genetics in cardiac arrhythmias - 대한심장학회 · pdf file ·...
TRANSCRIPT
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Genetics in cardiac arrhythmias
인제대학교
상계 백병원
송영환
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ECG and action potential (AP)
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Na channel
Ca channel
K channel
Ionic & molecular basis for AP
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Experiment of inherited arrhythmia (I)
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Experiment of inherited arrhythmia (II)
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Cardiomyocytes isolation
(collagenase)
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Patch clamp (electrophysiologic study)
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Inherited arrhythmias
• Long QT syndrome (LQTS)
• Short QT syndrome (SQTS)
• Brugada syndrome
• Catecholaminergic polymorphic ventricular tachycardia (CPVT)
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Long QT syndrome (LQTS)
Electrical dispersion
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Short QT syndrome (SQTS)
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Brugada syndrome
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Catecholaminergic polymorphic ventricular tachycardia (CPVT)
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Genes involved in inherited arrhythmias
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Genes involved in inherited arrhythmias
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Experiment of inherited arrhythmias with induced pluripotent stem cells
(iPSs)
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Modeling LQTS with iPSs
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Modeling CPVT with iPSs
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Summary of studies with iPSs
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R644C Mutation of Lamin A Causes Cardiac Fibroblasts Senescence
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• Lamin A/C • Nuclear membrane protein
• It affects cell proliferation
• Mutation cardiomyopathy, arrhythmia, progeria
Background
R644C mutation family
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▼
Homo sapiens DNLVTRSYLLGN 650
Pan troglodytes DNLVTRSYLLGT 650
Macaca mulatta DNLVTRSYLLGN 712
Sus scrofa DNLVTRSYLLGN 650
Canis familiaris DNLVTRSYLLGS 651
Mus muluscus DNLVTRSYLLGN 651
R644C (24050 C>T) R644C (Arg>Cys)
Lamin A, R644C mutation
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Methods
We generated recombinant adenoviruses and
expressed Flag-tagged wild type Lmna (LmnaWT)
and mutant Lmna R644C in fibroblasts isolated from
mouse hearts.
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0
0.5
1
1.5
2
2.5
3
3.5
4
0 hr 24 hr 48 hr 72 hr
NC
Vector
Wild
R644C
0
10
20
30
40
50
60
NC Vector Wild R644C
N=3 *p<0.05 *
N=3 *p<0.05
Cel
l D
ou
bli
ng T
ime
(hr)
Rel
ati
ve
Cel
l N
um
ber
0
0.2
0.4
0.6
0.8
1
1.2
NC Vector Wild R644C
Rel
ati
ve
Brd
U
Inco
rpo
rati
on
rate
s
N=3 *p<0.01
*
*
Lamin A Mutation (R644C) decreases Cell Proliferation In Mouse Cardiac Fibroblasts
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Cell Proliferation (Ki67 expression) is reduced in Lamin A Mutation (R644C) In Mouse Cardiac
Fibroblasts
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SA
-bet
a g
ala
ctosi
dase
-posi
tive
cell
s (%
) 0
0.1
0.2
0.3
0.4
0.5
0.6
NC Vector Wild R644C
Cells only Vector only
Lmna-R644C Lmna-WT
Lamin A Mutation (R644C) causes Cellular Senescence In Mouse Cardiac Fibroblasts (SA-beta galactosidase assay)
* N=3 *p<0.05
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Flag Emerin DAPI Merge
Wild
Cell
Vector
R644C
Lamin A Mutation (R644C) causes Nucleus Blebbing In Mouse Cardiac Fibroblasts
10 μm
0
0.2
0.4
0.6
0.8
1
1.2
NC Vector Wild R644C
Ble
bed
nu
clei
(%
)
*
N=3 *p<0.01
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Lamin A Mutation (R644C) causes prelamin expression In Mouse Cardiac Fibroblasts
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Normal
Mutant
Lamin A Mutation (R644C) reduces Rb phosphorylation In Mouse Cardiac Fibroblasts
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Normal Mutant lamin A/C
Lamin A Mutation (R644C) causes dissociation of binding proteins in Mouse Cardiac Fibroblasts
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Normal Mutant lamin A/C
Hypothesis
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L-type Ca2+ channel activation by ROS-induced activation of CaMKII.
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Introduction
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Cardiovascular System
Stress ROS
Cardiac Hypertrophy
Arrhythmia
Hypertension
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Cardiac Myocyte
ROS
L-type Ca2+ channel
Facilitation
Mechanism ?
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ROS L-type Ca2+ channel
CaMKII ?
1) ROS CaMKII
2) CDF: CaMKII mediated reaction
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Study Plan
• Effect of ROS on L-type Ca2+ channel
• CaMKII: Involvement & mechanism
• Role of Ca2+
• Comparison with CDF
• Long term potentiation: LTP
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Materials & Methods
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Chemicals and Solutions
• Normal Tyrode solution (mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2,
10 glucose, and 5 HEPES, titrated to pH 7.4 with NaOH.
