hERg Meeting Patologia 210315vfvfvfvfvfvf.pptx

Cellular basis of arrhythmogeneicity in
hypertrophic cardiomyopathy:
a dangerous interplay between anomalies of Ca2+ and Na+
homeostasis and ion channel remodeling
Raffaele Coppini
University of Florence
Florence
March 21 2015

2
hERG Meeting-----Florence
March 21 2015
SUDDEN CARDIAC DEATH IN THE
YOUNG (<40y)
• 10000 per year in Europe
• HCM is the first cause of SCD in
athletes
• Caused by lethal arrhythmias
(ventricular fibrillation)
SUDDEN CARDIAC DEATH IN THE YOUNG

3
March 21 2015
Arrhythmogenic myocardial substrate in HCM
Olivotto I et al. Circ Heart Fail. 2012;5:535-546
Maron B J Circulation. 2010;121:445-456
Cardiomyocyte disarray Thickened arteriolar wall Replacement Fibrosis
Altered Electrical Conduction Local Ischemia Anatomical obstacle

4
March 21 2015
Sources: -Olivotto I et al. Circ Heart Fail. 2012;5:535-546; -
http://health-fts.blogspot.it/2012/01/implantable-cardioverter-
defibrillators.html; -cvphysiology.com
Altered Electrical Conduction
Local Ischemia
Anatomical obstacle
REENTRY
SUSTAINED ARRHYTHMIAS
TRIGGER

5
March 21 2015
Sarcoplasmic
reticulum
Myofilaments
External
Sarcolemma
20 µm

ICaL
DHPR
INCX
NCX
SERCA
RYR2
DHPR
NCX
ICaL
DHPR
INCX
NCX
SERCA
RYR2
NaV1.5
K+
Channels

ICaL
DHPR
INCX
NCX
SERCA
RYR2
DHPR
NCX
ICaL
DHPR
INCX
NCX
SERCA
RYR2
Sarcomere Mutation
Ca2+
sensitivity
?
↑
↑ ATP
consumed
?
NaV1.5
K+
Channels

Sarcomere Mutation
Ca2+
sensitivity
?
↑
↑ ATP
consumed
?
PLB
Sarcoplasmic
reticulum
ATP
ase
Na+
overload
RyR
SERCA
Sarcolemma
T-tubule
ROS
ATP
produced
Na/K
ATPase
Ca2+
overload
Ca2+
Ca2+
Ca2+
Ca2+
Na+
Na+
K+
↑
Coppini et al. Circulation 2013

11
March 21 2015
Methods
Left
Atrium
Left
Ventricle
Aorta
HCM patients with Obstruction
Basal inter-ventricular septum
Turbulent blood flow in the LVOT
STUDY COHORT: 26 HCM patients with symptomatic obstruction: Mean Left
Ventricular Outflow Tract gradient (LVOT) 73 ± 30 mmHg
Diastole Systole
MYBPC3
MHY7
TNNT2
Exon
E258K
6
S212R
5
A392fs
13
K865R
25
insC1065
30
T1095M
R597Q
18
COOH
NH2 NH2
COOH
R278C
17
TNNI3
NH2
COOH
T119N
8
R694C
R869H
22
E927K
23
M982T
A1128T
29
NH2
COOH
19
Mutations sites
MYBPC3
MHY7
Multiple
Negative

12
March 21 2015
Left
Atrium
Left
Ventricle
Aorta
HCM patients with Obstruction
Trans-aortic septal myectomy
Surgical Sample
1 cm
Diastole Systole
CONTROLS: 8 non-failing surgical patients:
septal thickness< 14mm, ejection fraction
>55%, aortic valve disease
Basal inter-ventricular septum
Turbulent blood flow in the LVOT
Methods
STUDY COHORT: 26 HCM patients with symptomatic obstruction: Mean Left
Ventricular Outflow Tract gradient (LVOT) 73 ± 30 mmHg

13
March 21 2015
HCM
Control
Calibration bar = 1 mm
F/Fo
1 s
Action Potential
and Membrane
currents
500
pA
Intracellular
Ca2+ transients
1s
4
mN/mm
2
Isometric Force
Cardiomyocytes
Trabeculae
1 cm
Calibration bar = 10 µm
Methods

14
March 21 2015
-80
-40
0
40
Membrane
Voltage
(mV)
300 ms
Control HCM
-80
-40
0
40
Membrane
Voltage
(mV)
300 ms
Prolonged action potential at all stimulation frequences.
0.2 Hz
0.5 Hz
1 Hz
0.0
0.2
0.4
0.6
0.8
1.0
APD90%
(s)
** ** **
Results

