Download Figure 1 - Cardiac Mechanics Research Group

Structural Defects and Increased Mechanical Load Promote
Arrhythmias in the Isolated Murine Heart
1
Wright ,
1
Alice E.
Jeffrey H.
2,1
Omens ,
Robert S.
2,3
Ross
Andrew D.
1
McCulloch
Department of Bioengineering & The Whitaker Institute of Biomedical Engineering, UCSD, La Jolla, CA
2 Department of Medicine, UCSD School of Medicine, La Jolla, CA
3 Veterans Administration San Diego Health Care System, San Diego, CA
ABSTRACT
METHODS
INTRODUCTION
Optical Mapping
• Irregular myocardial mechanics are associated with an increased incidence of cardiac
arrhythmias and sudden cardiac death.1
Introduction: Cardiac structural abnormalities and altered wall mechanics are associated with
an increased incidence of arrhythmias and sudden cardiac death. Myocardial fibrosis and
heterogeneities in cell-cell coupling associated with a number of cardiomyopathies may
contribute to this proarrhythmic phenotype. Additionally, mechanical load has been shown to
induce changes in action potential propagation experimentally and altered extracellular matrix
deposition can change mechanical load within the myocardium of diseased hearts. Cardiac
myocyte specific knock-out of vinculin in mice results in disturbed intercalated disc structure
with altered adherens junctions and Cx 43 distribution, as well as mild fibrosis. Ventricular
arrhythmias with early sudden death and later development of a dilated cardiomyopathy are
seen. This research investigates the results of altered mechanical load and structural defects
on action potential propagation in the isolated murine heart.
• The murine heart is Langendorff
perfused with warm, oxygenated
modified Krebs-Henseleit solution.
• Mechanical load and structural abnormalities have been shown to alter cardiac action
potential propagation.2,3
• Immersed in optical bath chamber.
70 mmHg
• Reentry is the predominant mechanism of sustained arrhythmias and is promoted by
heterogeneous and slowed conduction.
• Cardiac myocyte specific knockout of the structural protein vinculin results in cardiac
structural and cellular remodeling, and develop ventricular arrhythmias and early sudden
death.4
Control
cVinKO
Vinculin
Cx43
Vin & Cx43
Methods: Isolated murine hearts were perfused and optically mapped with a high-speed CCD
camera. Fluorescence intensity was recorded and analyzed using custom filtering and
analysis algorithms. Epicardial mapping was conducted in vinculin deficient and wild-type
hearts during intrinsic rhythm and ventricular epicardial pacing. A fluid-filled balloon was
inserted into the left ventricle and volume load was exerted to examine the effects of
mechanical load on electrical propagation in the wild-type heart.
B
A
Figure 1) Previous studies show that
volume loading of the isolated rabbit
ventricle results in conduction slowing as
assessed by optical mapping.2
E
F
G
H
I
J
37°C
Adam T.
2,3
Zemljic-Harpf ,
Control
65
C
Activation Time
Unloaded
Loaded
D
Figure 2) Mice with cardiac specific knockout of vinculin (cVinKO) develop
structural abnormalities. Control ventricular myocytes show well-aligned
myofibrils inserted into preserved intercalated disc (ICD) structures (A and
C), while cVinKO myocytes display abnormal ICDs and gaps at insertion of
myofibrils (B and D). cVinKO hearts display redistribution of gap junctions
to the lateral walls (H, I, and J) compared with typical gap junction
localization at the ICDs in control myocytes (E, F, and G).4
0 ms
RESULTS
Electrophysiological Analysis of Vinculin Deficient Hearts
Mechanical Load Induced Changes in AP Propagation
8
B
B
18
Figure 4) Optical mapping of left
ventricular epicardium was performed in
8 week-old cVinKO compared to control.
Epicardial pacing of control (A) and
cVinKO (B) hearts was performed and
activation maps were constructed.
cVinKO hearts
displayed irregular
conduction wavefronts with regions of
greater negative curvature (-1.284 ±
0.435 mm-1 in cVinKO vs -0.751 ± 0.333
mm-1 in control; P<0.05; n=7 each).3
16
14
Activation Time
12
10
8
6
5
5
5
4
4
12
12
10
4
3
3
10
8
3
2
8
6
4
2
1.6
1
0
CONCLUSIONS
• Structural abnormalities in vinculin deficient myocardium are associated
with abnormal action potential conduction and development of ventricular
arrhythmias, as observed in the isolated heart preparation.
• Altered ventricular mechanical load slows conduciton velocity in both the
max and min directions.
1.0
1
0.8
0.6
CVmin
MaxCV
CV
max
1
0
0 ms
0.4
Initial Unloaded
IUL
Loaded
LD
• Volume loading of cVinKO hearts resulted in similar changes in
conduction velocity, but increased incidence of ventricular ectopic beats
and ventricular tacchycardia.
