1. Structural Defects and Increased Mechanical Load Promote Arrhythmias in the Isolated Murine Heart
Adam T. Wright1, Alice E. Zemljic-Harpf2,3, Jeffrey H. Omens2,1, Robert S. Ross2,3 Andrew D. McCulloch1
1 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
INTRODUCTION
METHODS
Optical Mapping
The murine heart is Langendorff perfused with warm, oxygenated modified Krebs-Henseleit solution.
Immersed in optical bath chamber.
Volume conducted ECG recorded.
Di-4-ANEPPS perfused and electrical propagation measured with CCD.
Recordings of LV epicardium taken during intrinsic rhythm and ventricular epicardial pacing.
Water-filled balloon inserted into left ventricle to exert volume load from the unloaded (0 mmHg) to loaded (30 mmHg) states.
Optical action potential propagation filtered and analyzed as previously described.5,3
Irregular myocardial mechanics are associated with an increased incidence of cardiac arrhythmias and sudden cardiac death.1
Mechanical load and structural abnormalities have been shown to alter cardiac action potential propagation.2,3
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
37°C
70 mmHg
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.
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.
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.
Control
cVinKO A B C D
Vinculin
Cx43
Vin & Cx43
Control
cVinKO E F G H I J
Figure 1) Previous studies show that volume loading of the isolated rabbit ventricle results in conduction slowing as assessed by optical mapping.2 65
0 ms
Unloaded
Loaded
Activation Time
Figure 3) Isolated murine heart perfusion and loading preparation. Maintained in warm bath during optical mapping.
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
CONCLUSIONS
RESULTS
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.
Return of conduction velocity to greater rate than before loading.
Volume loading of cVinKO hearts resulted in similar changes in conduction velocity, but increased incidence of ventricular ectopic beats and ventricular tacchycardia.
Electrophysiological Analysis of Vinculin Deficient Hearts
Mechanical Load Induced Changes in AP Propagation
1mm
0 ms
18 ms A B
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 ( ± mm-1 in cVinKO vs ± mm-1 in control; P<0.05; n=7 each).3
Activation Time C
0 ms
8 ms
Activation Time A B
Apparent Conduction Velocity
1.6
Normalized CV
1.0
CVmin
CVmax
0.4
IUL LD
FUL
FUTURE WORK
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.
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
12 ms
0 ms
Activation Time A B
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).
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). A
Control CVmax
cVinKO CVmax
Control CVmin
cVinKO CVmin
650
200
Conduction Velocity (mm/sec)
IUL
FUL LD
Apparent Conduction Velocity C
Time
4 sec
Time
2 sec B
REFERENCES
1. Dean, J.W. and M.J. Lab. Lancet, (8650): p Sung, D., et al. J Cardiovasc Electrophysiol, (7): p Gutstein, D.E., et al. Circulation, (10): p Zemljic-Harpf, A.E., et al. Genes & Dev In Submission
5. Sung, D., et al. Ann Biomed Eng, (10): p Mills, R.W. 2007, in revision 7. Woodman, S.E., et al. J Biol Chem, (41): p Gutstein, D.E., et al. Circ Res, (3): p
Supported by National Science Foundation Grant BES and Grant BES ; the National Biomedical Computational Resource; National Institutes of Health Grant P41 RR and Grant 5 P01 HL