INTRACELLULAR CALCIUM RELEASE IN NORMAL AND DISEASED HEART

1. Sandor Gyorke DHLRI, 507 INTRACELLULAR CALCIUM RELEASE IN NORMAL AND DISEASED HEART

2. CARDIAC FACTS • The human heart beats about 100,000 times in one day and about 35 million times in a year. During an average lifetime, it will beat more than 2.5 billion times. • CVD total mention deaths (1,372,000 deaths in 2005) accounted for about 56 percent of all deaths in 2005. • Nearly 2,400 Americans die of CVD each day, an average of one death every 37 seconds. CVD claims about as many lives each year as cancer, chronic lower respiratory diseases, accidents and diabetes mellitus combined.

3. Ca2+ Control of Contraction-Relaxation in Cardiac Muscle Excitation [Ca2+] Contraction Ca2+ SR CSQ DHPR RyR ATP NCX Ca2+ Na+ Sarcolemma

4. INTRACELLULAR Ca2+ RELEASE AND CARDIAC DISEASE Normal CICR SR Ca Triggered arrhythmias ? ? Heart failure Ca SR SR Ca Spnontaneous Ca release Diminished Ca release (Catecholaminergic ventricular tachycardia and sudden death linked to mutations in RyR and CASQ)

5. PRESENTATION TOPICS • STUDY OF INTRCELLULAR Ca RELEASE • RyR STRUCTURE and REGULATION • CONTROL MECHANISMS of CICR • ABNORMAL CALCIUM RELEASE AND CARDIAC DISEASE

6. pA STUDY OF INTRCALLULAR Ca RELEASE Ca2+ measurements in intact hearts of transgenic mice expressing Ca2+ sensitive proteins Intracellular Ca2+ measurements Fluorescence Ca2+ dyes Spatially resolved Ca2+ imaging 3H-Ryanodine Binding 45Ca2+ Flux RyR activity in Planar Lipid Bilayers

7. Patch-Clamp/Microfluorometry RyR Ca DHPR Ca APs [Ca2+]Cyt Fluo-3 480 nm 530 nm

8. Patch-Clamp/Microfluorometry RyR Ca DHPR Ca APs [Ca2+]Cyt Fluo-3 Rhod-2 580 nm 480 nm [Ca2+]SR 530 nm

9. SPATIALLY RESOLVED Ca2+ IMAGING Principle of confocal microscope Detector Pinhole X-t (line-scan) Lense [Ca2+] Length Time

10. [Ca2+] 10 um 100 ms Ca2+ Sparks • Elementary events of Ca2+ signaling (Cheng et al., 1994) • Involve 8-30 individual RyR2s • Sum to form systolic Ca2+ • transients and • mediate diastolic • SR Ca2+ leak Ca spark

11. Local Controls of Ca2+ Release [Ca2+] time DHPR RyRs RyRs RyRs Ca local Ca local RyRs RyRs RyRs

12. Ca2+ Waves

13. Imaging Cytosolic and Luminal Ca2+ Signals 3 F/F0 1 [Ca2+]Cyt (Rhod-2) 50 mm [Ca2+]SR (Fluo-5n) 50 mM Ca2+ sparks [Ca2+]Cyt (Rhod-2) [Ca2+]SR (Fluo-5n) Ca2+ blinks

14. The Lipid Bilayer Technique Open pA Closed Mean Open Time Open probability = Total Recording Time

15. RyR2 STRUCTURE AND REGULATION

16. THE SR Ca2+ RELEASE CHANNEL/RYANODINE RECEPTOR • 4 x ~500,000 DA • 3 isoforms (RyR1&3 • skeletal; RyR2 cardiac) FKBP CaM ~500,000 DA 3 isoforms Imperotoxin A CaM TM

17. REGULATION OF SR CA2+ RELEASE BY INTRACELLULAR LIGANDS • Cytosolic Ca2+ • Cytosolic Mg2+ • Luminal Ca2+ Mg2+ Open probability 0.1 1 10 0.1 1 10 Cytosolic [Ca2+] [mM] Luminal [Ca2+] mM

