Download Determinants of Conduction Slowing During Ventricular

Determinants of Conduction Slowing
During Ventricular Volume Loading in
the Healthy and Failing Heart
Adam Wright
Thesis Proposal submitted to Senate Qualifying Committee:
Dr. Andrew McCulloch, Chair – Bioengineering
Dr. Wayne Giles – Bioengineering
Dr. Sanjiv Narayan – Medicine
Dr. Jeffrey Omens – Medicine/Bioengineering
Dr. Robert Ross – Medicine
Dr. Gabriel Silva – Bioengineering
Tuesday, January 9th, 2007
Powell-Focht Bioengineering Hall 291, 10:00 AM
University of California, San Diego
Mechanically Induced Arrhythmogenesis
Cardiac arrhythmias associated with irregular
wall mechanics
Mechanical load contributes to changes in
electrical activity that may promote arrhythmias


Induce triggered events
Reentry
Predominant mechanism of
sustained ventricular
arrhythmias associated with
irregular mechanics
(Taggart and Sutton, 1999)
(Franz, et al, 1992)
(London, et al, 2003)
Mechanically Induced Arrhythmogenesis
Reentry promoted by:


Increased dispersion of refractory period
Decreased cardiac wavelength
Shortened ERP


CV
*
ERP
Slowed Conduction

Changes in conduction
velocity with stretch
(Zabel, et al, 1996)
Observed Effects of Mechanical Load on
Conduction Velocity
Inconsistent Observations




Atrial and ventricular strips displayed increased/no
change in CV with stretch (Penefsky and Hoffman,
1963)
Rat papillary muscles displayed decrease in CV with
stretch (Spear and Moore, 1972)
QRS prolongation in in situ canine heart during
increased LV pressure (Sideris, et al, 1994)
No change in CV during ventricular loading of intact
rabbit heart (Reiter, et al, 1997)
Studies use contact electrodes
Different experimental preparations
Effect of Volume Load on Conduction Velocity
and Passive Myocardial Electrical Properties
Value Normalized by Initial Unloaded Value
1.25
1.2
Value Normalized by Initial Unloaded Value
Our group has demonstrated conduction slowing in the loaded
isolated rabbit heart (Sung, et al, 2003)
Conduction slowing still present in presence of SAC blockers
streptomycin and Gd3+
Associated with changes in space and time constant during load
(Mills, et al, 2006)
CV
CV with 50M Gd3+
APD30
APD30 with 50M Gd3+
1.1
1.05
1
0.95
0.9
0.85
0.8
Initial Unloaded
Loaded
Final Unloaded
Conduction Velocity
X-fiber Space Constant
X-fiber Time Constant
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
1.15
0.75
1.8
Initial Unloaded
Loaded
Final Unloaded
Objective
Mechanical load has been shown to alter
cardiac electrophysiology. This feedback may
contribute to arrhythmia in patients suffering
from structural heart defects. Our group has
recently shown that a slowing of conduction in
the volume loaded rabbit left ventricle is due, in
part, to alterations in passive electrical
properties of the myocardium under mechanical
stress. The goals of this research are to
investigate the cellular mechanisms behind
observed changes of these passive electrical
properties during mechanical loading.
Specific Aims
1) To investigate the effects of mechanical load on
action potential propagation and passive myocardial
electrical properties of the myocardium.
2) To investigate the role of caveolae in changes in
action potential propagation and passive electrical
properties during mechanical load.
3) To investigate the role of gap junctions in changes in
action potential propagation and passive electrical
properties during mechanical load.
Specific Aims
1) To investigate the effects of mechanical load on
action potential propagation and passive myocardial
electrical properties of the myocardium.
1-a) To design experimental and analytical techniques
to measure conduction velocity and passive
myocardial electrical properties in the freely beating
isolated murine heart.
Experimental Preparation
70 mmHg
• Langendorff isolated mouse heart
• Perfused with oxygenated
modified Krebs-Henseleit saline
• Flow rate, ECG, etc. is monitored
• Di-4-ANEPPS loaded
37°C
• Excited with LED lamps
• Emission filtered and collected
CCD-Camera
Optical Bath
Chamber
(Mills defense, 2005)
Conduction Velocity Analysis
Activation Time (ms)
22 ms
22
20
18
16
14
12
10
Activation time calculated
at max upstroke (dF/dt)
8
6
4
2
Local conduction velocity
vectors calculated as
reciprocal gradient of
activation time
0 ms
Time Series of
Fluorescence Images
Filtered Optical
Action Potentials
(Filtering, Sung, 2001)
(Algorithm, Bayly, 1998)
Determinants of Conduction Velocity
Conduction Velocity
Active Cellular Properties
Passive Tissue Properties
(Ionic Model)
(Bidomain Model)
Ion Channel Dynamics
Ion Pump Activity
Calcium Handling
Membrane Conductance
Extracellular Conductance
Intracellular Conductance
Membrane Capacitance
Bidomain Model – Coupled Equations Governing Intracellular and Extracellular Potentials
 2i
 2 i
 2 i 