15
March 21 2015
Iso prolongs APD in HCM, while shortening APD in controls.
*p<0.01, unpaired
APD 90%
-30
-20
-10
0
10
20
30
40
%
change
upon
Iso
*
200 ms
40
mV
+ iso
+ iso
Results

16
March 21 2015
TQ
APD, QT interval and TQ interval
QT
TQ
QT
TQ
QT
QT TQ
Control HCM
Rest
Exercise
Rest
Exercise
ΔQTc
Results

17
March 21 2015
ECG features at rest and during exercise
Control (n=20) HCM (n=60) P vs. Control
Age 41.8±5 41.7±10 0.962
Females 9 (45%) 27 (45%) 0.981
RR rest (ms) 898±136 920±180 0.619
RR ex. (ms) 398±49 460±83 0.002
QTc rest (ms) 401±16 451±29 <0.0001
QTc ex. (ms) 399±17 481±42 <0.0001
ΔQTc (ms) - 3±22 + 28±44 0.002
Results

18
March 21 2015
Inward currents
(depolarizing)
Outward currents
(repolarizing)
EAD Inward currents
Outward currents

19
March 21 2015
2 s
40
mV
Stimoli
= Early After Depolarization, EAD EADs
** =p<0.01
0
20
40
60
Occurence
(%
of
cells)
**
Control
HCM
0.4 0.8 1.2
0.0
0.2
0.4
0.6
0.8
1.0
Number
of
patients
(%)
Average APD90% (s) EADs
0
20
40
60
HCM NSVT-
Occurence
(%
of
cells)
*
Control HCM NSVT+
Results
*

20
March 21 2015
0 200 400 600 800
-8
-4
0
Time (ms)
-40 -20 0 20 40 60 80
-8
-6
-4
-2
0
I
CaL
density
(pA/pF)
Membrane Potential (mV)
0 200 400 600 800
-8
-4
0
-80
-40
0
40
80
400 ms
Current
density
(pA/pF)
100 ms
Normalized
0,5 1,0 1,5 2,0 2,5
-1,0
-0,5
0,0
Current
density
(pA/pF)
Time (s)
0
50
100
150
200
250
I
NaL
integral
(A*ms*F
-1
)
**
-120
-60
0
1s
Control HCM
INaL
ICaL
0.0
0.5
1.0
1.5
Nav
1.5
/GAPDH
* =p<0.05
** =p<0.01
*
0,0
0,5
1,0
1,5
2,0
2,5
Cav
1.2/GAPDH
Coppini al. Circ 2013.
Inward currents
Results

21
March 21 2015
0 200 400 600 800
-8
-4
0
Time (ms)
0 200 400 600 800
-8
-4
0
-80
-40
0
40
80
400 ms
Current
density
(pA/pF)
0,5 1,0 1,5 2,0 2,5
-1,0
-0,5
0,0
Current
density
(pA/pF)
Time (s)
0
50
100
150
200
250
I
NaL
integral
(A*ms*F
-1
)
**
-120
-60
0
1s
Control HCM
INaL
ICaL
Results
p-LTCCb2-Thr498
CaMKII
p-CaMKII
p-LTCCb2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Protein
levels
(relative
to
GAPDH)
Nav1.5
p-CaMKII
CaMKII
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
CamKII
co-IP
protein
levels
(relative
to
GAPDH)
CaMKII 60 kDa
CTL HCM
1 2 3 4 5 1 2 3 4 5
p-CaMKII-Thr287 50 kDa
75 kDa
GAPDH 37 kDa
Nav1.5
GAPDH
CaMKII
260 kDa
50 kDa
37 kDa
CTL HCM
1 2 3 4 5 1 2 3 4 5
p-CaMKII 50 kDa
--------- CaMKII co-IP ----------
** **
**
**
* =p<0.05
** =p<0.01

22
March 21 2015
HERG1a
HERG1b
MIRP-1
KVLQT1
minQ
Kv4.3
KChIP
Kir2.1
Kir2.2
0.0
0.5
1.0
1.5
mRNA
expression
(

Ct)
(relative
to
GAPDH)
-80
-40
0
40
80
400 ms
50 100 150 200
0
5
10
Time (ms)
-40 -20 0 20 40 60 80
0
2
4
6
I
to
density
(pA/pF) Membrane Potential (mV)
50 100 150 200
0
5
10
Current
density
(pA/pF)
-20
-10
0
0 1 2 3
-20
-10
0
Time (s)
Barium
-100
-50
0
50
1s
-100 -50 0
-20
-15
-10
-5
0
I
k1
density
(pA/pF)
Membrane Potential (mV)
*
*
*
*
Current
density
(pA/pF)
* * * * ** *
Results
Ito
Ik1
Control HCM
* =p<0.05
** =p<0.01
Outward K currents
KVLQT1
*

