Project Title

Decreased Reactive Oxygen Species Buffering Capacity May Underlie Arrhythmia Susceptibility in the Scn1b-/- Mouse Model of Dravet Syndrome

Authors' Affiliations

Jessa L. Aldridge, Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University Emily Davis Alexander, Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University Chad R. Frasier, Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University

Location

Culp Room 303

Start Date

4-6-2022 10:00 AM

End Date

4-6-2022 11:00 AM

Faculty Sponsor’s Department

Biomedical Sciences

Name of Project's Faculty Sponsor

Chad Frasier

Classification of First Author

Graduate Student-Doctoral

Competition Type

Competitive

Type

Oral Presentation

Project's Category

Cardiovascular Disease

Abstract or Artist's Statement

Dravet syndrome (DS) is a severe pediatric-onset epilepsy disorder that mostly arises from loss-of-function mutations in voltage-gated sodium channel genes. Patients with DS have an elevated risk (~17%) of Sudden Unexpected Death in Epilepsy (SUDEP), a fatal complication of seizure disorders. While the exact physiological mechanisms underlying SUDEP remain unclear, cardiac arrhythmias have been heavily implicated. Previous work indicates that reactive oxygen species (ROS) accumulation during oxidative stress, a condition where ROS generation exceeds antioxidant capacity, can lead to mitochondrial instability and cardiac arrhythmias. The purpose of this investigation was to determine how ROS scavenging and antioxidant levels may be altered in the Scn1b-/- mouse model of DS. In the heart, the primary antioxidant pathway is the glutathione (GSH) system. This study tested the hypothesis that Scn1b-/- (KO) mice are more susceptible to arrhythmias due to a decreased ability to buffer ROS accumulations during prolonged oxidative stress through the GSH system. First, qPCR analysis was conducted on hearts from KO and Scn1b+/+ (WT) mice to determine differences in gene expression of key enzymes in the GSH system, glutathione peroxidase (Gpx) and glutathione reductase (Gsr). Gpx is responsible for the reduction of H2O2 to H2O. Gsr maintains GSH in the reduced state. In P17 (after seizure onset) KO mice, Gpx expression was decreased (0.27-fold; p = 0.03) but Gsr remained unchanged. The enzymatic activity of GPx and GR were also measured. Counter to the qPCR data, the results suggested that there are no differences in enzyme activity in KO mice. To determine if there were differences at the cellular level, isolated cardiac cells were subjected to a prolonged oxidative challenge. Fluorescence microscopy was used to assess changes in the signal of CM-DCF, a fluorescent ROS indicator, following the addition of 80 mM diamide to cells. Diamide causes oxidative stress by depleting intracellular stores of GSH. Cells isolated from KO mice subject to prolonged oxidative stress die at much faster rates than cells isolated from WT mice. In addition, death occurs at earlier timepoints (as early as 1 min). The mean time to death in KO cells was significantly shorter (p = 0.03) and occurred on average 4.25 minutes faster than in WT cells. In addition, CM-DCF signals intensified shortly before cell death, indicating ROS accumulation precedes cell death. Finally, to test for arrhythmia susceptibility at the whole organ level, isolated hearts were perfused with diamide and scored for arrhythmia susceptibility and severity. Over the first 15 and 30 minutes of perfusion KO hearts had significantly higher scores. In conclusion, the results of this study indicate that hearts from Scn1b-/- mice have a decreased capability to handle ROS accumulations during prolonged oxidative stress. This may be the result of deficits in the GSH system that compromise ROS scavenging in the heart.

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Apr 6th, 10:00 AM Apr 6th, 11:00 AM

Decreased Reactive Oxygen Species Buffering Capacity May Underlie Arrhythmia Susceptibility in the Scn1b-/- Mouse Model of Dravet Syndrome

Culp Room 303

Dravet syndrome (DS) is a severe pediatric-onset epilepsy disorder that mostly arises from loss-of-function mutations in voltage-gated sodium channel genes. Patients with DS have an elevated risk (~17%) of Sudden Unexpected Death in Epilepsy (SUDEP), a fatal complication of seizure disorders. While the exact physiological mechanisms underlying SUDEP remain unclear, cardiac arrhythmias have been heavily implicated. Previous work indicates that reactive oxygen species (ROS) accumulation during oxidative stress, a condition where ROS generation exceeds antioxidant capacity, can lead to mitochondrial instability and cardiac arrhythmias. The purpose of this investigation was to determine how ROS scavenging and antioxidant levels may be altered in the Scn1b-/- mouse model of DS. In the heart, the primary antioxidant pathway is the glutathione (GSH) system. This study tested the hypothesis that Scn1b-/- (KO) mice are more susceptible to arrhythmias due to a decreased ability to buffer ROS accumulations during prolonged oxidative stress through the GSH system. First, qPCR analysis was conducted on hearts from KO and Scn1b+/+ (WT) mice to determine differences in gene expression of key enzymes in the GSH system, glutathione peroxidase (Gpx) and glutathione reductase (Gsr). Gpx is responsible for the reduction of H2O2 to H2O. Gsr maintains GSH in the reduced state. In P17 (after seizure onset) KO mice, Gpx expression was decreased (0.27-fold; p = 0.03) but Gsr remained unchanged. The enzymatic activity of GPx and GR were also measured. Counter to the qPCR data, the results suggested that there are no differences in enzyme activity in KO mice. To determine if there were differences at the cellular level, isolated cardiac cells were subjected to a prolonged oxidative challenge. Fluorescence microscopy was used to assess changes in the signal of CM-DCF, a fluorescent ROS indicator, following the addition of 80 mM diamide to cells. Diamide causes oxidative stress by depleting intracellular stores of GSH. Cells isolated from KO mice subject to prolonged oxidative stress die at much faster rates than cells isolated from WT mice. In addition, death occurs at earlier timepoints (as early as 1 min). The mean time to death in KO cells was significantly shorter (p = 0.03) and occurred on average 4.25 minutes faster than in WT cells. In addition, CM-DCF signals intensified shortly before cell death, indicating ROS accumulation precedes cell death. Finally, to test for arrhythmia susceptibility at the whole organ level, isolated hearts were perfused with diamide and scored for arrhythmia susceptibility and severity. Over the first 15 and 30 minutes of perfusion KO hearts had significantly higher scores. In conclusion, the results of this study indicate that hearts from Scn1b-/- mice have a decreased capability to handle ROS accumulations during prolonged oxidative stress. This may be the result of deficits in the GSH system that compromise ROS scavenging in the heart.