Vladimir G. Fast

Role of wavefront curvature in propagation of cardiac impulse

It is traditionally assumed that impulse propagation in cardiac muscle is determined by the combination of two factors: (1) the active properties of cardiac cell membranes and (2) the passive electrical characteristics of the network formed by cardiac cells. However, advances made recently in the theory of generic excitable media suggest that an additional factor-the geometry of excitation wavefronts -may play an important role. In particular, impulse propagation strongly depends on the wavefront curvature on a small spatial scale. In the heart, excitation wavefronts have pronounced curvatures in several situations including waves initiated by small electrodes, waves emerging from narrow tissue structures, and waves propagating around the sharp edges of anatomical obstacles or around a zone of functional conduction block during spiral wave rotation. In this short review we consider the theoretical background relating impulse propagation to wavefront curvature and we estimate the role of wavefront curvature in electrical stimulation, formation of conduction block, and the dynamic behavior of spiral waves.

Cardiac tissue geometry as a determinant of unidirectional conduction block: assessment of microscopic excitation spread by optical mapping in patterned cell cultures and in a computer model.

OBJECTIVE Unidirectional conduction block (UCB) and reentry may occur as a consequence of an abrupt tissue expansion and a related change in the electrical load. The aim of this study was to evaluate critical dimensions of the tissue necessary for establishing UCB in heart cell culture. METHODS Neonatal rat heart cell cultures with cell strands of variable width emerging into a large cell area were grown using a technique of patterned cell growth. Action potential upstrokes were measured using a voltage sensitive dye (RH-237) and a linear array of 10 photodiodes with a 15 microns resolution. A mathematical model was used to relate action potential wave shapes to underlying ionic currents. RESULTS UCB (block of a single impulse in anterograde direction - from a strand to a large area - and conduction in the retrograde direction) occurred in narrow cell strands with a width of 15(SD 4) microns (1-2 cells in width, n = 7) and there was no conduction block in strands with a width of 31(8) microns (n = 9, P < 0.001) or larger. The analysis of action potential waveshapes indicated that conduction block was either due to geometrical expansion alone (n = 5) or to additional local depression of conduction (n = 2). In wide strands, action potential upstrokes during anterograde conduction were characterised by multiple rising phases. Mathematical modelling showed that two rising phases were caused by electronic current flow, whereas local ionic current did not coincide with the rising portions of the upstrokes. CONCLUSIONS (1) High resolution optical mapping shows multiphasic action potential upstrokes at the region of abrupt expansion. At the site of the maximum decrement in conduction, these peaks were largely determined by the electrotonus and not by the local ionic current. (2) Unidirectional conduction block occurred in strands with a width of 15(4) microns (1-2 cells).

Transmural Heterogeneity and Remodeling of Ventricular Excitation-Contraction Coupling in Human Heart Failure

Background— Excitation-contraction (EC) coupling is altered in end-stage heart failure. However, spatial heterogeneity of this remodeling has not been established at the tissue level in failing human heart. The objective of this article was to study functional remodeling of excitation-contraction coupling and calcium handling in failing and nonfailing human hearts. Methods and Results— We simultaneously optically mapped action potentials and calcium transients in coronary perfused left ventricular wedge preparations from nonfailing (n=6) and failing (n=5) human hearts. Our major findings are the following. First, calcium transient duration minus action potential duration was longer at subendocardium in failing compared with nonfailing hearts during bradycardia (40 bpm). Second, the transmural gradient of calcium transient duration was significantly smaller in failing hearts compared with nonfailing hearts at fast pacing rates (100 bpm). Third, calcium transient in failing hearts had a flattened plateau at the midmyocardium and exhibited a 2-component slow rise at the subendocardium in 3 failing hearts. Fourth, calcium transient relaxation was slower at the subendocardium than at the subepicardium in both groups. Protein expression of sarcoplasmic reticulum Ca2+-ATPase 2a was lower at the subendocardium than the subepicardium in both nonfailing and failing hearts. Sarcoplasmic reticulum Ca2+-ATPase 2a protein expression at subendocardium was lower in hearts with ischemic cardiomyopathy compared with those with nonischemic cardiomyopathy. Conclusions— For the first time, we present direct experimental evidence of transmural heterogeneity of excitation-contraction coupling and calcium handling in human hearts. End-stage heart failure is associated with the heterogeneous remodeling of excitation-contraction coupling and calcium handling.

