Jan P. Kucera

Slow Conduction in Cardiac Tissue, I: Effects of a Reduction of Excitability Versus a Reduction of Electrical Coupling on Microconduction

It was the aim of this study to characterize the spread of activation at the cellular level in cardiac tissue during conduction slowing, a key element of reentrant arrhythmias; therefore, activation patterns were assessed at high spatiotemporal resolution in narrow (70 to 80 microm) and wide (230 to 270 microm) linear strands of cultured neonatal rat ventricular myocytes, using multiple site optical recording of transmembrane voltage. Slow conduction was induced by graded elevation of [K+]o, by applying tetrodotoxin, or by exposing the preparations to the gap junctional uncouplers palmitoleic acid or 1-octanol. The main findings of the study are 4-fold: (1) gap junctional uncoupling reduced conduction velocity (range, 37 to 47 cm/s under control conditions) to a substantially larger extent before block (</=1 cm/s; ultra-slow conduction) than did a reduction of excitability (range, approximately 10 to 15 cm/s); (2) activation wavefronts during uncoupling meandered within the boundaries of the preparations, resulting in a pronounced additional slowing of conduction in wide cell strands; (3) at the cellular level, propagation during uncoupling-induced ultra-slow conduction was sustained by sequentially activated tissue patches, each of which consisted of a few cells being activated simultaneously; and (4) depending on the uncoupler used, maximal action potential upstroke velocities during ultra-slow conduction were either slightly (palmitoleic acid) or highly (1-octanol) depressed. Thus, depolarizing inward currents, the spatial pattern and degree of gap junctional coupling, and geometrical factors all contribute in a concerted manner to conduction slowing, which, at its extreme (0.25 cm/s measured over 1 mm), can reach values low enough to permit, theoretically, reentrant excitation to occur in minuscule areas of cardiac tissue (<<1 mm2).

Localization of Sodium Channels in Intercalated Disks Modulates Cardiac Conduction

Abstract— It is well known that the sodium current (INa) and the degree of gap-junctional electrical coupling are the key determinants of action potential (AP) conduction in cardiac tissue. Immunohistochemical studies have shown that sodium channels (NaChs) are preferentially located in intercalated disks (IDs). Using dual immunocytochemical staining, we confirmed the colocalization of NaChs with connexin43 in cultures of neonatal rat ventricular myocytes. In mathematical simulations of conduction using the Luo-Rudy dynamic model of the ventricular AP, we assessed the hypothesis that conduction could be modulated by the preferential localization of NaChs in IDs. Localization of INa at the ID caused a large negative potential in the intercellular cleft, which influenced conduction in two opposing ways, depending on the degree of electrical coupling: (1) for normal and moderately reduced coupling, the negative cleft potential led to a large overshoot of the transmembrane potential resulting in a decreased driving force for INa itself (self-attenuation), which slowed conduction; (2) for greatly reduced coupling (<10%), the negative cleft potential induced by INa in the prejunctional membrane led to suprathreshold depolarization of the postjunctional membrane, which facilitated and accelerated conduction. When cleft potential effects were not incorporated, conduction was not significantly affected by the ID localization of INa. By enhancing conduction through the establishment of cleft potentials, the localization of NaChs in IDs might protect the myocardium from conduction block, very slow conduction, and microreentry under conditions of greatly reduced coupling. Conversely, by supporting moderately slow conduction, this mechanism could also promote arrhythmias.

Effects of Chronic Baroreceptor Stimulation on the Autonomic Cardiovascular Regulation in Patients With Drug-Resistant Arterial Hypertension

In patients with drug-resistant hypertension, chronic electric stimulation of the carotid baroreflex is an investigational therapy for blood pressure reduction. We hypothesized that changes in cardiac autonomic regulation can be demonstrated in response to chronic baroreceptor stimulation, and we analyzed the correlation with blood pressure changes. Twenty-one patients with drug-resistant hypertension were prospectively included in a substudy of the Device Based Therapy in Hypertension Trial. Heart rate variability and heart rate turbulence were analyzed using 24-hour ECG. Recordings were obtained 1 month after device implantation with the stimulator off and after 3 months of chronic electric stimulation (stimulator on). Chronic baroreceptor stimulation decreased office blood pressure from 185±31/109±24 mm Hg to 154±23/95±16 mm Hg (P<0.0001/P=0.002). Mean heart rate decreased from 81±11 to 76±10 beats per minute−1 (P=0.001). Heart rate variability frequency-domain parameters assessed using fast Fourier transformation (FFT; ratio of low frequency:high frequency: 2.78 versus 2.24 for off versus on; P<0.001) were significantly changed during stimulation of the carotid baroreceptor, and heart rate turbulence onset was significantly decreased (turbulence onset: −0.002 versus −0.015 for off versus on; P=0.004). In conclusion, chronic baroreceptor stimulation causes sustained changes in heart rate variability and heart rate turbulence that are consistent with inhibition of sympathetic activity and increase of parasympathetic activity in patients with drug-resistant systemic hypertension; these changes correlate with blood pressure reduction. Whether the autonomic modulation has favorable cardiovascular effects beyond blood pressure control should be investigated in further studies.

