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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 50M Gd3+ APD30 APD30 with 50M 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 2i 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 2i 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)