Block of impulse propagation at an abrupt tissue expansion: evaluation of the critical strand diameter in 2- and 3-dimensional computer models

OBJECTIVE Unidirectional conduction block in the heart can occur at a site where the impulse is transmitted from a small to a large tissue volume. The aim of this study was to evaluate the occurrence of conduction block in a 2-dimensional and 3-dimensional computer model of cardiac tissue consisting of a narrow strand abruptly emerging into a large area. In this structure, the strand diameter critical for the occurrence of block, hc, was evaluated as a function of changes in the active and passive electrical properties of both the strand and the large medium. METHODS The effects of changes in the following parameters on hc were analysed: (1) maximum sodium conductance (gNamax), (2) longitudinal (Rx) and transverse (Ry) intracellular resistivities, and (3) inhomogeneities in gNamax and Rx and Ry between the strand and the large area. Three ionic models for cardiac excitation described by Beeler-Reuter, Ebihara-Johnson, and Luo-Rudy ionic current kinetics were compared. RESULTS In the 2-dimensional simulations, hc was 175 microns in Ebihara-Johnson and Beeler-Reuter models and 200 microns in the Luo-Rudy model. At the critical strand diameter, the site of conduction block was located beyond the transition, i.e. a small circular area was activated in the large medium, whereas with narrower strands conduction block occurred within the strands. The decrease of gNamax resulted in a large increase of hc. This increase was mainly due to the change of gNamax in the large area, while hc was almost independent of gNamax in the strand. Changing Rx had no effect on hc, whereas the increase of Ry decreased hc and reversed conduction block. Inhomogeneous changes of Rx and Ry in the strand versus the large medium had opposite effects on hc. When the resistivities of the strand alone were increased, hc also increased. In contrast, the increase of the resistivities in the large area reduced hc. In the 3-dimensional model, hc was 2.7 times larger than the corresponding 2-dimensional values at the various levels of gNamax and resistivity. CONCLUSIONS (1) At physiological values for active and passive electrical properties, hc in the 2D simulations is close to 200 microns in all three ionic models. In the 3-dimensional simulations, hc is 2.7 larger than in the 2-dimensional models. (2) The excitable properties of the large area but not of the strand modify hc. The decrease of intercellular coupling in the large medium facilitates impulse conduction and reduces hc, while the same change in the strand increases hc. (3) Occurrence of conduction block at an abrupt geometrical transition can be explained by both the impedance mismatch at the transition site and the critical curvature beyond the transition.

Intramural Virtual Electrodes During Defibrillation Shocks in Left Ventricular Wall Assessed by Optical Mapping of Membrane Potential

Background—It is believed that defibrillation is due to shock-induced changes of transmembrane potential (&Dgr;Vm) in the bulk of ventricular myocardium (so-called virtual electrodes), but experimental proof of this hypothesis is absent. Here, intramural shock-induced &Dgr;Vm were measured for the first time in isolated preparations of left ventricle (LV) by an optical mapping technique. Methods and Results—LV preparations were excised from porcine hearts (n=9) and perfused through a coronary artery. Rectangular shocks (duration 10 ms, field strength E ≈2 to 50 V/cm) were applied across the wall during the action potential plateau by 2 large electrodes. Shock-induced &Dgr;Vm were measured on the transmural wall surface with a 16×16 photodiode array (resolution 1.2 mm/diode). Whereas weak shocks (E≈2 V/cm) induced negligible &Dgr;Vm in the wall middle, stronger shocks produced intramural &Dgr;Vm of 2 types. (1) Shocks with E>4 V/cm produced both positive and negative intramural &Dgr;Vm that changed their sign on changing shock polarity, possibly reflecting large-scale nonuniformities in the tissue structure; the &Dgr;Vm patterns were asymmetrical, with &Dgr;V−m>&Dgr;V+m. (2) Shocks with E>34 V/cm produced predominantly negative &Dgr;Vm across the whole transmural surface, independent of the shock polarity. These relatively uniform polarizations could be a result of microscopic discontinuities in tissue structure. Conclusions—Strong defibrillation shocks induce &Dgr;Vm in the intramural layers of LV. During action potential plateau, intramural &Dgr;Vm are typically asymmetrical (&Dgr;V−m>&Dgr;V+m) and become globally negative during very strong shocks.

Simultaneous Optical Mapping of Transmembrane Potential and Intracellular Calcium in Myocyte Cultures

Simultaneous Mapping of Vm and Cai2+. Introduction: Fast spatially resolved measurements of transmembrane potential (Vm) and intracellular calcium (Cai2+) are important for studying mechanisms of arrhythmias and defibrillation. The goals of this work were (1) to develop an optical technique for simultaneous multisite optical recordings of Vm and Cai2+, and (2) to determine the relationship between Vm and Cai2+ during normal impulse propagation in myocyte cultures.