Impulse Propagation in Synthetic Strands of Neonatal Cardiac Myocytes With Genetically Reduced Levels of Connexin43

Abstract —Connexin43 (Cx43) is a major determinant of the electrical properties of the myocardium. Closure of gap junctions causes rapid slowing of propagation velocity (&thgr;), but the precise effect of a reduction in Cx43 levels due to genetic manipulation has only partially been clarified. In this study, morphological and electrical properties of synthetic strands of cultured neonatal ventricular myocytes from Cx43+/+ (wild type, WT) and Cx+/− (heterozygote, HZ) mice were compared. Quantitative immunofluorescence analysis of Cx43 demonstrated a 43% reduction of Cx43 expression in the HZ versus WT mice. Cell dimensions, connectivity, and alignment were independent of genotype. Measurement of electrical properties by microelectrodes and optical mapping showed no differences in action potential amplitude or minimum diastolic potential between WT and HZ. However, maximal upstroke velocity of the transmembrane action potential, dV/dtmax, was increased and action potential duration was reduced in HZ versus WT. &thgr; was similar in the two genotypes. Computer simulation of propagation and dV/dtmax showed a relatively small dependence of &thgr; on gap junction coupling, thus explaining the lack of observed differences in &thgr; between WT and HZ. Importantly, the simulations suggested that the difference in dV/dtmax is due to an upregulation of INa in HZ versus WT. Thus, heterozygote‐null mutation of Cx43 produces a complex electrical phenotype in synthetic strands that is characterized by both changes in ion channel function and cell‐to‐cell coupling. The lack of changes in &thgr; in this tissue is explained by the dominating role of myoplasmic resistance and the compensatory increase of dV/dtmax. (Circ Res. 2003;92:1209–1216.)

Ionic charge conservation and long-term steady state in the Luo-Rudy dynamic cell model.

It has been postulated that cardiac cell models accounting for changes in intracellular ion concentrations violate a conservation principle, and, as a result, computed parameters (e.g., ion concentrations and transmembrane potential, V(m)) drift in time, never attaining steady state. To address this issue, models have been proposed that invoke the charge conservation principle to calculate V(m) from ion concentrations (algebraic method), rather than from transmembrane current (differential method). The aims of this study are to compare model behavior during prolonged periods of pacing using the algebraic and differential methods, and to address the issue of model drift. We pace the Luo-Rudy dynamic model of a cardiac ventricular cell and compare the time-dependent behavior of computed parameters using the algebraic and differential methods. When ions carried by the stimulus current are taken into account, the algebraic and differential methods yield identical results and neither shows drift in computed parameters. The present study establishes the proper pacing protocol for simulation studies of cellular behavior during long periods of rapid pacing. Such studies are essential for mechanistic understanding of arrhythmogenesis, since cells are subjected to rapid periodic stimulation during many arrhythmias.

Slow Conduction in Cardiac Tissue, II: Effects of Branching Tissue Geometry

In cardiac tissue, functional or structural current-to-load mismatches can induce local slow conduction or conduction block, which are important determinants of reentrant arrhythmias. This study tested whether spatially repetitive mismatches result in a steady-state slowing of conduction. Patterned growth of neonatal rat heart cells in culture was used to design unbranched cell strands or strands releasing branches from either a single point or multiple points at periodic intervals. Electrical activation was followed optically using voltage-sensitive dyes under control conditions and in elevated [K+]o (5.8 and 14.8 mmol/L, respectively; in the latter case, propagation was carried by the L-type Ca2+ current). Preparations with multiple branch points exhibited discontinuous and slow conduction that became slower with increasing branch length and/or decreasing inter-branch distance. Compared with unbranched strands, conduction was maximally slowed by 63% under control conditions (from 44.9+/-3.4 to 16.7+/-1.0 cm/s) and by 93% in elevated [K+]o (from 15.7+/-2.3 to 1.1+/-0.2 cm/s). Local activation delays induced at a single branch point were significantly larger than the delays per branch point in multiple branching structures. Also, selective inactivation of inward currents in the branches induced conduction blocks. These 2 observations pointed to a dual role of the branches in propagation: whereas they acted as current sinks for the approaching activation thus slowing conduction (pull effect), they supplied, once excited, depolarizing current supporting downstream activation (push effect). This pull and push action resulted in a slowing of conduction in which the safety was largely preserved by the push effect. Thus, branching microarchitectures might contribute to slow conduction in tissue with discontinuous geometry, such as infarct scars and the atrioventricular node.