23
March 21 2015
200 ms
2
mN/mm
2
Control HCM
0
1
2
3
4
5
6
Tension
(mN/mm
2
)
300
350
400
450
500
Twitch
duration
at
50%
relax
(ms)
*
Results
Force Twitches

24
March 21 2015
200 ms
2
mN/mm
2
0
1
2
3
4
5
6
Tension
(mN/mm
2
)
300
350
400
450
500
Twitch
duration
at
50%
relax
(ms)
*
Control HCM
β stimulus:
isoproterenol 10 µM
Results

25
March 21 2015
200 ms
2
mN/mm
2
0
1
2
3
4
5
6
Tension
(mN/mm
2
)
300
350
400
450
500
Twitch
duration
at
50%
relax
(ms)
*
+ iso + iso
Results

26
March 21 2015
200 ms
2
mN/mm
2
300
350
400
450
500
Twitch
duration
at
50%
relax
(ms)
*
+ iso + iso
0
5
10
15
20
25
Tension
(mN/mm
2
)
Peak time
RT50%
RT90%
-50
-40
-30
-20
-10
0
%
change
upon
Iso
Control
HCM
Results

Sarcomere Mutation
PLB
Sarcoplasmic
reticulum
ATP
ase
RyR
SERCA
Sarcolemma
T-tubule Na/K
ATPase
Ca2+
Ca2+
Ca2+
Ca2+
Na+
Na+
K+
β1
β2
β2
Adenylyl
Cyclase
TnI
Gs
Gi
Gs
Gs
ATP
cAMP
Adenylyl
Cyclase
ATP
cAMP

Sarcomere Mutation
PLB
Sarcoplasmic
reticulum
ATP
ase
RyR
SERCA
Sarcolemma
T-tubule Na/K
ATPase
Ca2+
Ca2+
Ca2+
Ca2+
Na+
Na+
K+
β1
β2
β2
Adenylyl
Cyclase
TnI
Gs
Gi
Gs
Gs
ATP
cAMP
Adenylyl
Cyclase
ATP
cAMP
200 ms
40
mV
+ iso
Control

Sarcomere Mutation
PLB
Sarcoplasmic
reticulum
ATP
ase
RyR
SERCA
Sarcolemma
T-tubule Na/K
ATPase
Ca2+
Ca2+
Ca2+
Ca2+
Na+
Na+
K+
β1
β2
β2
Adenylyl
Cyclase
TnI
Gs
Gi
Gs
Gs
ATP
cAMP
Adenylyl
Cyclase
ATP
cAMP
+ iso
HCM

30
March 21 2015
0.2 Hz 0.5Hz 1Hz
Control
HCM
Control
HCM
500 ms
150
nM
25 sec
F/Fo 0.1
25 sec
100
nM
0.0
0.1
0.2
0.3

F/F
max
1 Hz
0.2 Hz
TP
T50%
T90%
0
400
800
1200
Duration
(ms)
TP
T50%
T90%
1 Hz
0.2 Hz
* ** ** * ** **
Ca2+ transients amplitude
HCM: 26 cells/9 pts
Ctrl: 14 cells/5 pts
* =p<0.05
** =p<0.01
0.2 0.5 1
100
150
200
250
Diastolic
[Ca
2+
]
i
(nM)
Pacing rate (Hz)
** ** **
Results
Prolonged Ca2+ transients kinetics
Increased diastolic [Ca2+ ]i

31
March 21 2015
0.2 Hz 0.5Hz 1Hz
Control
HCM
Control
HCM
500 ms
150
nM
25 sec
F/Fo 0.1
25 sec
100
nM
0.0
0.1
0.2
0.3

F/F
max
1 Hz
0.2 Hz
TP
T50%
T90%
0
400
800
1200
Duration
(ms)
TP
T50%
T90%
1 Hz
0.2 Hz
* ** ** * ** **
Ca2+ transients amplitude
Prolonged Ca2+ transients kinetics
HCM: 26 cells/9 pts
Ctrl: 14 cells/5 pts
* =p<0.05
** =p<0.01
0.2 0.5 1
100
150
200
250
Diastolic
[Ca
2+
]
i
(nM)
Pacing rate (Hz)
Increased diastolic [Ca2+ ]i
** ** **
Results
50
mV
150
nM
1 s
DADs
0
10
20
30
40
Occurence
(%
of
cells)
**
DADs

