intracellular calcium release in normal and diseased heart sandor gyorke dhlri, 507
TRANSCRIPT
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 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.
CARDIAC FACTS
ATP
SR
RyRDHPR
Sarcolemma
Ca2+
Ca2+ Control of Contraction-Relaxation in Cardiac Muscle
NCX
Na+
Ca2+
CSQ
Excitation [Ca2+] Contraction
SR
SRCa
Normal CICR
INTRACELLULAR Ca2+ RELEASE ANDCARDIAC DISEASE
(Catecholaminergicventricular tachycardia and sudden death linked to mutations in RyR and CASQ)
?
SR
Heart failure ?
Triggered arrhythmias
Ca
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+ measurementsFluorescence Ca2+ dyesSpatially resolved Ca2+ imaging
Ca2+ measurements in intact heartsof transgenic miceexpressing Ca2+ sensitiveproteins
45Ca2+Flux
3H-RyanodineBinding RyR activity in
Planar Lipid Bilayers
Patch-Clamp/Microfluorometry
480 nm
530 nm
Ca
Ca
DHPR
RyR
Fluo-3
[Ca2+]Cyt
APs
Patch-Clamp/Microfluorometry
480 nm
530 nm
Ca
Ca
DHPR
RyR
Fluo-3
580 nm
Rhod-2
[Ca2+]Cyt
[Ca2+]SR
APs
SPATIALLY RESOLVED Ca2+ IMAGING
Time
Pinhole
Leng
th
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
RyRs
Ca local
DHPRRyRs
RyRs
Ca local
Local Controls of Ca2+ Release
time
RyRs
RyRs
[Ca2+]
Ca2+ Waves
F/F
0
1
3
50
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 TimeOpen 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
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 Ca2+
0.1 1 10
Luminal [Ca2+] mM
0.1 1 10
Cytosolic [Ca2+] [mM]
Op
en p
rob
abili
ty
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
Ca2+
Ca2+
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-OH S-NO
S-Nitrosilation
S- Glutathionylation
S-SG S
Disulfide formation
S
ROS (superoxide, H2O2 etc) ~90 cysteinsIncreased 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 Ca2+
CASQ2
The RyR2 Ca2+ 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 Ca2+
CASQ2
The RyR2 Ca2+ Release Channel Complex
Abnormal Ca2+ Regulation and Cardiac Disease
SR
SRCa
Normal CICR
INTRACELLULAR Ca2+ RELEASE ANDCARDIAC DISEASE
SR
Heart failure Triggered arrhythmias
Ca
Ca
Spontaneous Ca2+ release 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
F/F0
1
CASQR33Q Control
80 m
V
CASQ2DEl
20 μ
m
41 s
F/F
0
1
3
Antisense
Ctr Ctras WT
CASQ2
Ctr WT R33Q
CASQ2
CELLULAR MECHANISMS OF TRIGGEREDARRYTHMIA
NCX3Na+
Ca2+
Spontaneous Ca2+release
DADs and extrasystolic APs
Impaired control by [Ca]sr
[Ca2+]Cyt
MP
50 m
2 s
CytosolRhod-2
SR lumenFluo-5n
SR lumenFluo-5n
SR store Ca2+ content is reduced in heart failure
Ca2+-inducedCa2+ release
SE
RC
A
CaHF
time
RyR
Control HF
Increased Arrhythmogenesis Causes Electro-mechanical Dissociation in Late HF Stage Myocytes
4 F/F01
F/F
080
mV
40
M AP
[Ca]c(Fluo-3)
1
2
3 s
15
M
Shortening