Nonlinear changes of transmembrane potential caused by defibrillation shocks in strands of cultured myocytes

Organization of cardiac tissue into cell strands and layers has been implicated in changes of transmembrane potential (DeltaV(m)) during defibrillation. To determine the shock-induced DeltaV(m) in such structures, cell strands of variable width [strand width (SW) = 0.15-2 mm] were grown in culture. Uniform-field shocks with variable strength [shock strength (SS) = 2-50 V/cm] were applied across strands during the action potential (AP) plateau, and DeltaV(m) were measured optically. Three different types of DeltaV(m) were observed. Small DeltaV(m) [<40%AP amplitude (APA)] were linearly dependent on SS and SW and were symmetrically distributed about a strand centerline with maximal positive and negative DeltaV(m) on opposite strand sides being equal. Intermediate DeltaV(m) (<200%APA) were strongly asymmetric with negative DeltaV(m) > positive DeltaV(m) because of a negative time-dependent shift of V(m) at the depolarized side of the strands. For large DeltaV(m) (>200%APA), a second time-dependent shift of V(m) to more positive levels was observed in the hyperpolarized portions of strands, causing reduction of the DeltaV(m) asymmetry. We conclude that during application of shocks to cell strands during the AP plateau, passive changes of V(m) were followed by two voltage- and time-dependent shifts of V(m), possibly reflecting changes of ionic currents or membrane electroporation.     

Scaling Up ITO‐Free Solar Cells

Indium‐tin‐oxide‐free (ITO‐free) polymer solar cells with composite electrodes containing current‐collecting grids and a semitransparent poly(3,4‐ethylenedioxythiophene):polystyrenesulfonate) (PEDOT:PSS) conductor are demonstrated. The up‐scaling of the length of the solar cell from 1 to 6 cm and the effect of the grid line resistance are explored for a series of devices. Laser‐beam‐induced current (LBIC) mapping is used for quality control of the devices. A theoretical modeling study is presented that enables the identification of the most rational cell dimension for the grids with different resistances. The performance of ITO‐free organic solar cells with different dimensions and different electrode resistances are evaluated for different light intensities. The current generation and electric potential distribution are found to not be uniformly distributed in large‐area devices at simulated 1 Sun illumination. The generated current uniformity increases with decreasing light intensities.     

Chamber properties from transmitral flow: prediction of average and passive left ventricular diastolic stiffness.

A chamber stiffness (K(LV))-transmitral flow (E-wave) deceleration time relation has been invasively validated in dogs with the use of average stiffness [(DeltaP/DeltaV)(avg)]. K(LV) is equivalent to k(E), the (E-wave) stiffness of the parameterized diastolic filling model. Prediction and validation of 1) (DeltaP/DeltaV)(avg) in terms of k(E), 2) early rapid-filling stiffness [(DeltaP/DeltaV)(E)] in terms of k(E), and 3) passive (postdiastasis) chamber stiffness [(DeltaP/DeltaV)(PD)] from A waves in terms of the stiffness parameter for the Doppler A wave (k(A)) have not been achieved. Simultaneous micromanometric left ventricular (LV) pressure (LVP) and transmitral flow from 131 subjects were analyzed. (DeltaP)(avg) and (DeltaV)(avg) utilized the minimum LVP-LV end-diastolic pressure interval. (DeltaP/DeltaV)(E) utilized DeltaP and DeltaV from minimum LVP to E-wave termination. (DeltaP/DeltaV)(PD) utilized atrial systolic DeltaP and DeltaV. E- and A-wave analysis generated k(E) and k(A). For all subjects, noninvasive-invasive relations yielded the following equations: k(E) = 1,401. (DeltaP/DeltaV)(avg) + 59.2 (r = 0.84) and k(E) = 229.0. (DeltaP/DeltaV)(E) + 112 (r = 0.80). For subjects with diastasis (n = 113), k(A) = 1,640. (DeltaP/DeltaV)(PD) - 8.40 (r = 0.89). As predicted, k(A) showed excellent correlation with (DeltaP/DeltaV)(PD); k(E) correlated highly with (DeltaP/DeltaV)(avg). In vivo validation of average, early, and passive chamber stiffness facilitates quantitative, noninvasive diastolic function assessment from transmitral flow.     