Nonlinear Changes of Transmembrane Potential During Defibrillation Shocks Role of Ca2+ Current

Defibrillation shocks induce complex nonlinear changes of transmembrane potential (&Dgr;Vm). To elucidate the ionic mechanisms of nonlinear &Dgr;Vm, we studied the effects of ionic channel blockers on &Dgr;Vm in geometrically defined myocyte cultures. Experiments were carried out in cell strands with widths of 0.2 mm (narrow strands) and 0.8 mm (wide strands) produced using a technique of directed cell growth. Uniform-field shocks were applied across strands during the action potential (AP) plateau, and the distribution of shock-induced &Dgr;Vm was measured using an optical mapping technique. Nifedipine and 4-aminopyridine were applied to inhibit the L-type calcium current (ICa) and the transient outward current (Ito), respectively. In control conditions, the distribution of &Dgr;Vm across cell strands was highly asymmetrical with a large ratio of negative to positive &Dgr;Vm (&Dgr;V−m/&Dgr;V+m) measured at the opposite strand borders. Application of nifedipine caused a large increase of &Dgr;V+m and a decrease of &Dgr;V−m/&Dgr;V+m, indicating involvement of ICa in the asymmetrical &Dgr;Vm, likely as a result of the outward flow of ICa when Vm exceeded the ICa reversal potential. &Dgr;V−m decreased in the narrow strands but remained unchanged in the wide strands, indicating that the changes of &Dgr;V−m were caused by electrotonic interaction with an area of depolarization. 4-Aminopyridine did not change &Dgr;V−m/&Dgr;V+m. These results provide evidence that (1) the asymmetry of shock-induced &Dgr;Vm during the AP plateau is due to outward flow of ICa in the depolarized portions of the strands, (2) Ito is not involved in the mechanism of &Dgr;Vm asymmetry, and (3) the effects of drugs on &Dgr;Vm are modulated by the tissue geometry.

Optical Mapping of Impulse Propagation in Engineered Cardiac Tissue

Cardiac tissue engineering has a potential to provide functional, synchronously contractile tissue constructs for heart repair, and for studies of development and disease using in vivo-like yet controllable in vitro settings. In both cases, the utilization of bioreactors capable of providing biomimetic culture environments is instrumental for supporting cell differentiation and functional assembly. In the present study, neonatal rat heart cells were cultured on highly porous collagen scaffolds in bioreactors with electrical field stimulation. A hallmark of excitable tissues such as myocardium is the ability to propagate electrical impulses. We utilized the method of optical mapping to measure the electrical impulse propagation. The average conduction velocity recorded for the stimulated constructs (14.4 +/- 4.1 cm/s) was significantly higher than that of the nonstimulated constructs (8.6 +/- 2.3 cm/s, p = 0.003). The measured electrical propagation properties correlated to the contractile behavior and the compositions of tissue constructs. Electrical stimulation during culture significantly improved amplitude of contractions, tissue morphology, and connexin-43 expression compared to the nonsimulated controls. These data provide evidence that electrical stimulation during bioreactor cultivation can improve electrical signal propagation in engineered cardiac constructs.

Myocardial Tissue Engineering with Cells Derived from Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold

Rationale: Conventional 3-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. Objective: Here, we used multiphoton-excited 3D printing to generate a native-like extracellular matrix scaffold with submicron resolution and then seeded the scaffold with cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human-induced pluripotent stem cells to generate a human-induced pluripotent stem cell–derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction. Methods and Results: The scaffold was seeded with ≈50 000 human-induced pluripotent stem cell–derived cardiomyocytes, smooth muscle cells, and endothelial cells (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced myocardial infarction, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. Conclusions: Thus, the novel multiphoton-excited 3D printing technique produces extracellular matrix–based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

Nonlinear Changes of Transmembrane Potential During Electrical Shocks Role of Membrane Electroporation

Abstract— Defibrillation shocks induce nonlinear changes of transmembrane potential (&Dgr;Vm) that determine the outcome of defibrillation. As shown earlier, strong shocks applied during action potential plateau cause nonmonotonic negative &Dgr;Vm, where an initial hyperpolarization is followed by Vm shift to a more positive level. The biphasic negative &Dgr;Vm can be attributable to (1) an inward ionic current or (2) membrane electroporation. These hypotheses were tested in cell cultures by measuring the effects of ionic channel blockers on &Dgr;Vm and measuring uptake of membrane-impermeable dye. Experiments were performed in cell strands (width ≈0.8 mm) produced using a technique of patterned cell growth. Uniform-field shocks were applied during the action potential plateau, and &Dgr;Vm was measured by optical mapping. Shock-induced negative &Dgr;Vm exhibited a biphasic shape starting at a shock strength of ≈15 V/cm when estimated peak &Dgr;V−m was ≈−180 mV; positive &Dgr;Vm remained monophasic. Application of a series of shocks with a strength of 23±1 V/cm resulted in uptake of membrane-impermeable dye propidium iodide. Dye uptake was restricted to the anodal side of strands with the largest negative &Dgr;Vm, indicating the occurrence of membrane electroporation at these locations. The occurrence of biphasic negative &Dgr;Vm was also paralleled with after-shock elevation of diastolic Vm. Inhibition of If and IK1 currents that are active at large negative potentials by CsCl and BaCl2, respectively, did not affect &Dgr;Vm, indicating that these currents were not responsible for biphasic &Dgr;Vm. These results provide evidence that the biphasic shape of &Dgr;Vm at sites of shock-induced hyperpolarization is caused by membrane electroporation.