gix

g

g


G
(



)


C
(



)
iy
iz
e
m
i
e   Ii
 m i
x 2
y 2
z 2
t

 2 e
 2 e
 2 e



g ex

g

g



G
(



)


C
(



)
ey
ez
e
m
i
e   Ie
 m i
x 2
y 2
z 2
t

Intra or extracellular current
Transmembrane Current
External
Applied Current
Passive Electrical Properties
Electrical Space Constants
Inversely proportional to intracellular and extracellular resistance
Proportional to transmembrane resistance
L 
T 
giL g eL

g

g

G
 iL eL  m
Rm
 riL  reL  
giT g eT

 giT  geT   Gm
Rm
 riT  reT  
Greater space constant would result
in faster conduction
Electrical Time Constant
Proportional to membrane capacitance and transmembrane resistance

Cm
 Cm Rm
Gm
Greater time constant would result
in slower conduction
Measuring Effective Space and Time
Constants
Apply Non-Excitatory Stimulus
Electrical Space Constants
Fit spatial exponential decay of
transmembrane voltage at steady-state
(Akar et al, 2001)
Measuring Effective Space and Time
Constants
Apply Non-Excitatory Stimulus
Electrical Space Constants
Normalized Signal Amplitude
Normalized Signal Amplitude
Fit spatial exponential decay of
transmembrane voltage at steady-state
1
0.5
0
-0.5
600
800
1000 1200
milliseconds
1400
1
0.5
0
-0.5
600
800
1000 1200
milliseconds
1400
• Apply stimulus during refractory period of the previous beat and hold
(Mills et al, 2006)
• Subtract common mode signal
• Fit steady-state potential map to analytical solution of the bidomain equations

 3

 3 3   3cos     1  
  R,    M1  e R  M 8 e R   2  e R 1   2  
 

R
R
R
2






 

2
 x   z  
R       
 T   L  
2
2
1
2
 x   z 
  tan 1      
  T   L  
Measuring Effective Space and Time
Constants
Apply Non-Excitatory Stimulus
Electrical Space Constants
Fit spatial exponential decay of transmembrane voltage at steady-state
Electrical Time Constant
Fit transient exponential rise of transmembrane voltage during
application of stimulus
Specific Aims
1) To investigate the effects of mechanical load on
action potential propagation and passive myocardial
electrical properties of the myocardium.
1-b) To investigate the effects of balloon volume
loading on conduction velocity and passive
myocardial electrical properties in the murine heart.
Hypothesis: Alterations in passive electrical properties
contribute to the slowing of action potential
conduction during loading.
Experimental Preparation
• Langendorff perfused murine heart
37°C
70 mmHg
Optical Bath
Chamber
• Plastic balloon inserted
into the left ventricle
• Volume infusion loads
the left ventricle
• LV pressure is monitored
Pressure transducer
(Mills defense, 2005)
Preliminary Results in the Mouse
10
10
10
10
10
9
8
8
8
7
7
7 7
6
6
6 6
5
5
9
5
9
8
5
4
4
4
4
3
3
3
3
2
2
2
2
Act. Time (ms)
9
1
1
1
0
570
CV (mm/s)
Conduction Velocity (mm/sec)
Conduction Velocity (mm/sec)
540
530
520
510
500
500
CVmin
CVmin
320
550
0
330
CVmax
560
CV (mm/s)
0
330
CVmax
570
310
300
290
280
270
Initial Unloaded
IUL
Loaded
LD
Final Unloaded
FUL
260
260
Initial Unloaded
IUL
0
1
0
Loaded
LD
Final Unloaded
FUL
Specific Aims
1) To investigate the effects of mechanical load
on action potential propagation and passive
myocardial electrical properties of the
myocardium.
1-c) Develop mathematical model to validate
relationship between changes in passive
myocardial electrical properties and
conduction velocity
Mathematical Model
Anisotropic Bidomain Model
Incorporate Bondarenko Ionic Model
 2i
 2 i
 2 i 


