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Excitation-contraction coupling, Ca2+ and Na+ regulation in the normal and diseased heart;
Cellular bases of triggered ventricular arrhythmias
Research Interest
Sanda Despa, PhDDepartment of Pharmacology
Cardiac excitation-contraction coupling
Sarcolemma
Ca
T-T
ub
ule
Ca
3Na
NCXATP
Na
3Na
2K
PLM ATP
ICa
Ca
CaSR
PLB ATP
RyR
3Na
CaNCX
Cardiac excitation-contraction coupling
Sarcolemma
Ca
T-T
ub
ule
Ca
3Na
NCXATP
Na
3Na
2K
PLM ATP
ICa
Ca
CaSR
PLB ATP
RyR
3Na
CaNCX
Sarcolemma
CaT
-Tu
bu
le
Ca
3Na
NCXATP
Na
3Na
2K
PLM ATP
ICa
Ca
CaSR
PLB ATP
RyR
3Na
CaNCX
Contraction of the heart
Relaxation of the heart
Sarcolemma
Ca
Ca
T-T
ub
ule
Ca
ATP
Na
3Na
2K
PLM ATP
ICa
Ca
CaSR
PLB ATPR
yR
3Na
CaNCX
3Na
NCX
Ca2+ and contraction-relaxation of a cardiac myocyte
Rat ventricular myocyte loaded with a Ca2+-sensitive fluorescent indicator
Project 1: Ca dysregulation and arrhythmias induced by loss-of-function of ankyrin B
Ankyrin-B = multivalent “adaptor” protein that targets select membrane proteins to the cytoskeleton
Ankyrin-B loss of function mutations lead to long QT4 syndrome and ventricular
arrhythmias in humans
Ventricular arrhythmias
E1425G mutation
Long QT4 syndrome
DII DIII
(Schott JJ et al., Am. J. Hum. Genet. 1994)
1 sec
Q-T
* * *
QTc= 450 ms in symptomatic patients(normal QTc<420 ms in men; <440 ms in women)
Long QT syndromes in humans
1 sec
Decreased NCX and NKA expression, particularly at the T-tubules, in AnkB+/-
myocytes
(Mohler et al., Nature 2003)AnkB+/- mice = mice heterozygous for a null mutation in
ankyrin-B gene.
0
4
8
WT
An
kB+
/-
*
Caf
fein
e
(s)
WT
AnkB+/-
5 s
Caffeine
Reduced NCX and NKA function in AnkB+/-
myocytes NCX function
0 10 20 30 40
0
2
4
6WT
AnkB+/-
K0.5
[Na]i (mM)
-d[N
a]i/d
t (m
M/m
in)
0 10 20 300
20
40
60NT K- free 4 K, 0 Na
Time (min)
[Na]
i(m
M)
0
4
8
WT
An
kB+
/-
*
Vm
ax (
mM
/min
)
NKA function
Camors, …, Despa. JMCC, 2012
0
4
8
12
16W
T
An
kB+
/-
[Na]
i(m
M)
50
100
Diasto
lic [Ca]i (n
M)
Similar [Na]i and diastolic [Ca]i in myocytes from AnkB+/- and WT mice
[Na]i Diastolic [Ca]i
Resting Pacing
Camors, …, Despa. JMCC, 2012
0
2
4
6
8
2 sCaffeine
AnkB+/-
WT
F/F
0
0
1
2
6
8
10
*
*
Twitch Caffeine
WT
An
kB+
/-
[Ca]
i(
F/F
0)
Larger Ca transients, SR Ca load & fractional release in AnkB+/- mice
0
10
20
30
40
WT
An
kB+
/-
*
Fra
ctio
nal
SR
Ca
Rel
ease
(%
)
Camors, …, Despa. JMCC, 2012
0
1
2
3
WT
An
kB+
/-
* *
*
0.5 1 2
Frequency (Hz)
Ca
Sp
ark-
Fre
qu
ency
(100
µm
-1se
c-1)
Enhanced Ca spark frequency in intact AnkB+/- mice
200 ms
10
µm
0
250
40
µm
AnkB+/-
0 Na/ 0 Ca Tyrode1 mM CaTyrode
Caffeine
WT
Caffeine1 s0
250
Camors, …, Despa. JMCC, 2012
More pro-arrhythmic Ca waves in AnkB+/- myocytes
0
10
20
30
40
Ca wavesNo waves
*
WT AnkB+/-
Nu
mb
er o
f cel
ls
0
10
20
30
40
**
WT AnkB+/-
Nu
mb
er o
f cel
ls20 %
60 %
10 %
Control condition ISO (1 µM)
AnkB+/-
Camors, …, Despa. JMCC, 2012
WT AnkB+/-
T-tu
bule
NKA-α1
Cleft
Ca
Na
CaNCX
KNKA-2
CaATP
Ca
Ca
RyR
diastolic Ca
B56
Nuclearenvelope
IP3R
IP3 R
AnkB
Cleft
[Ca]
T-tu
bule
CaCaATP
Ca
Ca
RyR
diastolic Ca
Nuclear envelope
P
1. What causes the increased propensity for Ca sparks and waves in AnkB+/- myocytes? Altered cytosolic RyR regulation?
Work in progress; Questions:
Work in progress; Questions:2. Does AnkB proteolysis by calpain lead to a cardiac phenotype similar to that caused by genetic AnkB loss-of-function?
AnkB protein but not mRNA is reduced in the infarct border zone after MI
Hundt et al., Cardiovasc Res. 2009;81:742
Protein expression mRNA level
Work in progress; Questions:2. Does AnkB proteolysis by calpain lead to a cardiac phenotype similar to that caused by genetic AnkB loss-of-function?
AnkB & NKA protein expression are reduced following ischemia/reperfusion;
the effect is prevented by calpain inhibition
Inserte et al., Circ Res 2005;97:465.
NKA
How does calpain activation affect the protein expression, subcellular distribution and function of AnkB, NCX and NKA?
What is the role of AnkB proteolysis by calpain in the structural and electrical remodeling of the heart following ischemia/reperfusion?
How is Ca cycling altered in diabetic heart disease? Timeline!
Is [Na]i altered in diabetic heart disease? Does this further alter the cardiac metabolism?
ROS production → slowly inactivating INa → [Na]i → [Ca]m → ATP
Electrical remodeling & occurrence of arrhythmias in diabetic hearts
Long QT
Project 2: Electrical remodeling and arrhythmias in diabetic heart disease
0.0 0.5 1.0 1.5 2.0
1
2
3
** * * ***
Ctl
HIP
Frequency (Hz)
Am
pli
tud
e
F/F
0
0.0 0.5 1.0 1.5 2.02
3
4
5
Ctl
HIP
* *
Frequency (Hz)
Am
pli
tud
e
F/F
0Pre-diabetic stage Diabetic stage
Acknowledgments
University of California DavisEmmanuel CamorsKevin VoelkerFlorin DespaSamuel GaliceJeffrey ElliotKaleena JacksonBrian KochDonald M. BersKenneth GinsburgKhana Dao
Ohio State UniversityPeter Mohler
University of California Los AngelesEnrico StefaniYong Wu
University of CincinnatiJerry B. Lingrel
University of ManchesterFabian Brette
Funding from NIH & AHA
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