1.  Excitation-contraction coupling, Ca 2+ and Na + regulation in the normal and diseased heart;  Cellular bases of triggered ventricular arrhythmias Research Interest Sanda Despa, PhD Department of Pharmacology

2. Cardiac excitation-contraction coupling Sarcolemma Ca T-Tubule Ca 3Na NCX ATP Na 3Na 2K PLM ATP I Ca Ca SR PLB ATP RyR 3Na Ca NCX

3. Cardiac excitation-contraction coupling Sarcolemma Ca T-Tubule Ca 3Na NCX ATP Na 3Na 2K PLM ATP I Ca Ca SR PLB ATP RyR 3Na Ca NCX

4. Sarcolemma Ca T-Tubule Ca 3Na NCX ATP Na 3Na 2K PLM ATP I Ca Ca SR PLB ATP RyR 3Na Ca NCX Contraction of the heart

5. Relaxation of the heart Sarcolemma Ca T-Tubule Ca ATP Na 3Na 2K PLM ATP I Ca Ca SR PLB ATP RyR 3Na Ca NCX 3Na NCX

6. Ca 2+ and contraction-relaxation of a cardiac myocyte Rat ventricular myocyte loaded with a Ca 2+ -sensitive fluorescent indicator

7. Project 1: Ca dysregulation and arrhythmias induced by loss-of-function of ankyrin B Ankyrin-B = multivalent “adaptor” protein that targets select membrane proteins to the cytoskeleton

8. Ankyrin-B loss of function mutations lead to long QT4 syndrome and ventricular arrhythmias in humans Ventricular arrhythmias E1425G mutation Long QT4 syndrome DII DIII (Schott JJ et al., Am. J. Hum. Genet. 1994) 1 sec Q-T *** QTc= 450 ms in symptomatic patients (normal QTc<420 ms in men; <440 ms in women) Long QT syndromes in humans 1 sec

9. Decreased NCX and NKA expression, particularly at the T-tubules, in AnkB+/- myocytes (Mohler et al., Nature 2003) AnkB+/- mice = mice heterozygous for a null mutation in ankyrin-B gene.

10. Caffeine Reduced NCX and NKA function in AnkB+/- myocytes NCX function NKA function Camors, …, Despa. JMCC, 2012

11. Similar [Na] i and diastolic [Ca] i in myocytes from AnkB+/- and WT mice [Na] i Diastolic [Ca] i RestingPacing Camors, …, Despa. JMCC, 2012

12. Larger Ca transients, SR Ca load & fractional release in AnkB+/- mice Camors, …, Despa. JMCC, 2012

13. Enhanced Ca spark frequency in intact AnkB+/- mice 200 ms 10 µm 0 250 40 µm AnkB+/- 0 Na/ 0 Ca Tyrode 1 mM Ca Tyrode Caffeine WT Caffeine 1 s 0 250 Camors, …, Despa. JMCC, 2012

14. More pro-arrhythmic Ca waves in AnkB+/- myocytes 20 % 60 % 10 % Control conditionISO (1 µM) AnkB+/- Camors, …, Despa. JMCC, 2012

15. WTAnkB+/- T-tubule NKA-α1 Cleft Ca Na Ca NCX K NKA-  2 Ca ATP Ca RyR diastolic Ca B56  Nuclear envelope IP 3 R AnkB Cleft [Ca]  T-tubule Ca ATP Ca RyR diastolic Ca Nuclear envelope P 1.What causes the increased propensity for Ca sparks and waves in AnkB+/- myocytes?  Altered cytosolic RyR regulation? Work in progress; Questions:

16. 2. Does AnkB proteolysis by calpain lead to a cardiac phenotype similar to that caused by genetic AnkB loss-of-function? AnkB protein but not mRNA is reduced in the infarct border zone after MI Hundt et al., Cardiovasc Res. 2009;81:742 Protein expression mRNA level

17. Work in progress; Questions: 2. Does AnkB proteolysis by calpain lead to a cardiac phenotype similar to that caused by genetic AnkB loss-of-function? AnkB & NKA protein expression are reduced following ischemia/reperfusion; the effect is prevented by calpain inhibition Inserte et al., Circ Res 2005;97:465. NKA  How does calpain activation affect the protein expression, subcellular distribution and function of AnkB, NCX and NKA?  What is the role of AnkB proteolysis by calpain in the structural and electrical remodeling of the heart following ischemia/reperfusion?

18.  How is Ca cycling altered in diabetic heart disease? Timeline!  Is [Na] i altered in diabetic heart disease? Does this further alter the cardiac metabolism?  ROS production →  slowly inactivating I Na →  [Na] i →  [Ca] m →  ATP  Electrical remodeling & occurrence of arrhythmias in diabetic hearts Long QT Project 2: Electrical remodeling and arrhythmias in diabetic heart disease Pre-diabetic stage Diabetic stage

19. Acknowledgments University of California Davis Emmanuel Camors Kevin Voelker Florin Despa Samuel Galice Jeffrey Elliot Kaleena Jackson Brian Koch Donald M. Bers Kenneth Ginsburg Khana Dao Ohio State University Peter Mohler University of California Los Angeles Enrico Stefani Yong Wu University of Cincinnati Jerry B. Lingrel University of Manchester Fabian Brette Funding from NIH & AHA