Examination of stimulation mechanism and strength-interval curve in cardiac tissue

Understanding the basic mechanisms of excitability through the cardiac cycle is critical to both the development of new implantable cardiac stimulators and improvement of the pacing protocol. Although numerous works have examined excitability in different phases of the cardiac cycle, no systematic experimental research has been conducted to elucidate the correlation among the virtual electrode polarization pattern, stimulation mechanism, and excitability under unipolar cathodal and anodal stimulation. We used a high-resolution imaging system to study the spatial and temporal stimulation patterns in 20 Langendorff-perfused rabbit hearts. The potential-sensitive dye di-4-ANEPPS was utilized to record the electrical activity using epifluorescence. We delivered S1-S2 unipolar point stimuli with durations of 2-20 ms. The anodal S-I curves displayed a more complex shape in comparison with the cathodal curves. The descent from refractoriness for anodal stimulation was extremely steep, and a local minimum was clearly observed. The subsequent ascending limb had either a dome-shaped maximum or was flattened, appearing as a plateau. The cathodal S-I curves were smoother, closer to a hyperbolic shape. The transition of the stimulation mechanism from break to make always coincided with the final descending phase of both anodal and cathodal S-I curves. The transition is attributed to the bidomain properties of cardiac tissue. The effective refractory period was longer when negative stimuli were delivered than for positive stimulation. Our spatial and temporal analyses of the stimulation patterns near refractoriness show always an excitation mechanism mediated by damped wave propagation after S2 termination.     

A generalized activating function for predicting virtual electrodes in cardiac tissue.

To fully understand the mechanisms of defibrillation, it is critical to know how a given electrical stimulus causes membrane polarizations in cardiac tissue. We have extended the concept of the activating function, originally used to describe neuronal stimulation, to derive a new expression that identifies the sources that drive changes in transmembrane potential. Source terms, or virtual electrodes, consist of either second derivatives of extracellular potential weighted by intracellular conductivity or extracellular potential gradients weighted by derivatives of intracellular conductivity. The full response of passive tissue can be considered, in simple cases, to be a convolution of this "generalized activating function" with the impulse response of the tissue. Computer simulations of a two-dimensional sheet of passive myocardium under steady-state conditions demonstrate that this source term is useful for estimating the effects of applied electrical stimuli. The generalized activating function predicts oppositely polarized regions of tissue when unequally anisotropic tissue is point stimulated and a monopolar response when a point stimulus is applied to isotropic tissue. In the bulk of the myocardium, this new expression is helpful for understanding mechanisms by which virtual electrodes can be produced, such as the hypothetical "sawtooth" pattern of polarization, as well as polarization owing to regions of depressed conductivity, missing cells or clefts, changes in fiber diameter, or fiber curvature. In comparing solutions obtained with an assumed extracellular potential distribution to those with fully coupled intra- and extracellular domains, we find that the former provides a reliable estimate of the total solution. Thus the generalized activating function that we have derived provides a useful way of understanding virtual electrode effects in cardiac tissue.     

Interactions in vitro and in vivo between rat serum protease inhibitors and anodal and cathodal rat trypsin and chymotrypsin.

Reaction mixtures of increasing amounts of the pancreatic homologous proteases, anodal and cathodal chymotrypsin and trypsin, respectively, and normal rat serum were analyzed by immunoelectrophoretic methods in order to determine their distribution on serum protease inhibitors. This paper concerns three proteins occurring in normal serum and capable of binding protease viz. alpha1-macroglobulin, alpha1-antitrypsin and alpha1-inhibitor 3. The distribution of the enzymes among these protease inhibitors differed significantly from one protease to another. The distribution of the proteases among the serum protease inhibitors following intravenous injection of 125I-labelled proteases corresponded to that in vitro. Complexes formed with alpha1-macroglobulin and alpha1-inhibitor 3 were quickly eliminated irrespective of the enzyme species used, whereas those formed with alpha1-antitrypsin persisted much longer in the circulation.     