gix
 giy
 giz
   Gm ( i   e )   Cm ( i   e )   I i
2
2
2
x
y
z
t


 2 e
 2 e
 2 e



g ex

g

g



G
(



)


C
( i   e )   I e
ey
ez
m
i
e
m
2
2
2

x
y
z
t


(Bondarenko et al, 2004)
Specific Aims
2) To investigate the role of caveolae on changes in
electrical passive properties and conduction velocity
in the isolated murine heart during volume loading
using a murine model lacking caveolae in the
cardiomyocyte membranes.
Hypothesis: Caveolae alter their conformation under
stretch and contribute to changes in membrane
capacitance, and thus contribute to conduction velocity
and time constant changes during loading. Myocardium
lacking caveolae would have a smaller change in
capacitance under load and thus a different change in
conduction velocity.
Caveolae Unfold with Stretch
Caveolae are invaginated lipid rafts in sarcolemma
associated with cav-3
Caveolae account for ~30% of plasmalemmal area of
rabbit ventricular cardiomyocytes (Levin and Page, 1980)
These structures unfold and incorporate with
sarcolemma under ventricular load
Unloaded
Loaded
(Kohl et al, 2003)
Cavolae Deficient Mice
Cav-3 deficient mice lack caveolae in cardiomyocytes

Develop cardiomyopathy after 2 months
If load induced increase in time constant is dependent on
caveolae unfolding, myocardium lacking these structures
will display a diminished increase in time constant, and
thus a diminished slowing of conduction
(Woodman et al, 2002)
Specific Aims
3) To investigate the role of gap junctions on changes
in conduction velocity and intercellular coupling in
the isolated murine heart during volume loading
using a murine model with diminished expression of
connexin-43 in the myocardium.
Hypothesis: Conformational changes of gap junctions in
myocardium under stretch play a role in changes of
intercellular resistance and, thus, myocardial space
constants and conduction velocity in the volume loaded
heart. Hearts deficient in connexin-43 will display a
diminished response in intercellular resistance during
volume loading, and thus a different change in
conduction velocity.
Gap Junctions
Gap junctions are intercellular channels



Allow current to flow between cells
Composed of aligned hemichannels, each hexamers of connexin
proteins (primarily Cx-43 in adult ventricular myocardium)
Located primarily at intercalated discs
Gap junction conductance is primary regulator of
intercellular resistance
Increase in connexin expression is associated with
stretch in vitro (Zhuang, 2000)
Connexin hemichannels open in response to shear
stress (Cherian, 2005) and membrane stretch (Bao,
2004)
Connexin-43 Deficient Myocardium
Connexin-43 deficiency reduces intercellular coupling
Cardiac specific KO of connexin-43 results in slowed
conduction


Mice develop spontaneous arrhythmias and die early
No mechanical dysfunction
If the increase in space constant during load are a result
of gap junction conformational change, then myocardium
lacking functional gap junctions will display a diminished
change in space constant and conduction velocity during
load.
(Gutstein et al, 2001)