Abstract:AIMS: Gene therapy to protect the heart from ischemic reperfusion injury is clinically attractive. We investigated if delivery of DNA encoding for Carbonic Anhydrase 9 (CA9) to HL-1 cells is protective against H2O2 stimulation in vitro.MAIN METHODS: Plasmid DNA with CA9 incorporated (PcDNA 3,1 CA9 2,1) had been pre-made and incorporated into a bacteria. We grew the bacteria and isolated the pcDNA for transfection experiments with HL-1 cells. CA9 protein were detected after transfection using Western blot, and band density was meassured and compared to HL-1 cells transfected with empty vector. Transfection rate was evaluated using EGFP and Hoechst staining of cells, and calculated. A model of cell injury using H2O2 was established. PcDNA 3,1 CA9 2,1 transfected HL-1 cells and empty vector transfected HL-1 cells were injured with 300mM H2O2 , and cell death were analyzed using Flow Cytometry (propidium iodid) and meassurements of lactate dehydrogenase (LDH) release into the medium. KEY FINDINGS: Lipofectamin transfected HL-1 cells had a transfection rate of 22% as evaluated by EGFP. Transfection lead to a 50 times increase of CA9 expression as evaluated by western blot. H2O2 injury of HL-1 cells increased the number of propidium iodide positive cells, and increased release of LDH. Weather or not CA9 protects against H2O2 stimulation still remains unknown. More experiments have to be done.SIGNIFICANCE: This observation do not have clinical potential at this point. Too many questions remain unanswered. However we have collected some of the pieces in the puzzle and laid grounds for completing the scientific question which was raised.