Noninvasive assessment of local myocardium repolarization changes using high resolution surface ECG mapping

A method using body surface potential maps for assessment of myocardium lesions with changed repolarization is presented and suitable mapping system is introduced. Differences between normal and altered QRST integral maps together with torso volume conductor model were used to determine the equivalent dipole representing the lesion. Performance of the method was studied on simulated data. Changed repolarization was modeled by shortening of myocyte action potentials in regions typical for stenosis of the main coronary arteries. The equivalent dipole estimated the positions of small lesions with a mean error of 9+/-4 mm (17+/-14 mm for larger transmural lesions). The subepicardial or subendocardial character of the lesions was reflected in the dipole orientation. Tests of the method on patients after myocardial infarction that underwent coronary intervention on a single coronary vessel showed that in 7 of 8 successfully treated patients the dipole position matched well with the treated vessel. A small dipole moment in another patient indicated unsuccessful treatment. The method was implemented in a new 128-channel mapping system. Its active electrodes, battery powered measuring unit and optical computer interface help to minimize noise in ECG and guarantee patient's safety. The results suggest that the method and mapping system offer useful tools for noninvasive identification of local repolarization changes in the myocardium.     

Roles of electric field and fiber structure in cardiac electric stimulation.

This study investigated roles of the variation of extracellular voltage gradient (VG) over space and cardiac fibers in production of transmembrane voltage changes (DeltaV(m)) during shocks. Eleven isolated rabbit hearts were arterially perfused with solution containing V(m)-sensitive fluorescent dye (di-4-ANEPPS). The epicardium received shocks from symmetrical or asymmetrical electrodes to produce nominally uniform or nonuniform VGs. Extracellular electric field and DeltaV(m) produced by shocks in the absolute refractory period were measured with electrodes and a laser scanner and were simulated with a bidomain computer model that incorporated the anterior left ventricular epicardial fiber field. Measurements and simulations showed that fibers distorted extracellular voltages and influenced the DeltaV(m). For both uniform and nonuniform shocks, DeltaV(m) depended primarily on second spatial derivatives of extracellular voltages, whereas the VGs played a smaller role. Thus, 1) fiber structure influences the extracellular electric field and the distribution of DeltaV(m); 2) the DeltaV(m) depend on second spatial derivatives of extracellular voltage.     

Current Collecting Grids for ITO‐Free Solar Cells

Indium‐tin‐oxide (ITO) free polymer solar cells prepared by ink jet printing a composite front electrode comprising silver grid lines and a semitransparent PEDOT:PSS conductor are demonstrated. The effect of grid line density is explored for a large series of devices and a careful modeling study enabling the identification of the most rational grid structure is presented. Both optical and light beam induced current (LBIC) mapping of the devices are used to support the power loss model and to follow the evolution of the performance over time. Current generation is found to be evenly distributed over the active area initially progressing to a larger graduation in areas with different performance. Over time coating defects also become much more apparent in the LBIC images.     

Mapping action potentials and calcium transients simultaneously from the intact heart

Intracellular calcium handling plays an important role in cardiac electrophysiology. Using two fluorescent indicators, we developed an optical mapping system that is capable of measuring calcium transients and action potentials at 256 recording sites simultaneously from the intact guinea pig heart. On the basis of in vitro measurements of dye excitation and emission spectra, excitation and emission filters at 515 +/- 5 and >695 nm, respectively, were used to measure action potentials with di-4-ANEPPS, and excitation and emission filters at 365 +/- 25 and 485 +/- 5 nm, respectively, were used to measure calcium transients with indo 1. The percent error due to spectral overlap was small when action potentials were measured (1.7 +/- 1.0%, n = 3) and negligible when calcium transients were measured (0%, n = 3). Recordings of calcium transients, action potentials, and isochrone maps of depolarization time and the time of calcium transient onset indicated negligible error due to fluorescence emission overlap. These data demonstrate that the error due to spectral overlap of indo 1 and di-4-ANEPPS is sufficiently small, such that optical mapping techniques can be used to measure calcium transients and action potentials simultaneously in the intact heart.     

