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