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 Shi 2, 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

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 K ATP Channels

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

Cardiac sarcK ATP Channels Under normal metabolic conditions closed. During metabolic stress they open - cellular metabolic sensors. SUR2A Kir6.2 COO - NH 2 ATP ADP H + - +

Cardiac K ATP Channels Mitochondrial K ATP channel K+K+ K+K+ K+K+ K+K+ Sarcolemmal K ATP channel K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+

The Mechanism of Protection Ca 2+ Extracellular K+K+ SarcK ATP Channel

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

Suppression of mitochondrial Ca 2+ overload Proposed mechanisms of protection ΔΨ m -200 mVΔΨ m depolarized Ca 2+ Mitochondrial K + channels open Ca 2+ K+K+ Mitochondrial swelling and improved oxidative phosphorylation ATP Mitochondrial K + channels open H+H+ A-A- Pi-Pi- K+K+ K+K+ OH -

Freshly isolated cardiac myocytes –Effect on mitochondrial membrane potential (ΔΨ m ) Isolated cardiac mitochondria –Mitochondrial bioenergetics –Ca 2+ loading –Resistance to stress Experimental Design

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:

Mitochondrial membrane potential

Mitochondrial swelling

Resistance to Ca 2+ loading

Effect of hypoxia-reoxygenation on mitochondrial respiration

Myocite Resistance to metabolic inhibition

Conclusions SUR2 mutant mitochondria had more depolarized ΔΨ m compared to wild type Tolerance to Ca 2+ 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

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