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.
Solid tumors such as hepatocellular carcinoma are very often not amenable to chemotherapy and radiotherapy. Local ablation methods, including chemical ablation with absolute ethanol, are therefore an option for treatment but lack of information about the mechanism of devitalization leading to cell death is a hindrance to further adoption. Systemic toxicity also has limited the amount of ethanol that can be used in a single treatment session. Therefore we evaluated the mechanism of urea, a denaturant with little or no systemic toxicity, for potential use in chemical ablation. In this study we report on the use of three methods to analyze the effects in cell culture with a view towards eventual clinical application. Human hepatoma HuH-7 cells were analyzed at several time points after treatment using FTIR, DSC, and Raman microspectroscopy based on MTT and PI-exclusion viability assays. Time course fractional denaturation data plotted against viability show that a 50% viability drop occurs after only a 10-20% drop in overall protein denaturation. Other methods of cell death such as apoptosis may also be operative, but this result implies that protein denaturation is one of the major mechanisms of cell death. This is in line with what has been previously suggested for purely thermal methods, and opens the way to mechanism-based improvements in chemical ablation of solid tumors.
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