Extracellular HCO(3)(-) dependence of electrogenic Na/HCO(3) cotransporters cloned from salamander and rat kidney

We studied the extracellular [HCOabstract (3) (-)] dependence of two renal clones of the electrogenic Na/HCO(3) cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (DeltaV(m)) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (DeltaI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract (3 )(-)/CO(2) (0.33-99 mM HCOabstract (3)(-), pH(o) 7.5) elicited an immediate, DIDS (4, 4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na(+)-dependent hyperpolarization (or outward current). In DeltaV(m) experiments, the apparent K(m ) for HCOabstract (3)(-) of akNBC (10. 6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCOabstract (3)(-) of rkNBC was less (6.5 mM). Because it has been reported that SOabstract (3)(=)/HSO abstract (3)(-) stimulates Na/HCO(3 ) cotransport in renal membrane vesicles (a result that supports the existence of a COabstract (3)(=) binding site with which SOabstract (3)(=) interacts), we examined the effect of SOabstract (3)(=)/HSO abstract (3)(-) on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract (4)(=) nor 33 mM SOabstract (3) (=)/HSOabstract (3)(-) substantially affects the apparent K(m) for HCO abstract (3)(-). We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract (3 )(-)/0.5% CO(2). We found that SO abstract (3)(=)/HSOabstract (3 )(-) did not significantly affect the DIDS-sensitive component of the pH(i) recovery from the initial CO(2 )-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO(2)](o), pH(o), and [COabstract (3)(=)](o) at a fixed [HCOabstract (3)(-)](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract (3)(=)](o ). However, a pH titration curve nicely fitted the data expressed as current versus pH(o). Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract (3 )(=), HSOabstract (3)(-), or COabstract (3)(=).     

Packing properties of 1-alkanols and alkanes in a phospholipid membrane

We have used vibrating tube densitometry to investigate the packing properties of four alkanes and a homologous series of ten alcohols in fluid-phase membranes of dimyristoyl phosphatidylcholine (DMPC). It was found that the volume change of transferring these compounds from their pure states into the membrane, DeltaV(m)(pure-->mem), was positive for small (C4-C6) 1-alkanols while it was negative for larger alcohols and all alkanes. The magnitude of DeltaV(m)(pure-->mem) ranged from about +4 cm3/mol for alcohols with an alkyl chain about half the length of the fatty acids of DMPC, to -10 to -15 cm3/mol for the alkanes and long chain alcohols. On the basis of these observations, previously published information on the structure of the membrane-solute complexes and the free volume properties of (pure) phospholipid membranes, we suggest that two effects dominate the packing properties of hydrophobic solutes in DMPC. First, perturbation of the tightly packed interfacial zone around the ester bonds and first few methylene groups of DMPC brings about a positive contribution to DeltaV(m)(pure-->mem). This effect dominates the volume behavior for alcohols like 1-butanol, 1-pentanol and 1-hexanol. More hydrophobic solutes penetrate into the membrane core, which is loosely packed. In this region, they partially occupy interstitial (or free-) volume, which bring about a denser molecular packing and generate a negative contribution to DeltaV(m)(pure-->mem).     

Multipole representations of current generators in a volume conductor

As far as the potential distribution outside the current generators is concerned, any current source distribution may be replaced by a suitable collection of multipoles. If these current generators lie close to the geometrical center of the volume conductor, a central dipole is a good approximation for potentials at surface points which are at considerable distances from the center. For better accuracy and for points close to the center, additional singularities such as a central quadrupole, a central octopole, etc., should be included. Potential expressions due to such multipoles in a spherical conductor can be obtained in closed forms by means of the “interior sphere theorem”. This paper presents a method for determining successively better multipole representations of the current generators in a homogeneous conducting sphere by measuring surface potentials at a successively increasing number of points. It is shown that Einthoven's triangle and Wilson's tetrahedron in the theory of electrocardiography are first and second approximations of this method. This concept also applies to conductors of other shapes. This investigation was supported by The National Heart Institute under a research grant H-2263(c).     

