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-Tu

bule

Ca

3Na

NCXATP

Na

3Na

2K

PLM ATP

ICa

Ca

CaSR

PLB ATP

RyR

3Na

CaNCX

Cardiac excitation-contraction coupling

Sarcolemma

Ca

T-Tu

bule

Ca

3Na

NCXATP

Na

3Na

2K

PLM ATP

ICa

Ca

CaSR

PLB ATP

RyR

3Na

CaNCX

Sarcolemma

CaT-

Tubu

leCa

3Na

NCXATP

Na

3Na

2K

PLM ATP

ICa

Ca

CaSR

PLB ATP

RyR

3Na

CaNCX

Contraction of the heart

Relaxation of the heart

Sarcolemma

CaCa

T-Tu

bule

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 syndromeDII 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

AnkB

+/-

*

Caffe

ine

(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

60 NT K- free 4 K, 0 Na

Time (min)

[Na]

i(m

M)

0

4

8

WT

Ank

B+/-

*

V max

(mM

/min

)

NKA function

Camors, …, Despa. JMCC, 2012

0

4

8

12

16W

TA

nkB+

/-

[Na]

i(m

M)

50

100

Diastolic [Ca]i (nM)

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+/-WTF/

F 0

0

1

2

6

8

10

*

*

Twitch Caffeine

WT

Ank

B+/-

[Ca]

i(

F/F 0

)

Larger Ca transients, SR Ca load & fractional release in AnkB+/- mice

0

10

20

30

40

WT

AnkB

+/-

*

Frac

tiona

l SR

CaRe

leas

e (%

)

Camors, …, Despa. JMCC, 2012

0

1

2

3

WT

AnkB

+/-

* *

*

0.5 1 2Frequency (Hz)

Ca S

park

-Fre

quen

cy(1

00 µ

m-1

sec-1

)

Enhanced Ca spark frequency in intact AnkB+/- mice

200 ms

10 µm

0

250

40 µ

m

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+/-

Num

ber o

f cel

ls

0

10

20

30

40

**

WT AnkB+/-

Num

ber 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 envelopeP

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

plitu

de

F/F 0

0.0 0.5 1.0 1.5 2.02

3

4

5

Ctl

HIP

* *

Frequency (Hz)

Am

plitu

de

F/F 0

Pre-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