32
March 21 2015
* =p<0.05
** =p<0.01
-80
0
Caffeine
100
pA
1 s
500 ms
Normalized
Amplitude
Caffeine 20 mM
300
nM
Results
Reduced forward mode of NCX: decreased Ca2+ extrusion.
NCX1
0.0
0.5
1.0
1.5
Protein
levels
(relative
to
GAPDH)
0.0
0.5
1.0
1.5
2.0
2.5
Caff.
tr.
decay

(s)
**
*
Control HCM

33
March 21 2015
-80
0
Caffeine
100
pA
1 s
500 ms
Normalized
Amplitude
Caffeine 20 mM
300
nM
Results
Increased [Na+]i
0.2 0.5 1
0.30
0.35
0.40
0.45
0.50
Diastolic
[Na
+
]
(F/F
max
)
Pacing rate (Hz)
HCM
Control
Asante Natrium Green 2
fluorescence
**
Reduced forward mode of NCX: decreased Ca2+ extrusion.

34
March 21 2015
Increased INaL in cardiac disease
Valdivia CR et al. Increased late sodium current in myocytes from a
canine heart failure model and from failing human heart. JMCC. 2005
Maltsev VA et al.Novel, ultraslow inactivating sodium current in human
ventricular cardiomyocytes. Circulation. 1998
Human HF Canine HF
Human HF
Xi Y et al. Increased late sodium currents are related to
transcription of neuronal isoforms in a pressure-
overload model. Eur J Heart Fail. 2009
0,5 1,0 1,5 2,0 2,5
-1,0
-0,5
0,0
Current
density
(pA/pF)
Time (s)
0
50
100
150
200
250
I
NaL
integral
(A*ms*F
-1
)
**
-120
-60
0
1s
Human HCM
Rat Aortic Stenosis
Systolic dysfunction
Diastolic dysfunction
Results

Ca2+
sensitivity
↑
↑ ATP
consumed
PLB
Sarcoplasmic
reticulum
ATP
ase
Na+
overload
RyR
SERCA
Sarcolemma
T-tubule
ATP
produced
Na/K
ATPase
Ca2+
overload
Ca2+
Ca2+
Ca2+
Ca2+
Na+
Na+
K+
↑
CaMKII
Coppini et al. Circ 2013

37
March 21 2015
0.2 Hz
0.5 Hz
1 Hz
0.0
0.2
0.4
0.6
0.8
1.0
APD90%
(s)
-80
-40
0
40
80
Membrane
Voltage
(Vm)
200 ms
HCM Basal
HCM + Ran
*
*
**
500ms
50
pA
-120
0
1s
HCM Basal
HCM + Ran
HCM + TTX
B
a
s
a
l
R
a
n
0
100
200
I
Na-L
integral
(A*ms*F
-1
)
**
Transmembrane
Current

38
March 21 2015
500 ms
200
nM
Ampl.
0
100
200
300
400
[
Ca
2+
]
i
(nM)
**
** **
0.2 0.5 1
100
150
200
250
Diastolic
[Ca
2+
]
i
(nM)
Pacing rate (Hz)
**
TP T50% T90%
0.0
0.4
0.8
1.2
Duration
(s)
**
* **
HCM Basal HCM + Ran

39
March 21 2015
0.0
0.5
1.0
1.5
2.0
2.5
Caff.
tr.
decay

(s)
*
*
0
40
80
120
SR
Ca
2+
load
(mmol/l)
0%
100%
Ca
2+
Current
0%
100%
2s
0,4
0,5
Intracellular
Sodium
(F/Fmax)
20 s
HCM Basal HCM + Ran
0.2 0.5 1
0.30
0.35
0.40
0.45
0.50
Diastolic
[Na
+
]
(F/F
max
)
Pacing rate (Hz)
* * **
Ranolazine decreases [Na+]i
* =p<0.05
** =p<0.01
Ranolazine increases forward mode of NCX

40
March 21 2015
1 s
50
mV
0.5 s
40mV
EAD
DAD
0
20
40
60
Occurrence
(%
of
cells)
** **
Coppini et al. Circ 2013
= Early After Depolarization (EAD)
= Delayed After Depolarization (DAD)
HCM Basal HCM +Ran

41
March 21 2015
Clinical Study with Ranolazine

42
March 21 2015
40 Sites in 7 Countries: USA, Italy, UK, France, Germany, Netherlands, Israel
Clinical Study with Novel INaL inhibitor

• Cellular arrhythmogenesis in patients with HCM is
mediated by a complex electrophysiological/ EC-
coupling remodeling, in which increased late Na+
current plays a major role
• Reduction of K currents account for the anomalous
effect of beta adrenergic stimulation on AP duration
and render HCM myocytes more susceptible to the
effects of late Na current increase
• Ranolazine reduces cellular arrhythmias in human
HCM cardiomyocytes
CONCLUSIONS

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