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

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 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. CARDIAC FACTS

ATP SR RyR DHPR Sarcolemma Ca 2+ Ca 2+ Control of Contraction-Relaxation in Cardiac Muscle NCX Na + Ca 2+ CSQ Excitation [Ca 2+ ] Contraction

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

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

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

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

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

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

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 [Ca2+] 100 ms 10 um

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

Ca2+ Waves

F/F  m 50  M [Ca2+]Cyt (Rhod-2) [Ca2+]SR (Fluo-5n) [Ca2+]Cyt (Rhod-2) [Ca2+]SR (Fluo-5n) Ca2+ sparks Ca2+ blinks Imaging Cytosolic and Luminal Ca2+ Signals

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

RyR2 STRUCTURE AND REGULATION

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

REGULATION OF SR CA2+ RELEASE BY INTRACELLULAR LIGANDS Cytosolic Ca2+ Cytosolic Mg2+ Luminal Ca Luminal [Ca2+] mM Cytosolic [Ca2+] [mM] Open probability Mg2+

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

CASQ2 Triadin Junctin RyR2 Ca 2+ FKBP12.6 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 Modulation by Associated Proteins

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

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

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

CASQ2 monomers inhibit RyR2 channel at low luminal Ca2+ Triadin Junctin RyR2 _ (Gyorke et al., BJ 2004; Qin et al. BJ 2009 ) Molecular Basis of CICR Modulation by Luminal Ca 2+ CASQ2 The RyR2 Ca 2+ Release Channel Complex

CASQ2 monomers inhibit RyR2 channel at low luminal Ca2+ This inhibition is relieved at high luminal Ca2+ Triadin Junctin RyR2 (Gyorke et al., BJ 2004; Qin et al. BJ 2009 ) Molecular Basis of CICR Modulation by Luminal Ca 2+ CASQ2 The RyR2 Ca 2+ Release Channel Complex

Abnormal Ca2+ Regulation and Cardiac Disease

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

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)

Arrhythmogenic Ca Oscillations in Myocytes Expressing CPVT CASQ2 Mutants F/F 0 1 CASQ R33Q Control 80 mV CASQ2 DEl 20 μm 4 1 s F/F Antisense Ctr asWT CASQ2 CtrWTR33Q CASQ2

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

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

Increased Arrhythmogenesis Causes Electro- mechanical Dissociation in Late HF Stage Myocytes 4F/F01 80 mV 40  M AP [Ca]c (Fluo-3) s 15  M Shortening