18. Modulation by Drugs • Ryanodine (and certain scorpion toxins) locks • RyR in a subcondactant open state open closed open closed open closed • RyR is inhibited by ruthenium red, • Tetracaine and dandrolene

19. Modulation by Associated Proteins • FKBP12.6 Stabilizes RyR2 in Closed State • Triadin and Junctin Link CASQ to RyR2 • CASQ2 inhibits RyR2 at low luminal [Ca2+] and may serve as luminal Ca2+ sensor for RyR2 FKBP12.6 RyR2 Triadin Junctin CASQ2 Ca2+ Ca2+

20. Regulation of RyR by Phosphorylation/Dephosphorylation • Multiple phosphorylation sites for PKA (1 or 2) and CAMK2 (up to 5) • Functional effects of PKA phosphorylation are controversial; most studies report increase in RyR activity; Reported to result in dissociation of FKBP12.6 from RyR leading to increased RyR open probability • CAMK2 phosphorylation increases RyR activity

21. Modification of –SH Residues by ROS Oxidation S-Nitrosilation S-OH S-O2H S-NO S- Glutathionylation Disulfide formation S-SG S S ROS (superoxide, H2O2 etc) ~90 cysteins Increased RyR activity

22. Termination of SR Ca2+ Release and Ca2+ Signaling Refractoriness Store-dependent deactivation

23. _ Molecular Basis of CICR Modulation by Luminal Ca2+ The RyR2 Ca2+ Release Channel Complex • CASQ2 monomers inhibit RyR2 channel at low luminal Ca2+ RyR2 Triadin Junctin CASQ2 • (Gyorke et al., BJ 2004; Qin et al. BJ 2009 )

24. Molecular Basis of CICR Modulation by Luminal Ca2+ The RyR2 Ca2+ Release Channel Complex • CASQ2 monomers inhibit RyR2 channel at low luminal Ca2+ • This inhibition is relieved at high luminal Ca2+ RyR2 Triadin Junctin CASQ2 • (Gyorke et al., BJ 2004; Qin et al. BJ 2009 )

25. Abnormal Ca2+ Regulation and Cardiac Disease

26. INTRACELLULAR Ca2+ RELEASE AND CARDIAC DISEASE Normal CICR SR Ca Triggered arrhythmias Heart failure SR Ca SR Ca Spontaneous Ca2+ release Diminished Ca2+ release

27. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) • Adrenergically mediated polymorphic ventricular tachyarrhythmias leading to syncope and sudden cardiac death • The episodes of tachyarrhythmia are typically triggered by physical exercise or emotional stress • Linked to Mutations in RyR2 (~80) and CASQ2 (~10) • (Laitinen et al. Ann Med 2004; Napolitano & Priori SG. Heart Rhythm 2007)

28. Arrhythmogenic Ca Oscillations in Myocytes Expressing CPVT CASQ2 Mutants Antisense CASQR33Q CASQ2DEl Control 80 mV 20 μm 3 F/F0 1 1 4 1 s Ctr as Ctr WT Ctr WT R33Q F/F0 CASQ2 CASQ2

29. CELLULAR MECHANISMS OF TRIGGERED ARRYTHMIA DADs and extrasystolic APs MP NCX 3Na+ Ca2+ Spontaneous Ca2+ release [Ca2+]Cyt Impaired control by [Ca]sr

30. 50 m SR store Ca2+ content is reduced in heart failure Control HF Ca2+-induced Ca2+ release SR lumen Fluo-5n RyR SERCA Cytosol Rhod-2 Ca HF time SR lumen Fluo-5n 2 s

31. 40 mM 2 1 Shortening 15 mM 3 s Increased Arrhythmogenesis Causes Electro-mechanical Dissociation in Late HF Stage Myocytes AP 80 mV [Ca]c (Fluo-3) 1 4 F/F0 F/F0