Direct evidence IF1 preserves mitochondrial ATP during hypoxia
Publication Date
July 2019
Location
LSU Health Medical Education Building
Document Type
Abstract
Start Date
26-7-2019 9:00 AM
End Date
26-7-2019 12:00 PM
Description
Adenosine triphosphate (ATP) is an organic chemical that provides energy in living cells. Mitochondria generate over 90% of ATP via oxidative phosphorylation driven by proton motive force (PMF). The PMF drives the forward rotation of ATP synthase to synthesize ATP. Under hypoxic conditions, ATP is hydrolyzed, driving the backward rotation of ATP synthase, which pumps back proton to maintain mitochondrial membrane potential. A natural occurring inhibitor (ATPase inhibiting factor 1, IF1) has long been shown to be an ATP synthase regulator, but it remains unclear that IF1 inhibits the backward rotation or the entire rotation of ATP synthase. An upregulation of IF1 has been found in many cancer cells, failing hearts and diabetic skeletal muscle. However, the exact role of IF1 on ATP synthase activities remain controversial, due in part to the lack of tools to directly monitor the spatiotemporal ATP contents in cells. In this work, we used an imaging approach to investigate how IF1 determines the changes of mitochondrial ATP in normoxia and hypoxia to understand the role of IF1 in regulating ATP synthase. We transfected mouse embryonic stem cells (MEF) derived from IF1 knock-out, overexpression, and wild-type mice with a plasmid carrying a mitochondria-specific dye that visualizes mitochondrial ATP production (mitoMaLionR). The plasmid was delivered by the lipid-base transfection system (Lipofectamine). Cells were incubated for 24h with the DNA/Lipofectamine followed by a medium change and recovery for 24h. Images were taken using Cytation 5 with 20x and 40x objective lens. To determine if mitoMaLionR is localized in the mitochondria, cells were double-stained with MitoTracker Green FM. The double staining showed that mitoMaLionR was localized in the mitochondria with minimal cytosolic fluorescence. No difference in mitoMaLionR was found among the three experimental groups under normoxia. After switching to hypoxia (2% O2) for 3h, MEF with IF1 overexpression exhibited higher fluorescent intensity than that of control cells, indicating that ATP was preserved. In the IF1 knock-out cells, the fluorescent intensity was significantly reduced compared to the control. In conclusion, the used mitochondrial detection probe is a powerful tool to visualize the mitochondrial ATP. The mitochondrial ATP imaging results support that IF1 inhibits the hydrolysis of the ATP synthesis to preserve ATP under the hypoxic condition.
Recommended Citation
Dante, Dean John Hendrix T., "Direct evidence IF1 preserves mitochondrial ATP during hypoxia" (2019). Summer Research Internship Program. 8.
https://digitalscholar.lsuhsc.edu/srip/2019/undergrad/8
Direct evidence IF1 preserves mitochondrial ATP during hypoxia
LSU Health Medical Education Building
Adenosine triphosphate (ATP) is an organic chemical that provides energy in living cells. Mitochondria generate over 90% of ATP via oxidative phosphorylation driven by proton motive force (PMF). The PMF drives the forward rotation of ATP synthase to synthesize ATP. Under hypoxic conditions, ATP is hydrolyzed, driving the backward rotation of ATP synthase, which pumps back proton to maintain mitochondrial membrane potential. A natural occurring inhibitor (ATPase inhibiting factor 1, IF1) has long been shown to be an ATP synthase regulator, but it remains unclear that IF1 inhibits the backward rotation or the entire rotation of ATP synthase. An upregulation of IF1 has been found in many cancer cells, failing hearts and diabetic skeletal muscle. However, the exact role of IF1 on ATP synthase activities remain controversial, due in part to the lack of tools to directly monitor the spatiotemporal ATP contents in cells. In this work, we used an imaging approach to investigate how IF1 determines the changes of mitochondrial ATP in normoxia and hypoxia to understand the role of IF1 in regulating ATP synthase. We transfected mouse embryonic stem cells (MEF) derived from IF1 knock-out, overexpression, and wild-type mice with a plasmid carrying a mitochondria-specific dye that visualizes mitochondrial ATP production (mitoMaLionR). The plasmid was delivered by the lipid-base transfection system (Lipofectamine). Cells were incubated for 24h with the DNA/Lipofectamine followed by a medium change and recovery for 24h. Images were taken using Cytation 5 with 20x and 40x objective lens. To determine if mitoMaLionR is localized in the mitochondria, cells were double-stained with MitoTracker Green FM. The double staining showed that mitoMaLionR was localized in the mitochondria with minimal cytosolic fluorescence. No difference in mitoMaLionR was found among the three experimental groups under normoxia. After switching to hypoxia (2% O2) for 3h, MEF with IF1 overexpression exhibited higher fluorescent intensity than that of control cells, indicating that ATP was preserved. In the IF1 knock-out cells, the fluorescent intensity was significantly reduced compared to the control. In conclusion, the used mitochondrial detection probe is a powerful tool to visualize the mitochondrial ATP. The mitochondrial ATP imaging results support that IF1 inhibits the hydrolysis of the ATP synthesis to preserve ATP under the hypoxic condition.
Comments
Mentors: Lothar Lauterboeck and Qinglin Yang, Cardiovascular Center of Excellence