Final Unloaded
FUL
FUTURE WORK
2
0
1mm
0
A
12
12
Figure 5) Following isolation and prior to
dye loading, 6 of 7 cVinKO and 0 of 7
control
hearts
displayed
ventricular
arrhythmias
observed
by
surface
electrocardiogram (C
) (P<0.005).
Ventricular ectopic events were observed
optically in cVinKO hearts (B) as beats with
abnormal breakthrough sites and delayed
activation compared with intrinsic beats in
control hearts (A).
8
6
4
2
Activation Time
10
12 ms
B
12
10
10
8
8
6
6
Figure 6) Volume loading slows conduction. Activation maps of the left ventricle in the volume unloaded (A) and
loaded (B) states. Isochrones at 1 ms intervals. Note bunching of isochrones and longer activation in the loaded
state. Normalized conduction velocity in the max and min directions in the initial unloaded (IUL), loaded (LD), and
final unloaded (FUL) states (C). Slowing of conduction is observed in both the min and max directions (n=3) with
an overshoot of original CV upon unloading.
A 650
650
4
4
2
2
0
0
0 ms
0
C
Time
4 sec
Control CVmax
Control
CVmax
cVinKO CVmax
cVinKO
CVmax
Control
CVmin
Control CVmin
cVinKO
CVmin
cVinKO CVmin
550
500
450
400
B
350
300
250
200
200
0
Figure 7) Loading of cVinKO hearts results in conduction rate
response similar to that seen in control hearts (A, n=2 each).
Volume loading resulted in ventricular ectopic events in both
control and cVinKO hearts, but resulted in non-sustained VT in 2
of 2 cVinKO hearts and 0 of 2 control hearts. Example of VT
observed in one of the cVinKO hearts (B).
Apparent Conduction Velocity
600
Conduction Velocity (mm/s)
0 ms
Conduction Velocity (mm/sec)
0
Initial Unloaded
IUL
Loaded
LD
Final Unloaded
FUL
0
• Optical action potential propagation
filtered and analyzed as previously
described.5,3
1.2
0.4
0
Figure 3) Isolated murine heart perfusion
and loading preparation. Maintained in
warm bath during optical mapping.
• Water-filled balloon inserted into left
ventricle to exert volume load from
the unloaded (0 mmHg) to loaded (30
mmHg) states.
• Return of conduction velocity to greater rate than before loading.
1.4
MinCV
1
4
2
2
2
6
4
1.6
6
14
14
Apparent Conduction Velocity
6
16
16
C
7
6
18
8 ms
7
Normalized CV
18 ms
A
8
7
Activation Time
A
18
8
• Di-4-ANEPPS perfused and electrical
propagation measured with CCD.
• Recordings of LV epicardium taken
during intrinsic rhythm and ventricular
epicardial pacing.
cVinKO
Results and Discussion: Vinculin deficient hearts exhibited increased spontaneous
ventricular arrhythmias recorded by ECG and optical mapping. These hearts showed
disturbed activation wavefront propagation, quantified by a greater negative wavefront
curvature. Volume loading of the wild-type ventricle to 30 mmHg resulted in decreased
conduction velocity by approximately 10%. The heterogeneous distribution of conduction
velocity observed in the vinculin deficient hearts provides a proarrhythmic substrate. Altered
mechanical load within the diseased epicardium may also contribute to arrhythmogenesis as
increased load alters action potential conduction through the myocardium.
• Volume conducted ECG recorded.
Time
2 sec
• Our group had found that conduction slowing during ventricular loading in
the rabbit is due to changes in the passive myocardial electrical properties,
time and space constants.6
• Modify techniques to analyze changes in these passive electrical properties
in the murine heart.
• To investigate underlying cellular mechanisms responsible for these
changes with the use of transgenic mouse models:
• Caveolin-3 deficient mice: observe the importance of caveolae
unfolding on conduction slowing during load.6
• Cardiac-specific connexin-43 deficienct mice: observe the importance
of gap junction conductance changes on conduction slowing during
load.7
REFERENCES
1. Dean, J.W. and M.J. Lab. Lancet, 1989. 1(8650): p. 1309-12. 2. Sung, D., et al. J Cardiovasc Electrophysiol, 2003. 14(7): p. 739-749. 3. Gutstein, D.E., et al. Circulation, 2001. 104(10): p.1194-9 4. Zemljic-Harpf, A.E., et al. Genes & Dev. 2007 In Submission
5. Sung, D., et al. Ann Biomed Eng, 2001. 29(10): p.854-61 6. Mills, R.W. 2007, in revision 7. Woodman, S.E., et al. J Biol Chem, 2002. 277(41): p. 38988-97 8. Gutstein, D.E., et al. Circ Res, 2001. 88(3): p. 333-339.
Supported by National Science Foundation Grant BES-0086482 and Grant BES- 0506252; the National Biomedical Computational Resource; National Institutes of Health Grant P41 RR08065-11 and Grant 5 P01 HL46345-12.