Discipline: Biological Sciences
Courtnee' R. Bell - Alabama State University
Co-Author(s): Sparkle D. Williams, University of Alabama in Birmingham, AL; Brian Sims, University of Alabama in Birmingham, AL; Glenn C. Rowe, University of Alabama in Birmingham, AL; Gulnaz Javan, Alabama State University, AL; and Qiana L. Matthews, Alabama State University, AL
Cardiovascular disease (CVD) refers to conditions that involve the narrowing or blockage of blood vessels that can potentially lead to the onset of a heart attack, and may be the cause of sudden, suspicious, or unnatural death in criminal cases. CVDs are frequently concealed and discovered only at postmortem autopsy through the use of molecular investigations. Determining cause of death, and time since death, using conventional autopsy techniques relies on interpretation of physical changes that occur after death. Although common in their use, these physical changes cannot be documented in cases of advanced or prolonged decomposition. The need for more accurate alternative methods of postmortem investigation has sparked interest in the gene expression shifts that take place after death. The goal of this project is to identify potential exosomal markers for the identification of sudden cardiac death and determination of time of death.
Currently, we are quantifying the effect that alcohol administration has on exosomes biogenesis of human cardiomyocyte cell line, AC16. The AC16 cell line was cultured in exosome-free medium and was either not treated (control) or treated with 50mM or 100mM of alcohol for 24, 48, or 72 hours. AC16 cell viability was significantly decreased with treatments of 50 mM or 100 mM of alcohol. The presence of exosomes was confirmed using NanoSight analysis, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). The groups of exosomes were isolated using a series of high-speed ultracentrifugation and quantitated using the Lowry dilution method. By means of ELISA, various tetraspanins (CD9, CD63, CD81) were confirmed to be present on AC16-derived exosomes. Our findings show that alcohol administration on AC16 cell line significantly impacts actin and heat shock protein 70 within AC16-derived exosomes. In this study, we are also evaluating postmortem exosomes from mice and swine samples, at 0hr time of death. Blood was isolated and exosomes were extracted and confirmed by means of NanoSight, SDS-PAGE and ELISA. Our findings, identified a baseline of serum-derived exosome associated proteins (clathrin, actin, tubulin) that can be used in postmortem analysis in mouse and swine and animal model systems at a 0hr time of death.
The evaluation of the impact of alcohol on cardiac cell death and postmortem exosome biogenesis will yield new insight into the importance of exosomes in a variety of physiological and pathological settings including cardiovascular disease. Our future studies will include evaluating exosomes at other times of death as well as evaluating postmortem gene expression.
Funder Acknowledgement(s): NSF grant number NSF-HRD 1401075
Faculty Advisor: Qiana Matthews, email@example.com
Role: I performed all the Laboratory experiments: Cell culture, ELISA, SDS-PAGE, and NanoSight