THE DISTRIBUTION OF THE ACTION CURRENTS PRODUCED BY HEART MUSCLE AND OTHER EXCITABLE TISSUES IMMERSED IN EXTENSIVE CONDUCTING MEDIA

The action currents produced by heart muscle and other tissues immersed in or in contact with a large body of conducting material are distributed in accordance with the laws that govern the flow of electric currents in volume conductors. The curve obtained when one electrode (the exploring electrode) is placed very close to and the other (the indifferent electrode) very far from the active tissue may be regarded as representing the potential variations of the exploring electrode alone; the. potential of the indifferent electrode is by comparison nearly constant. Curves obtained by this method of leading from the surface of the mammalian auricle indicate that the electrical effects produced by the passage of the excitation wave along a single muscle fiber are nearly the same as those that would occur if the crest of this wave were immediately preceded by a source and followed by a sink. A study of the electric field of a polarized membrane immersed in a volume conductor shows that this conclusion may be derived on theoretical grounds from the membrane theory of Bernstein.     

A novel hydrogen peroxide biosensor based on the immobilization of hemoglobin on three-dimensionally ordered macroporous (3DOM) gold-nanoparticle-doped titanium dioxide (GTD) film

The three-dimensionally ordered macroporous gold-nanoparticle-doped titanium dioxide (3DOM GTD) film was modified on the indium-tin oxide (ITO) electrode surface. Hemoglobin (Hb) has been successfully immobilized on the 3DOM GTD film and the fabrication process was characterized by Raman and UV-vis spectra. The results indicated that the Hb immobilized on the film retained its biological activity and the secondary structure of Hb was not destroyed. The direct electrochemistry and electrocatalysis of Hb immobilized on this film have been investigated. The Hb/3DOM GTD/ITO electrode exhibited two couples of redox peaks corresponding to the Hb intercalated in the mesopores and adsorbed on the external surface of the film with the formal potential of -0.20 and -0.48 V in 0.1M PBS (pH7.0), respectively. The Hb/3DOM GTD/ITO electrode exhibits an excellent eletrocatalytic activity, a wide linear range for H(2)O(2) from 5.0 μM to 1.0mM with a limit of detection of 0.6μM, high sensitivity (144.5 μA mM(-1)), good stability and reproducibility. Compared with the TiO(2) nanoneedles modified electrode, the GTD modified electrode has higher sensitivity and response peak current. The 3DOM GTD provided a good matrix for bioactive molecules immobilization, suggesting it has the potential use in the fields of H(2)O(2) biosensors.     

Mitochondrial creatine kinase binding to phospholipids decreases fluidity of membranes and promotes new lipid-induced beta structures as monitored by red edge excitation shift, laurdan fluorescence, and FTIR

Structural modifications induced by the binding of mitochondrial creatine kinase (mtCK) to saturated and unsaturated phospholipids were monitored by using Laurdan, a membrane probe sensitive to the polarity of the environment. The abrupt change characteristic of a phase transition of lipids alone was attenuated by addition of mtCK. Generalized polarization spectra indicated that mtCK surface binding changed the phospholipid liquid-crystalline state to a more rigid state. Infrared spectra of lipids further strengthened these results: upon mtCK binding, the phospholipid methylene chains had a more rigid conformation than that observed without mtCK at the same temperature. After mtCK binding to vesicles of perdeuterated dimyristoylphosphatidylcholine and nondeuterated dimyristoylphosphatidylglycerol, no lateral phase separation was observed, suggesting that both lipids were rigidified. Moreover, mtCK bound to liposomes exhibited an uncommon red edge excitation shift of 19 nm, while that of the soluble enzyme was only 6 nm. These results indicated that the environment of some mtCK tryptophan residues was motionally restricted. Strong stabilization of the enzyme structure against heat denaturation was observed upon lipid binding. In addition, lipids promoted a new reversible protein-protein or protein-lipid interaction, as evidenced by infrared data showing a slight modification of the beta sheet over alpha helix ratio with formation of a new 1632-cm(-)(1) beta sheet instead of the soluble protein 1636-cm(-)(1) one. Such modifications, inducing a decrease in the fluidity of the mitochondrial membranes, may play a role in vesicle aggregation; they could be implicated in the appearance of contact sites between internal and external mitochondrial membranes.     

