Master of Science
Pathology and Laboratory Medicine
Cardiac ischemia-reperfusion injury (IRI) occurs intra-operatively during heart transplantation (HTx), underpinning graft survival. Past research implicated the PI3K/Akt1 & RhoA/ROCK pathways in IRI. Rho-GTPase activity/Akt regulates Deleted-in-liver-cancer 1 protein’s beta-isoform (DLC1β). Therefore, we hypothesized that DLC1β overexpression (OE) had anti-apoptotic effects. In vitro, HL-1 cells (mouse cardiomyocytes) received chamber hypoxia-oxygenation reperfusion (H/R) for 24H4R with/without DLC1β plasmid, before collection for protein/mRNA analyses. DLC1β-OE resulted in downregulated pro-apoptotic mRNA expression (Bax/Cycs/Casp3), upregulated protective mRNA targets (BCl2/Akt1) and less late/early apoptosis via flow cytometry. Results were confirmed via H9C2 (rat cardiomyocyte) cell lines. In vivo, C57/BL6 mice received heterotopic HTx with/without DLC1β-OE plasmid tail-vein injections. Grafts were analyzed POD1/7 via mRNA/protein/histopathology. Treatment with DLC1β-OE reduced IRI as evidenced by decreased cumulative injury via histopathology. In summary, future translational applications are of interest as DLC1β-OE demonstrated novel anti-apoptotic effects conferred via RT-qPCR in vitro; moreover, in vivo grafts demonstrated reduced neutrophilic infiltrate/fibrosis/overall injury.
Summary for Lay Audience
The conducted study focuses on how we can reduce injury to the heart during heart transplantation. Cardiac ischemia-reperfusion injury (IRI) occurs when transplanting a heart from donor to recipient. The “ischemia” phase of cardiac IRI occurs when the donor heart is being collected and blood/oxygen is not going to the heart. The surgery connects the recipients blood supply back to the heart, however, the rush of blood back to the heart can result in further damage. This is known as the “reperfusion” phase in cardiac IRI. In transplantation, the heart that is removed from the donor is not being pumped with blood anymore which is why many consider IRI to be inevitable. However, if we can understand how this damage happens, we may be able to develop strategies to avoid this injury in future. Furthermore, as cardiac IRI results in poor outcomes for the patient, we may be able to use research in this field to help future recipients. Recently, a few studies have shown that a protein known as deleted in liver cancer 1 beta (DLC1β) could have protective effects on transplanted hearts against IRI. Therefore, we aimed to study whether the increase of this protein could have protective effects against IRI. This study focuses specifically on a unique form of DLC1 known as DLC1β, which has been found in heart tissue. To date, research available on this topic is currently limited. However, we wanted to conduct this study in hopes of finding targets that could be used for further research. By doing this, we hoped that this study would allow for clinical applications (human studies/drug trials) in the future. The big question that this study aimed to answer was, does manipulating DLC1β reduce I/R injury? And which pathways does DLC1β interact with? By analyzing these questions, this research might be useful in humans to potentially reduce the rate of injury during heart transplantation. To study IRI, we had to perform mouse heart transplants. We conducted the transplants on mice after they received an injection of DLC1β (or not); and then their hearts were stored in a fridge for 24hrs. This temporarily decreases the amount of oxygen the heart receives and simulates a clinical transplant-like model. Then we transplanted the hearts into new recipients and sacrificed the mice after 24hrs or 7 days to see how the heart reacted. To conclude, it is believed that this study could have potential extensions into human populations if we discover that DLC1β is protective to the heart.
Collings, Samantha L., "The role of DLC1β in attenuating cardiac ischemia-reperfusion injury during heart transplantation" (2023). Electronic Thesis and Dissertation Repository. 9444.
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