular thresholds and protein-based mechanisms of action that can be used to improve focal therapy application for atrial fibrillation.

Engineering T cell response to cancer antigens by choice of focal therapeutic conditions

“…In the case of Heat, very little protein is released compared to Cryo and IRE ( Figure 3), and it is almost completely denatured as would be expected in a lethal heat exposure above 50 C [11]. In contrast, Cryo at À80 C releases large quantities of protein, but denatures very little presumably due to membrane rupture [11]. Finally, IRE releases large amounts of protein due to membrane rupture [11].…”

“…Cancer cell protein release after exposure to different focal therapeutic conditions is dramatically different, as summarized in Table 1. In the case of Heat, very little protein is released compared to Cryo and IRE ( Figure 3), and it is almost completely denatured as would be expected in a lethal heat exposure above 50 C [11]. In contrast, Cryo at À80 C releases large quantities of protein, but denatures very little presumably due to membrane rupture [11].…”

“…In contrast, Cryo at À80 C releases large quantities of protein, but denatures very little presumably due to membrane rupture [11]. Finally, IRE releases large amounts of protein due to membrane rupture [11]. However, unlike Cryo, IRE shows a large amount of denaturation of the released protein likely caused by the interaction of the high electric field with the charged amino acid residues of proteins and with solvent molecules [10].…”

“…The protein release measurement was modified from the cellular protein loss measurement described in [11]. In this study, we define protein release as the content of protein within the lysate, i.e., released from the cells after exposure to the focal therapy conditions and the gentle 200 g 10 min spin.…”

“…Despite increased use, focal therapies can fail to completely destroy cancer [4,5], resulting in local [6] and/or systemic recurrence or spread [7]. To address these shortcomings investigators have studied the mechanism of action at the molecular and cellular level to improve local cancer destruction by thermal therapy (Heat) [8], cryotherapy (Cryo) [9] and IRE [10,11]. Further studies have shown the ability to physically enhance these destructive mechanisms [12,13] and/or combine them to achieve synergistic destruction of local cancer [14,15].…”