Transcript Sanda Despa, Ph.D., Deparment of Pharmacology

Research Interest
Sanda Despa, PhD
Department of Pharmacology
 Excitation-contraction coupling, Ca2+ and Na+ regulation in the normal
and diseased heart;
 Cellular bases of triggered ventricular arrhythmias
Cardiac excitation-contraction coupling
Ca
Ca
SR
NCX
T-Tubule
3Na
Ca
PLB
RyR
Ca
ICa
ATP
Ca
ATP
NCX
Na
2K
PLM
Sarcolemma
3Na
3Na
ATP
Cardiac excitation-contraction coupling
Ca
Ca
SR
NCX
T-Tubule
3Na
Ca
PLB
RyR
Ca
ICa
ATP
Ca
ATP
NCX
Na
2K
PLM
Sarcolemma
3Na
3Na
ATP
Contraction of the heart
3Na Na
Ca
Ca
SR
NCX
T-Tubule
3Na
Ca
PLB
RyR
Ca
ICa
ATP
Ca
NCX
PLM
SarcolemmaATP
2K
ATP
3Na
Relaxation of the heart
3Na
T-Tubule
3Na
Ca
PLB
Ca
SR
NCX
NCX
Ca
RyR
Ca
ICa
ATP
Ca
ATP
Ca
2K
PLM
Sarcolemma
Na
3Na
ATP
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
E1425G mutation
Long QT4 syndrome
DII
Q-T
Ventricular arrhythmias
DIII
1 sec
QTc= 450 ms in symptomatic patients
(normal QTc<420 ms in men; <440 ms in women)
Long QT syndromes in humans
*
*
*
1 sec
(Schott JJ et al., Am. J. Hum. Genet. 1994)
Decreased NCX and NKA expression, particularly at
the T-tubules, in AnkB+/- myocytes
AnkB+/- mice = mice heterozygous for a null mutation in
ankyrin-B gene.
(Mohler et al., Nature 2003)
Reduced NCX and NKA function in AnkB+/- myocytes
WT
5s
4
Caffeine
WT
AnkB+/-
*
8
0
AnkB+/-
Caffeine  (s)
NCX function
4 K, 0 Na
40
20
0
0
10
20
Time (min)
30
6
WT
4
AnkB+/2
K0.5
0
0
10
8
4
0
20
30
*
AnkB+/-
K- free
WT
NT
Vmax (mM/min)
[Na]i (mM)
60
-d[Na]i/dt (mM/min)
NKA function
40
[Na]i (mM)
Camors, …, Despa. JMCC, 2012
Similar [Na]i and diastolic [Ca]i in myocytes
from AnkB+/- and WT mice
[Na]i
100
12
8
0
50
AnkB+/-
4
WT
[Na]i (mM)
Diastolic [Ca]i
Resting
Diastolic [Ca]i (nM)
16
Pacing
Camors, …, Despa. JMCC, 2012
Larger Ca transients, SR Ca load &
fractional release in AnkB+/- mice
WT
2
0
2s
2
*
1
Caffeine
0
Twitch
Caffeine
*
30
20
10
0
AnkB+/-
4
6
40
WT
AnkB+/-
8
AnkB+/-
6
*
WT
[Ca]i (F/F0)
F/F0
8
Fractional SR Ca
Release (%)
10
Camors, …, Despa. JMCC, 2012
Enhanced Ca spark frequency in intact AnkB+/- mice
250
0
250
WT
1s
Caffeine
0
1 mM Ca
Tyrode
Caffeine
3
*
200 ms
*
*
1
0
0.5
1
Frequency (Hz)
AnkB+/-
2
WT
Ca Spark-Frequency
(100 µm-1sec-1)
0 Na/ 0 Ca Tyrode
10 µm
40 µm
AnkB+/-
2
Camors, …, Despa. JMCC, 2012
More pro-arrhythmic Ca waves in AnkB+/- myocytes
AnkB+/-
ISO (1 µM)
Control condition
30
*
20
10
0
40
No waves
Ca waves
20 %
WT
AnkB+/-
Number of cells
Number of cells
40
30
**
20
60 %
10
10 %
0
WT
AnkB+/-
Camors, …, Despa. JMCC, 2012
Work in progress; Questions:
1. What causes the increased propensity for Ca sparks and waves in AnkB+/- myocytes?
 Altered cytosolic RyR regulation?
WT
AnkB+/-
diastolic Ca
diastolic Ca
NKA-α1
NCX
Ca
ATP
Ca
P
Ca
Ca
Ca
T-tubule
Ca
Ca
[Ca]
Cleft
Ca
Na
K
RyR
Ca
RyR
ATP
B56
NKA-2
Cleft
T-tubule
AnkB
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
Protein expression
mRNA level
Hundt et al., Cardiovasc Res. 2009;81:742
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
NKA
Inserte et al., Circ Res 2005;97:465.
 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?
Project 2: Electrical remodeling and arrhythmias in
diabetic heart disease
 How is Ca cycling altered in diabetic heart disease? Timeline!
5
3
* *
HIP
4
Ctl
3
2
0.0
Diabetic stage
Amplitude F/F0
Amplitude F/F0
Pre-diabetic stage
0.5
1.0
1.5
Frequency (Hz)
2.0
Ctl
2
** *
* HIP ***
1
0.0
0.5
1.0
1.5
2.0
Frequency (Hz)
 Is [Na]i altered in diabetic heart disease? Does this further alter the cardiac
metabolism?
Long QT
ROS production →  slowly inactivating INa →  [Na]i →  [Ca]m → ATP
 Electrical remodeling & occurrence of arrhythmias in diabetic hearts
Acknowledgments
University of California Davis
Emmanuel Camors
Kevin Voelker
Florin Despa
Samuel Galice
Jeffrey Elliot
Kaleena Jackson
Brian Koch
Donald M. Bers
Kenneth Ginsburg
Khana Dao
Ohio State University
Peter Mohler
University of California Los Angeles
Enrico Stefani
Yong Wu
University of Cincinnati
Jerry B. Lingrel
University of Manchester
Fabian Brette
Funding from NIH & AHA