Pravdić D. 1,3 , Aggarwal N. 2 , Mcnally M. E. 4 , Bošnjak Ž. 3 , Nian-Qing Shi 2 , Makielski J. 2

1. Disruption of sulfonylurea receptor-2 protects against ischemia and reperfusion injury via modulation of mitochondrial bioenergetic phenotype Pravdić D.1,3, Aggarwal N.2, Mcnally M. E.4, Bošnjak Ž.3, Nian-Qing Shi2, Makielski J.2 Mostar School of Medicine, Mostar, BIH 1 University of Wisconsin, Madison, Madison, WI, USA 2 Medical College of Wisconsin, Milwaukee, WI, USA 3 University of Chicago, Chicago, IL, USA 4

2. KATP Channels • First discovered in the heart by Noma (Nature 1983; 305: 147-8). • “An outward current of unknown nature increases significantly when cardiac cells are treated with cyanide or subjected to hypoxia, and decreases on intracellular injection of ATP.” • ATP-regulated K+ channels

3. KATP Channels • Following studies identified KATP channels in other tissues: • Pancreatic β-cells • Vascular smooth muscle • Skeletal muscle • Brain • Kidney • etc.

4. Cardiac sarcKATP Channels SUR2A Kir6.2 • Under normal metabolic conditions closed. • During metabolic stress they open - cellular metabolic sensors. NH2 - + ATP COO- ADP H+

5. K+ K+ K+ K+ K+ K+ Cardiac KATP Channels Sarcolemmal KATP channel K+ K+ K+ K+ K+ Mitochondrial KATP channel

6. Ca2+ The Mechanism of Protection Extracellular SarcKATP Channel K+

7. Diazo +5-HD Vehicle Diazo Fig. 4 MitoKATP channels and cellular protection MitoKATP channel activator diazoxide reduced the severity of ischemia/ reperfusion damage in rat hearts (Garlid et al., Circ Res 1997;81:1072-82) Diazoxide protected rabbit cardiomyocytes from ischemia in a 5-HD dependent manner (Liu et al., Circulation 1998;97:2463-9)

8. Proposed mechanisms of protection Suppression of mitochondrial Ca2+ overload Ca2+ Ca2+ Mitochondrial K+ channelsopen Ca2+ Ca2+ K+ ΔΨm -200 mV ΔΨm depolarized Mitochondrial swelling and improved oxidative phosphorylation A- H+ Mitochondrial K+ channelsopen ATP K+ OH- K+ Pi-

9. Experimental Design • Freshly isolated cardiac myocytes • Effect on mitochondrial membrane potential (ΔΨm) • Isolated cardiac mitochondria • Mitochondrial bioenergetics • Ca2+ loading • Resistance to stress

10. Animals • A transgenic mouse was created previously (SUR2 mutant) where the gene encoding SUR2 was disrupted by removing exons 14 to 18 encoding the first nucleotide binding domain. • These SUR2 mutant mice lack pinacidil, diazoxide and glybenclamide sensitive sarcKATP currents in the cardiac, smooth and skeletal muscle and they are hypertensive, arrhythmic, and exhibit coronary vasospasm and sudden cardiac death • SUR2 mutant mice have increased protection against both acute adrenergic stress and ischemia compared to control Stoller D, Kakkar R, Smelley M, Chalupsky K, Earley JU, Shi NQ, Makielski JC, McNally EM. Mice lacking sulfonylurea receptor 2 (SUR2) ATP-sensitive potassium channels are resistant to acute cardiovascular stress. J Mol Cell Cardiol. 2007;43:445-454.

11. Mitochondrial membrane potential

12. Mitochondrial swelling

13. Resistance to Ca2+ loading

14. Effect of hypoxia-reoxygenation on mitochondrial respiration

15. Myocite Resistance to metabolic inhibition

16. Conclusions • SUR2 mutant mitochondria had more depolarized ΔΨm compared to wild type • Tolerance to Ca2+ loading was increased in SUR2 mutant mitochondria • Mitochondria swelling, an indicator of K+ influx, was greater in SUR2 mutants • SUR2 mutant mitochondria recovered better from hypoxia-reoxygenation injury than Wild type

17. Acknowledgements Zeljko J. Bosnjak, PhD Nitin Aggarwal, PhD Martin Bienengraeber, PhD Jonatan Makielski, MD Elizabeth McNally, MD Chiaki Kwok