Time-resolved absorption and photothermal measurements with recombinant sensory rhodopsin II from Natronobacterium pharaonis

Purified wild-type sensory rhodopsin II from Natronobacterium pharaonis (pSRII-WT) and its histidine-tagged analog (pSRII-His) were studied by laser-induced optoacoustic spectroscopy (LIOAS) and flash photolysis with optical detection. The samples were either dissolved in detergent or reconstituted into polar lipids from purple membrane (PML). The quantum yield for the formation of the long-lived state M(400) was determined as Phi(M) = 0.5 +/- 0.06 for both proteins. The structural volume change accompanying the production of K(510) as determined with LIOAS was DeltaV(R,1) /= Phi(M), indicating that the His tag does not influence this early step of the photocycle. The medium has no influence on DeltaV(R,1), which is the largest so far measured for a retinal protein in this time range (<10 ns). This confirms the occurrence of conformational movements in pSRII for this step, as previously suggested by Fourier transform infrared spectroscopy. On the contrary, the decay of K(510) is an expansion in the detergent-dissolved sample and a contraction in PML. Assuming an efficiency of 1.0, DeltaV(R,2) = -3 ml/mol for pSRII-WT and -4.6 ml/mol for pSRII-His were calculated in PML, indicative of a small structural difference between the two proteins. The energy content of K(510) is also affected by the tag. It is E(K) = (88 +/- 13) for pSRII-WT and (134 +/- 11) kJ/mol for pSRII-His. A slight difference in the activation parameters for K(510) decay confirms an influence of the C-terminal His on this step. At variance with DeltaV(R,1), the opposite sign of DeltaV(R,2) in detergent and PML suggests the occurrence of solvation effects on the decay of K(510), which are probably due to a different interaction of the active site with the two dissolving media.     

On Bioelectric Potentials in an Inhomogeneous Volume Conductor

Green's theorem is used to derive two sets of expressions for the quasi-static potential distribution in an inhomogeneous volume conductor. The current density in passive regions is assumed to be linearly related instantaneously to the electric field. Two equations are derived relating potentials to an arbitrary distribution of impressed currents. In one, surfaces of discontinuity in electrical conductivity are replaced by double layers and in the other, by surface charges. A multipole equivalent generator is defined and related both to the potential distribution on the outer surface of the volume conductor and to the current sources. An alternative result involves the electric field at the outer surface rather than the potential. Finally, the impressed currents are related to electrical activity at the membranes of active cells. The normal component of membrane current density is assumed to be equal at both membrane surfaces. One expression is obtained involving the potentials at the inner and outer surfaces of the membrane. A second expression involves the transmembrane potential and the normal component of membrane current.     

Refractoriness of cardiac muscle as affected by intercalated disks: a model study implications for fibrillation and defibrillation

It is generally agreed that inhomogeneities of the recovery process in cardiac tissue play an important role in the genesis of reentrant arrhythmias. Regarding cardiac muscle as an assembly of discrete cells connected by gap junctions, differences in recovery may result from a nonuniformity of membrane or cable properties. In this study, a computer model of a one-dimensional cardiac muscle fiber including a periodic intercalated disk structure is used to study the influence of disk resistance (Rj) and stimulus strength (J) on refractoriness. Stimulating currents are applied externally in a bipolar arrangement. The basic effect of a current pulse is local de- and hyperpolarizations at the ends of an individual cell. Polarization develops very rapidly and increases with increasing values of Rj or J so that an interaction with membrane current kinetics becomes possible. When a premature stimulus is applied during repolarization of a conditioning action potential, multiple Na currents can occur, either caused by depolarization of the cathodal end of a cell or in the form of anode break excitation at the hyperpolarized end. Those currents affect the response of a fiber such that, at a given value of J, the refractory period is shortened by an increase in Rj. In a ring fiber model with different Rj values in the two halves of ring an extrastimulus timed between the refractory periods of the two branches results in a sustained circus movement. Varying stimulus strength yields an upper limit of vulnerability characterized by a "synchronized extrasystole". The ring model also implies the suppression of circus movement by an external shock. The minimal shock strength required for suppression is close to the upper limit of vulnerability. The simulations suggest that discrete effects of junctional resistance may be involved in fibrillation and defibrillation.     

