Methods
Each trial consisted of isolating the hearts of two mice, aged anywhere between 8-15 weeks, with the strain FVB/NJ of the Mus genus. Eight trials were conducted throughout the duration of the experiment. No female mice were used for any of the trials because because the research in the lab concentrates on males, due to known differences in protein expression among the two sexes. The effect of the VU591 drug on mitochondrial KATP function in wildtype mice was measured by calculating the change in membrane potential, when compared with the negative control group, which consisted of the same wildtype mice before the addition of the ROMK2 inhibitor. A parallel study involving ROMK2 knockout mice and the VU591 drug was conducted simultaneously. …show more content…
(2007), but any modifications to the procedure have clearly been specified. The entire process was handled at cold temperatures, with all necessary tubes and reagents being stored on ice. For every sample, two mice hearts were sacrificed and transferred to a flat-bottomed 2 mL centrifuge tube. The hearts were minced with surgical scissors after 500 µL isolation buffer (50 mL sucrose, 200 mM mannitol, 5 mM KH2PO4, 1 mM EGTA, 5 mM MOPS, 0.1% bovine serum albumin, pH to 7.3) was added to the tube (Sigma-Aldrich, St. Louis, MO, USA). The minced heart solution was then homogenized using a Polytron PT 1200 E homogenizer 2-3 times for approximately 10-15 seconds each. After adding 1 mL protease buffer (1.25 µL proteinase K in 1 mL isolation buffer) to each sample, the samples were left to incubate on ice for 5 minutes (Sigma-Aldrich, St. Louis, MO, USA). Cells are digested into myocytes with the addition of the protease buffer. Rather than using a dounce homogenizer to homogenize the solution containing cardiac myocytes, the motorized Wheaton Overhead Stirrer was used to obtain a more uniform solution (Gostimskaya et al., 2010). The mixture was transferred to a 50 mL centrifuge tube and 10 mL protease inhibitor buffer (1 EDTA-free 20 mg protease inhibitor tablet dissolved in 10 mL of isolation buffer) was added to wash the suspended tissues into the larger …show more content…
By adding diazoxide, which is an activator of the potassium channel, the membrane would depolarize, thereby increasing the absorption. The blockage of the mitochondrial KATP channel with the VU591 inhibitor would prevent the membrane from depolarizing. However, the addition of the FCCP uncoupler would cause an instant depolarization. Oligomycin is an antibiotic that acts by binding ATP synthase to block the proton channel, but FCCP activates the proton conductance, causing the instant depolarization. Refer to Figure 1 for a visual representation of the expected outcomes. The graph summarizes a study conducted by Aggarwal et al,, and we expect our results to depict a similar pattern because our predictions are based off the research conducted by their group (2010). Figure 2 presents the absorption values for one of the trials we conducted, and the sudden decrease in absorption resulted from the addition of mitochondria and FCCP. Since this was a test run, the data has not yet been analyzed, but it is important to note that the decrease in absorption, upon the addition of FCCP, appears to be inconsistent with the previous
Each trial consisted of isolating the hearts of two mice, aged anywhere between 8-15 weeks, with the strain FVB/NJ of the Mus genus. Eight trials were conducted throughout the duration of the experiment. No female mice were used for any of the trials because because the research in the lab concentrates on males, due to known differences in protein expression among the two sexes. The effect of the VU591 drug on mitochondrial KATP function in wildtype mice was measured by calculating the change in membrane potential, when compared with the negative control group, which consisted of the same wildtype mice before the addition of the ROMK2 inhibitor. A parallel study involving ROMK2 knockout mice and the VU591 drug was conducted simultaneously. …show more content…
(2007), but any modifications to the procedure have clearly been specified. The entire process was handled at cold temperatures, with all necessary tubes and reagents being stored on ice. For every sample, two mice hearts were sacrificed and transferred to a flat-bottomed 2 mL centrifuge tube. The hearts were minced with surgical scissors after 500 µL isolation buffer (50 mL sucrose, 200 mM mannitol, 5 mM KH2PO4, 1 mM EGTA, 5 mM MOPS, 0.1% bovine serum albumin, pH to 7.3) was added to the tube (Sigma-Aldrich, St. Louis, MO, USA). The minced heart solution was then homogenized using a Polytron PT 1200 E homogenizer 2-3 times for approximately 10-15 seconds each. After adding 1 mL protease buffer (1.25 µL proteinase K in 1 mL isolation buffer) to each sample, the samples were left to incubate on ice for 5 minutes (Sigma-Aldrich, St. Louis, MO, USA). Cells are digested into myocytes with the addition of the protease buffer. Rather than using a dounce homogenizer to homogenize the solution containing cardiac myocytes, the motorized Wheaton Overhead Stirrer was used to obtain a more uniform solution (Gostimskaya et al., 2010). The mixture was transferred to a 50 mL centrifuge tube and 10 mL protease inhibitor buffer (1 EDTA-free 20 mg protease inhibitor tablet dissolved in 10 mL of isolation buffer) was added to wash the suspended tissues into the larger …show more content…
By adding diazoxide, which is an activator of the potassium channel, the membrane would depolarize, thereby increasing the absorption. The blockage of the mitochondrial KATP channel with the VU591 inhibitor would prevent the membrane from depolarizing. However, the addition of the FCCP uncoupler would cause an instant depolarization. Oligomycin is an antibiotic that acts by binding ATP synthase to block the proton channel, but FCCP activates the proton conductance, causing the instant depolarization. Refer to Figure 1 for a visual representation of the expected outcomes. The graph summarizes a study conducted by Aggarwal et al,, and we expect our results to depict a similar pattern because our predictions are based off the research conducted by their group (2010). Figure 2 presents the absorption values for one of the trials we conducted, and the sudden decrease in absorption resulted from the addition of mitochondria and FCCP. Since this was a test run, the data has not yet been analyzed, but it is important to note that the decrease in absorption, upon the addition of FCCP, appears to be inconsistent with the previous