Photolithographically defined deposition of attachment factors as a versatile method for patterning the growth of different cell types in culture

Spatially defined growth of cells in culture is a useful model for studies ranging from the characterization of cellular motility to the analysis of network behaviour in structurally defined ensembles of excitable cells. Current methodological approaches for obtaining patterned growth include sophisticated modifications of surface chemistry, stamping techniques and microfluidics. The implementation of most of these techniques requires the availability of highly specialized apparatus and some of the methods are specific for certain cell types and/or substrate materials. The goal of the present study was to develop a cell-patterning technique that can be implemented by any laboratory working with cell culture and that is highly adaptable in terms of cell types and substrate materials. The method is based on a photolithographic process that permits the patterned deposition of attachment factors of choice on surfaces previously coated with agar with a spatial resolution (maximal deviation from a straight line) of ±3xa0µm. Because agar efficiently prevents cell adhesion, patterned growth obtained with this technique displays virtually no off-pattern cell attachment. The method permitted the patterning of cardiomyocytes, fibroblasts and HeLa cells on either glass substrates or polymer-coated materials with a spatial resolution of a few micrometers.

Optical Recording System Based on a Fiber Optic Image Conduit: Assessment of Microscopic Activation Patterns in Cardiac Tissue

Optical recording of transmembrane voltage changes with the use of potentiometric dyes has opened the possibility of determining spatial patterns of electrical activity in excitable tissues. To follow such activation patterns on the cellular/subcellular level in heart cell cultures, a recording system was developed that features both high spatial resolution (4-200 microm) and high temporal resolution (uncertainty in the determination of delays between fast rising signals of +/-1 micros). Central to the system is a fiber optic image conduit consisting of 379 individual optical fibers. At one end the fibers are fused to form an input window that matches the size of the field of view of the microscope. At the other end, the fibers are loose, permitting a selectable subset to be connected to 80 discrete photodetectors. This design allows the sensitive area of the imager to be adapted to regions of interest in a given preparation, thus making optimal use of the limited number of detectors. Furthermore, by using a second fiber optic imager, individual photodetectors can be assigned to different optical ports, thus providing the means for fast and simultaneous dual-emission wavelength measurements. This feature permitted the elimination of motion artifacts arising from the myocytes without the use of contraction-suppressing drugs.

Power-Law Behavior of Beat-Rate Variability in Monolayer Cultures of Neonatal Rat Ventricular Myocytes

It is known that extracardiac factors (nervous, humoral, and hemodynamic) participate in the power-law behavior of heart-rate variability. To assess whether intrinsic properties of cardiac tissue might also be involved, beat-rate variability was studied in spontaneously beating cell cultures devoid of extracardiac influences. Extracellular electrograms were recorded from monolayer cultures of neonatal rat ventricular myocytes under stable incubating conditions for up to 9 hours. The beat-rate time series of these recordings were examined in terms of their Fourier spectra and their Hurst scaling exponents. A non-0 Hurst exponent was found in 21 of 22 preparations (0.29+/-0.09; range, 0.11 to 0.45), indicating the presence of fractal self-similarity in the beat-rate time series. The same preparations exhibited power-law behavior of the power spectra with a power-law exponent of -1.36+/-0.24 (range, -1.04 to -1.96) in the frequency range of 0.001 to 1 Hz. Furthermore, it was found that the power-law exponent was nonstationary over time. These results indicate that the power-law behavior of heart-rate variability is determined not only by extracardiac influences but also by components intrinsic to cardiac tissue. Furthermore, the presence of power-law behavior in monolayer cultures of cardiomyocytes suggests that beat-rate variability might be determined by the complex nonlinear dynamics of processes occurring at the level of the cellular network, eg, interactions among a large number of cell oscillators or metabolic regulatory systems.

Analyses of a novel SCN5A mutation (C1850S): conduction vs. repolarization disorder hypotheses in the Brugada syndrome.

AIMSnBrugada syndrome (BrS) is characterized by arrhythmias leading to sudden cardiac death. BrS is caused, in part, by mutations in the SCN5A gene, which encodes the sodium channel alpha-subunit Na(v)1.5. Here, we aimed to characterize the biophysical properties and consequences of a novel BrS SCN5A mutation.nnnMETHODS AND RESULTSnSCN5A was screened for mutations in a male patient with type-1 BrS pattern ECG. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in HEK293 cells. Sodium currents (I(Na)) were analysed using the whole-cell patch-clamp technique at 37 degrees C. The electrophysiological effects of the mutation were simulated using the Luo-Rudy model, into which the transient outward current (I(to)) was incorporated. A new mutation (C1850S) was identified in the Na(v)1.5 C-terminal domain. In HEK293 cells, mutant I(Na) density was decreased by 62% at -20 mV. Inactivation of mutant I(Na) was accelerated in a voltage-dependent manner and the steady-state inactivation curve was shifted by 11.6 mV towards negative potentials. No change was observed regarding activation characteristics. Altogether, these biophysical alterations decreased the availability of I(Na). In the simulations, the I(to) density necessary to precipitate repolarization differed minimally between the two genotypes. In contrast, the mutation greatly affected conduction across a structural heterogeneity and precipitated conduction block.nnnCONCLUSIONnOur data confirm that mutations of the C-terminal domain of Na(v)1.5 alter the inactivation of the channel and support the notion that conduction alterations may play a significant role in the pathogenesis of BrS.