Transcript Ca 2+
Sandor Gyorke DHLRI, 507 INTRACELLULAR CALCIUM RELEASE IN NORMAL AND DISEASED HEART 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. Ca2+ Control of Contraction-Relaxation in Cardiac Muscle Excitation [Ca2+] Contraction Ca2+ CSQ DHPR NCX SR RyR Ca2+ ATP Na+ Sarcolemma INTRACELLULAR Ca2+ RELEASE AND CARDIAC DISEASE Normal CICR SR Triggered arrhythmias ? Ca Ca ? Heart failure SR Ca Spnontaneous Ca release (Catecholaminergic ventricular tachycardia and sudden death linked to mutations in RyR and CASQ) SR 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 Ca2+ measurements in intact hearts of transgenic mice expressing Ca2+ sensitive proteins Intracellular Ca2+ measurements Fluorescence Ca2+ dyes Spatially resolved Ca2+ imaging 45Ca2+ 3H-Ryanodine Binding Flux pA RyR activity in Planar Lipid Bilayers Patch-Clamp/Microfluorometry RyR Ca DHPR Ca APs [Ca2+]Cyt Fluo-3 480 nm 530 nm Patch-Clamp/Microfluorometry RyR Ca DHPR Ca APs [Ca2+]Cyt Fluo-3 Rhod-2 580 nm 480 nm 530 nm [Ca2+]SR SPATIALLY RESOLVED Ca2+ IMAGING Principle of confocal microscope Detector Pinhole Lense X-t (line-scan) [Ca2+] 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 10 um [Ca2+] Local Controls of Ca2+ Release [Ca2+] time DHPR RyRs Ca local RyRs RyRs RyRs Ca local RyRs RyRs Ca2+ Waves Imaging Cytosolic and Luminal Ca2+ Signals 50 mm 1 [Ca2+]Cyt (Rhod-2) [Ca2+]SR (Fluo-5n) 50 mM F/F0 3 Ca2+ sparks [Ca2+]Cyt (Rhod-2) Ca2+ blinks [Ca2+]SR (Fluo-5n) The Lipid Bilayer Technique pA Open Closed Mean Open Time Open probability = Total Recording Time RyR2 STRUCTURE AND REGULATION THE SR Ca2+ RELEASE CHANNEL/RYANODINE RECEPTOR •4 x ~500,000 DA • 3 isoforms (RyR1&3 skeletal; RyR2 cardiac) FKBP CaM Imperotoxin A CaM TM ~500,000 DA 3 isoforms REGULATION OF SR CA2+ RELEASE BY INTRACELLULAR LIGANDS Open probability •Cytosolic Ca2+ •Cytosolic Mg2+ •Luminal Ca2+ Mg2+ 0.1 1 10 Cytosolic [Ca2+] [mM] 0.1 1 10 Luminal [Ca2+] mM 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 Modulation by Associated Proteins RyR2 FKBP12.6 Triadin •Triadin and Junctin Link CASQ to RyR2 Junctin Ca2+ •FKBP12.6 Stabilizes RyR2 in Closed State CASQ2 Ca2+ •CASQ2 inhibits RyR2 at low luminal [Ca2+] and may serve as luminal Ca2+ sensor for RyR2 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 Modification of –SH Residues by ROS Oxidation S-OH S-Nitrosilation S-O2H S- Glutathionylation S-SG S-NO Disulfide formation S S ROS (superoxide, H2O2 etc) ~90 cysteins Increased RyR activity Termination of SR Ca2+ Release and Ca2+ Signaling Refractoriness Store-dependent deactivation Molecular Basis of CICR Modulation by Luminal Ca2+ The RyR2 Ca2+ Release Channel Complex RyR2 Triadin •CASQ2 monomers inhibit RyR2 channel at low luminal Ca2+ Junctin CASQ2 (Gyorke et al., BJ 2004; Qin et al. BJ 2009 ) Molecular Basis of CICR Modulation by Luminal Ca2+ The RyR2 Ca2+ Release Channel Complex RyR2 Triadin Junctin CASQ2 •CASQ2 monomers inhibit RyR2 channel at low luminal Ca2+ •This inhibition is relieved at high luminal Ca2+ (Gyorke et al., BJ 2004; Qin et al. BJ 2009 ) Abnormal Ca2+ Regulation and Cardiac Disease INTRACELLULAR Ca2+ RELEASE AND CARDIAC DISEASE Normal CICR SR Triggered arrhythmias Ca Ca SR Spontaneous Ca2+ release Heart failure Ca SR Diminished 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 Antisense CASQR33Q CASQ2DEl 20 μm 80 mV Control F/F0 3 1 1 1s Ctr as Ctr WT CASQ2 Ctr CASQ2 WT R33Q 4 F/F0 CELLULAR MECHANISMS OF TRIGGERED ARRYTHMIA DADs and extrasystolic APs MP 3Na+ NCX Ca2+ Spontaneous Ca2+ release [Ca2+]Cyt Impaired control by [Ca]sr SR store Ca2+ content is reduced in heart failure Control HF Ca2+-induced Ca2+ release SR lumen Fluo-5n RyR Ca SERCA 50 mm Cytosol Rhod-2 HF time SR lumen Fluo-5n 2s 80 mV Increased Arrhythmogenesis Causes Electromechanical Dissociation in Late HF Stage Myocytes [Ca]c (Fluo-3) 40 mM AP 1 F/F0 2 1 15 mM Shortening 3s 4 F/F0