Research Papers

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

[+] Author and Article Information
Feng Liu

Department of Mechanical Engineering,
University of Minnesota,
111 Church Street SE,
Minneapolis, MN 55455
e-mail: liux3176@umn.edu

Priyatanu Roy

Department of Mechanical Engineering,
University of Minnesota,
111 Church Street SE,
Minneapolis, MN 55455
e-mail: royxx299@umn.edu

Qi Shao

Department of Mechanical Engineering,
University of Minnesota,
111 Church Street SE,
Minneapolis, MN 55455;
Institute for Engineering in Medicine,
University of Minnesota,
111 Church Street SE,
Minneapolis, MN 55455
e-mail: shaox070@umn.edu

Chunlan Jiang

Department of Mechanical Engineering,
University of Minnesota,
111 Church Street SE,
Minneapolis, MN 55455
e-mail: jiang240@umn.edu

Jeunghwan Choi

Department of Mechanical Engineering,
University of Minnesota,
Slay Hall, Library Drive,
Greenville, NC 27858;
Department of Engineering,
East Carolina University,
Slay Hall, Library Drive,
Greenville, NC 27858
e-mail: choijo14@ecu.edu

Connie Chung

Department of Mechanical Engineering,
University of Minnesota,
111 Church Street SE,
Minneapolis, MN 55455
e-mail: chung301@umn.edu

Dushyant Mehra

Department of Mechanical Engineering,
University of Minnesota,
111 Church Street SE,
Minneapolis, MN 55455
e-mail: mehr0096@umn.edu

John C. Bischof

Department of Mechanical Engineering,
University of Minnesota,
111 Church Street SE,
Minneapolis, MN 55455;
Institute for Engineering in Medicine,
University of Minnesota,
111 Church Street SE,
Minneapolis, MN 55455;
Department of Biomedical Engineering,
University of Minnesota,
Nils Hasselmo Hall,
312 Church St. SE,
Minneapolis, MN 55455
e-mail: bischof@umn.edu

1F. Liu and P. Roy contributed equally to this work.

2Corresponding author.

Manuscript received May 25, 2017; final manuscript received January 10, 2018; published online April 2, 2018. Assoc. Editor: Guy M. Genin.

J Biomech Eng 140(6), 061007 (Apr 02, 2018) (9 pages) Paper No: BIO-17-1226; doi: 10.1115/1.4039375 History: Received May 25, 2017; Revised January 10, 2018

Atrial fibrillation (AF) currently affects millions of people in the U.S. alone. Focal therapy is an increasingly attractive treatment for AF that avoids the debilitating effects of drugs for disease control. Perhaps the most widely used focal therapy for 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 in some settings. 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 propidium iodide (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 AF.

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Grahic Jump Location
Fig. 1

Schematics showing (a) cellular protein loss method with BCA; Equation (3) was used for protein loss calculation. (b) Protein denaturation measurement method with FTIR; Equation (5) was used for cellular protein denaturation fraction calculation.

Grahic Jump Location
Fig. 2

Total protein content in lysed cell suspensions (1 × 106 cells/ml) consisting of control and IRE at 2000 V/cm after sonication; PBS without any cells is also shown for comparison; n ≥ 3 for all measurements ±SD; P value = 0.550 indicates the average protein content in control and 2000 V/cm IRE group is not statistically different

Grahic Jump Location
Fig. 3

HL-1 viability after cryo, heating, and IRE exposure to primary treatment parameters: (a) viability after cells were cooled to increasingly cryogenic temperatures. (Reprinted with permission from Choi et al. [40]. Copyright 2017 by Elsevier.) (b) Viability after cells were heated to increasingly supra-physiological temperatures. (c) Viability of cell suspensions which underwent IRE at increasing electrical field strength. (Reprinted with permission from Jiang et al. [27]. Copyright 2015 by IEEE.) Each data point represents the average ±SD where each measurement included n ≥ 3 separate measurements on ≥100 cells in total.

Grahic Jump Location
Fig. 4

HL-1 viability, PLF and PDF after cryo, heating and IRE with varying primary parameter exposures; at increasing cryogenic end-temperatures ((a), (d) and (g)); at increasing supra-physiological end-temperatures ((b), (e), and (h)) (inset in (e) shows PLF for control on ice and at 25 °C); at increasing IRE field strengths of 250–2000 V/cm ((c), (f), and (i)); each data point represents the average of n ≥ 3 measurements ± SD. PDF in (h) is calculated based on correlated parameter fit [31]; PDF in (i) was calculated from Eq. (5). (a) Reprinted with permission from Choi et al. [40]. Copyright 2017 by Elsevier. (c) Reprinted with permission from Jiang et al. [27]. Copyright 2015 by IEEE.

Grahic Jump Location
Fig. 5

Variable protein content within cells, aggregates and supernatant after IRE; the line shows relative native protein remaining in cell pellet after treatment; n ≥ 3 for all measurements; p < 0.001 between all data sets



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