Investigating the Role of Uncoupling Proteins in Viability following nsPEF
College
College of Engineering & Technology (Batten)
Department
Frank Reidy Research Center for Bioelectrics
Graduate Level
Doctoral
Graduate Program/Concentration
Bioelectrical Engineering
Presentation Type
Poster Presentation
Abstract
Background: Nanosecond Pulsed Electric Fields (nsPEF) have been shown to induce mitochondrial stress responses such as depolarization of the mitochondrial membrane (ΔΨm) and the production of mitochondrial reactive oxygen species (mROS). However, the identity of the specific pore or proton transporter that helps stimulate these responses is still unknown.
Hypothesis: The hypothesis in this study is that the one of the mitochondrial uncoupling proteins (UCP1/2/3) is a proton transporter affecting these changes in mitochondrial function.
Methods: The experiments investigated nsPEF effects in H9c2 (rat cardiomyoblasts) and Jurkat (human T-lymphocytes) in the presence and absence of ADP (100-500 µM, ATPase-linked inhibitor of UCP2/3) and GDP (100-500uM, a known inhibitor of UCP1). Flow cytometry was used to assess changes in mitochondrial membrane potential (ΔΨm) and mROS production in nsPEF-treated samples, with and without ADP or GDP. Transepithelial Electrical Resistance (TEER) was measured via Electric cell-substrate Impedance Sensing (ECIS, Applied Biophysics) to understand how ADP and GDP influences nsPEF-induced changes in cellular adhesion.
Results: A dose-dependent decrease in ΔΨm and an increase in mROS production were observed in response to nsPEF exposure. In these experiments, ADP-treated samples exhibited significant dose-dependent preservation of mitochondrial membrane potential and reduction in mROS production, even under high nsPEF conditions. In contrast to this, GDP-treated samples displayed only minimal protection. In the TEER studies, NsPEF-treated H9c2 cells showed a reduction in cell adhesion in comparison to sham-treated samples. 100µM ADP appeared to protect cells from nsPEF-induced loss of adherence, while100µM GDP-treated cells seemed to further decrease in cellular adhesion.
Conclusion: Due to the limited effects of GDP in our trials, and that both H9c2 and Jurkat cells contain little to no presence of UCP1, the results of these experiments indicate that either UCP2 or UCP3 may be involved as a proton transporter. Further trials using Genipin (a UCP2 inhibitor) and Oleic Acid (a UCP3 activator) will identify the specific UCP involved in these nsPEF induced changes in mitochondrial function. Harnessing specific mechanisms underlying these mitochondrial processes may lead to new strategies in modulating mitochondrial bioenergetics.
Funding: this project was funded in-part by Old Dominion University, Frank Reidy Research Center for Bioelectrics, SEED grant.
Keywords
nsPEF- nanosecond pulsed electric fields, TEER- Transepithelial Electrical Resistance, ADP- Adenosine Diphosphate, GDP- Guanosine Diphosphate, UCP-Uncoupling Protein
Investigating the Role of Uncoupling Proteins in Viability following nsPEF
Background: Nanosecond Pulsed Electric Fields (nsPEF) have been shown to induce mitochondrial stress responses such as depolarization of the mitochondrial membrane (ΔΨm) and the production of mitochondrial reactive oxygen species (mROS). However, the identity of the specific pore or proton transporter that helps stimulate these responses is still unknown.
Hypothesis: The hypothesis in this study is that the one of the mitochondrial uncoupling proteins (UCP1/2/3) is a proton transporter affecting these changes in mitochondrial function.
Methods: The experiments investigated nsPEF effects in H9c2 (rat cardiomyoblasts) and Jurkat (human T-lymphocytes) in the presence and absence of ADP (100-500 µM, ATPase-linked inhibitor of UCP2/3) and GDP (100-500uM, a known inhibitor of UCP1). Flow cytometry was used to assess changes in mitochondrial membrane potential (ΔΨm) and mROS production in nsPEF-treated samples, with and without ADP or GDP. Transepithelial Electrical Resistance (TEER) was measured via Electric cell-substrate Impedance Sensing (ECIS, Applied Biophysics) to understand how ADP and GDP influences nsPEF-induced changes in cellular adhesion.
Results: A dose-dependent decrease in ΔΨm and an increase in mROS production were observed in response to nsPEF exposure. In these experiments, ADP-treated samples exhibited significant dose-dependent preservation of mitochondrial membrane potential and reduction in mROS production, even under high nsPEF conditions. In contrast to this, GDP-treated samples displayed only minimal protection. In the TEER studies, NsPEF-treated H9c2 cells showed a reduction in cell adhesion in comparison to sham-treated samples. 100µM ADP appeared to protect cells from nsPEF-induced loss of adherence, while100µM GDP-treated cells seemed to further decrease in cellular adhesion.
Conclusion: Due to the limited effects of GDP in our trials, and that both H9c2 and Jurkat cells contain little to no presence of UCP1, the results of these experiments indicate that either UCP2 or UCP3 may be involved as a proton transporter. Further trials using Genipin (a UCP2 inhibitor) and Oleic Acid (a UCP3 activator) will identify the specific UCP involved in these nsPEF induced changes in mitochondrial function. Harnessing specific mechanisms underlying these mitochondrial processes may lead to new strategies in modulating mitochondrial bioenergetics.
Funding: this project was funded in-part by Old Dominion University, Frank Reidy Research Center for Bioelectrics, SEED grant.