Volumetric behaviour of maltose-water, maltose-glycerol and starch-sorbitol-water systems mixtures in relation to structural relaxation

The densities of amorphous maltose-water, maltose-glycerol and starch-sorbitol-water mixtures were measured using a vibrating-tube density meter and pycnometry. The volumetric change on mixing was investigated through the calculation of the quantity DeltaV/V, the difference between (experimental) volume of the mixture and the linear composition weighted pure constituent volumes (ideal mixing). For all of the systems studied the quantity DeltaV/V was negative and approached a minimum of -0.03 and -0.015 at mass fractions of maltose in the region of 0.75 and 0.85 for the maltose-water and maltose-glycerol mixtures, respectively. Results are discussed in the context of volume change due to structural relaxation of vitreous materials and related to the phenomenon of antiplasticisation.     

A mathematical analysis of the action potential plateau duration of a human ventricular myocyte

The plateau phase of a human ventricular myocyte is analysed. The plateau duration is a function of the time required for a myocyte's transmembrane voltage to decrease by a certain voltage, DeltaV. The timing of the plateau is shown to be controlled by two slowly changing gate variables, the inactivation gate that controls the inward/depolarizing L-type calcium current and the inactivation gate that controls the outward/repolarizing slow rectifier potassium current. The amount of current controlled by these variables is a function of the net conductivity of the corresponding sodium and potassium channels. An equation is derived that relates action potential duration to these net conductivities and the time dependence of the slowly moving variables. This equation is used to estimate plateau duration for a given value of DeltaV. The initial conditions of the slowly moving inactivation variables are shown to affect plateau duration. These initial conditions depend on the amount of time that has elapsed between a previous repolarization and a current depolarization (diastolic interval). The analysis thus helps to quantify the characteristics of action potential duration restitution.     

Identification of transcription elements in the 5' intergenic region shared by LON and MDJ1 heat shock genes from the human pathogen Paracoccidioides brasiliensis. Evaluation of gene expression

The MDJ1/LON locus is conserved among pathogenic dimorphic fungi. We have mapped using DNase I footprinting and mobility shift assays three putative heat shock elements and one AP-1 binding domain (ARE) in the 5' intergenic region shared by PbMDJ1and PbLON (ML) from Paracoccidioides brasiliensis. The region bearing an ARE-like towards PbLON also has an opposite skn-1-like element. We studied genetically and pathogenically distinct isolates Pb18 and Pb3, where ML is polymorphic and the number of elements detected was higher. The functionality of the elements was suggested by the stimulatory response of both genes to heat shock and oxidative stress. Co-regulation occurred upon heat shock from 36 to 42 degrees C and, only in Pb3, also during mycelium to yeast transformation (26-36 degrees C). In Pb18, PbMDJ1 seemed to be preferentially expressed in yeast. Our study might help understand regulation of genes involved in fungal adaptation to the host.     

The role of vision and proprioception in the aversion of rats to the open arms of an elevated plus-maze

The elevated plus-maze test is usually run with a short edge surrounding the open arms in order to prevent the rats from falling. The present experiment investigated the role of transparent edges differing in heights: 1 (used as control), 5, 10, 20 and 40 cm, the latter the same height as the closed arm walls. Additionally, this 40-cm high transparent edge was also studied covered by white translucent or black opaque paper. The data show that the time spent in the open arms was significantly greater when the edge height was 5, 10 or 40 cm covered by the white or black paper. However, there were no differences from the 1-cm control edge when the height was 40 cm transparent. A similar effect was observed when entries in the open arms and total entries were analyzed. The facts that there were no differences when the open arms were surrounded by 1- or 40-cm transparent edges (which allow thigmotaxis) and that the same 40-cm edge caused increases in exploratory behavior when covered by papers indicate that vision triggers aversion to open spaces.