• Ca2+-free solution (mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 10 glucose, and
5 HEPES, titrated to pH 7.4 with NaOH.
• The high K+, low Cl- solution (mM): 70 KOH, 40 KCl, 50 L-glutamic acid,
20 taurine, 20 KH2PO4, 3 MgCl2, 10 glucose, 10 HEPES, and 0.5 EGTA.
• The pipette solution (mM): 100 CsOH, 110 gluconic acid, 10 NaCl, 20
HEPES, 20 tetraethylammonium-Cl, 4 Mg-ATP, 5 Na-phosphocreatine,
and 10 EGTA titrated to pH 7.3 with CsOH.
• Drugs were prepared as concentrated stock solutions either in distilled
water or dimethyl sulfoxide.
• All experiments were conducted at room temperature (22-25℃).
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Cell Isolation
• Male Sprgue-Dawley rat
• The removed heart was perfused with digestion
solution containing collagenase through
Langendorff system.
• The ventricles were cut into small pieces and
individual myocytes were obtained by gentle
trituration.
• The isolated cells were stored in the high K+, low Cl-
solution at 4℃ until used in experiments.
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Voltage Clamp Recording & Analysis
• Patch pipettes were pulled from borosilicate glass capillaries (Clark Electromedical Instruments, UK) using a pipette puller (model PP-83, Narishige Scientific Instrument Lab. Janpan) and were fire polished.
• Pipettes exhibited 3 to 4 MΩ resistance when filled with a pipette solution
• Voltage clamp was performed by using the conventional whole cell method.
• All recordings were initiated at least 10 min after rupture of membrane to allow complete dialysis of the cytoplasm.
• All the Ica,L recording were made at room temperature (22-25℃) using Axopatch amplifier (Axon Instruments, CA).
• Signals from the patch amplifier were filtered at 1 kHz and digitized with an A/D converter (PCI-6040E, National Instrument, USA) at a sampling rate of 1 kHz and stored on a hard disc installed in a personal computer using a software made in our laboratory (R-clamp, by SY Ryu) written with Delphi 6.0 (Borland Software Co.).
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Western Blot
• The removed rat heart was perfused with specific solutions
dependent on 4 each condition (+/- Ca2+, +/- H2O2) through
Langendorff system for 10 min.
• The ventricles were cut into small pieces and weighed about
300g and homogenized into the same solutions as perfused on
previous step.
• the total proteins was quantified by Brad-Ford assay
• 100 g of protein from the each samples was separated on 10%
SDS-polyacrylamide gel and electrophoretically transferred onto
the polyvinylidene difluoride membrane
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Results
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Action potential (AP) changes By H2O2
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Spontaneous SR Ca2+ spark
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H2O2 increases phosphorylation of
CaMKII
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H2O2 induced APD prolongation is
mediated by CaMKII
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CaMKII
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Activation
Catalytic activity Substrate
Mechanism of CaMKII
P
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Autophosphorylation
Oxidation
Activation of CaMKII
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Facilitation of ICa,L by H2O2
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Oxidation-dependent Facilitation of ICa,L (ODF)
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ODF mediated by CaMKII
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Occlusion of ODF with CDF (Ca2+ dependent facilitation)
(0.5 Hz)
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Ca2+ Source of ODF & CDF
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Ca2+ Source of ODF & CDF
Spontaneous SR Ca2+ release
dependent
CICR dependent
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Phosphorylation in ODF & CDF (Autophosphorylation, Catalytic activity)
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Phosphorylation in ODF & CDF
ODF; Autophosphorylation-independent non-catalytic reaction
CDF; Autophosphorylation-dependent non-catalytic reaction
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The effects of kinase inhibitors related to ROS on ODF
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Long term potentiation (LTP)
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The effct of DTT on LTP
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Langendorff perfusion with
Solutions (+/- Ca2+, +/- H2O2)
Cut & grind vent. tissue
Western blot
Oxidation-dependent Autophosphorylation of CaMKII
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The New Mechanism of CaMKII Activation
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CaMKII Oxidation alone in LTP
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--- SR Ca2+ release independent
--- SR Ca2+ release dependent
SR Ca2+ Dependency
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Summary
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The Effect of Duration of H2O2 Perfusion on LTP
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Discussion
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CDF
ODF
Activation of CaMKII
CICR
Spont. SR Ca2+ release Autophosphorylation
Independent
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Non-catalytic reaction
Interaction of CaMKII & L-type Ca2+ Channel
CaMKII
CaMKII
CaMKII
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The New Mechanism of CaMKII Activation
Long term potentiation: LTP
SR Ca2+ dependent SR Ca2+ independent SR Ca2+ independent
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