Poster Session 2 - C6
1,2Dylan Langburt, 1,2,3Scott Heximer
1 Department of Physiology, University of Toronto Faculty of Medicine; 2 Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research; 3 Heart and Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research
Ischemic heart disease (IHD) is the most prominent type of cardiovascular disease and a leading cause of death worldwide. Although reperfusion therapy has the potential to improve outcomes for IHD patients, it can also produce detrimental effects, collectively referred to as ischemia-reperfusion injury (IR injury). Recently, it was demonstrated that the outcome from induced IR injury in mice was associated with two highly expressed heterotrimeric G-protein alpha subunits, Gi2 and Gi3. Specifically, knockout of Gi2 worsened cardiac outcomes, whereas knockout of Gi3 improved the response to IR injury. Preliminary data from our lab points to the divergent roles of Gi2 and Gi3 in the regulation of metabolic homeostasis (autophagy) and the selective targeting of mitochondrial degradation (mitophagy). Additionally, we propose that the activity state of Gi3 (GDP-bound; inactive form or GTP-bound; active form) mediates an important switch between the mitophagic and autophagic signaling machinery (Vps34/Vps15/Atg14-containing complexes). My project is focused on elucidating Gi3’s role in IR injury, and assessing the mechanisms of the improved outcomes in the Gi3 KO mice. Complete functional (electrocardiogram, ultrasound, and PV-loop) and histologic (TTC/Evan’s Blue staining, immunohistochemistry) analyses will be performed at various time points after induced IR injury to determine the nature and timeline of the reperfusion injury in WT and Gi3 KO mice. IR-injury protocols will be repeated using inhibitors (3-methlyadenine) and activators (rapamycin) of autophagy to examine the extent to which altered autophagic activity may be driving the differences in IR injury outcomes. In parallel to the work described above, I am generating Gi3 KO human embryonic kidney cells (HEK) using Crisper/Cas9 targeting constructs. Together with WT and Gi3 KO mouse embryonic fibroblasts (MEF), I will have a complete set of cell-based genetic tools to study the role of Gi3 in cell metabolism, autophagy, and mitophagy in cultured cells. Successful Crisper/Cas9 KO of Gai3 in HEKs have been demonstrated through restriction enzyme digestion of genomic PCR samples, immunoblotting, and are currently being validated using sequencing. Developing a better understanding of Gai3’s role in regulating cardiac health during reperfusion therapy is a potential step toward designing therapeutic strategies that limit its damaging effects.