Effects of elevated extracellular potassium on the stimulation mechanism of diastolic cardiac tissue

During cardiac disturbances such as ischemia and hyperkalemia, the extracellular potassium ion concentration is elevated. This in turn changes the resting transmembrane potential and affects the excitability of cardiac tissue. To test the hypothesis that extracellular potassium elevation also alters the stimulation mechanism, we used optical fluorescence imaging to examine the mechanism of diastolic anodal unipolar stimulation of cardiac tissue under 4 mM (normal) and 8 mM (elevated) extracellular potassium. We present several visualization methods that are useful for distinguishing between anodal-make and anodal-break excitation. In the 4-mM situation, stimulation occurred by the make, or stimulus-onset, mechanism that involved propagation out of the virtual cathodes. For 8-mM extracellular potassium, the break or stimulus termination mechanism occurred with propagation out of the virtual anode. We conclude that elevated potassium, as might occur in myocardial ischemia, alters not only stimulation threshold but also the excitation mechanism for anodal stimulation.     

Cadherin-11 and cardiac fibrosis: A common target for a common pathology

Cardiac fibrosis represents an enormous health concern as it is prevalent in nearly every form of cardiovascular disease, the leading cause of death worldwide. Fibrosis is characterized by the activation of fibroblasts into myofibroblasts, a contractile cell type that secretes significant amounts of extracellular matrix components; however, the onset of this condition is also due to persistent inflammation and the cellular responses to a changing mechanical environment. In this review, we provide an overview of the pro-fibrotic, pro-inflammatory, and biomechanical mechanisms that lead to cardiac fibrosis in cardiovascular diseases. We then discuss cadherin-11, an intercellular adhesion protein present on both myofibroblasts and inflammatory cells, as a potential link for all three of the fibrotic mechanisms. Since experimentally blocking cadherin-11 dimerization prevents fibrotic diseases including cardiac fibrosis, understanding how this protein can be targeted for therapeutic use could lead to better treatments for patients with heart disease.     

Benzo[a]pyrene impairs beta-adrenergic stimulation of adipose tissue lipolysis and causes weight gain in mice. A novel molecular mechanism of toxicity for a common food pollutant

Benzo[a]pyrene (B[a]P) is a common food pollutant that causes DNA adduct formation and is carcinogenic. The report of a positive correlation between human plasma B[a]P levels and body mass index, together with B[a]P's lipophilicity, led us to test for possible adverse effects of B[a]P on adipose tissue. In ex vivo experiments using primary murine adipocytes, B[a]P rapidly (within minutes) and directly inhibited epinephrine-induced lipolysis (up to 75%) in a dose-dependent manner. Half-maximum inhibition was obtained with a B[a]P concentration of 0.9 mg.L(-1) (3.5 microm). Lipolysis induced by beta(1)-, beta(2)- and beta(3)-adrenoreceptor-specific agonists, as well as ACTH, were also significantly inhibited by B[a]P, whereas forskolin-induced lipolysis was not B[a]P-sensitive. Similar inhibition of catecholamine-induced lipolysis by B[a]P was also seen in isolated human adipocytes; half-maximum inhibition of lipolysis was achieved with a B[a]P concentration of 0.02 mg.L(-1) (0.08 microm). In vivo treatment of C57Bl/6J mice with 0.4 mg.kg(-1) B[a]P inhibited epinephrine-induced release of free fatty acids by 70%. Chronic exposure of mice to B[a]P (0.5 mg.kg(-1) injected i.p. every 48 h) for 15 days also decreased lipolytic response to epinephrine and induced a 43% higher weight gain compared with controls (B[a]P: 2.23 +/- 0.12 g versus control: 1.56 +/- 0.18 g, P < 0.01) due to increased fat mass. The weight gain occurred consistently without detectable changes in food intake. These results reveal a novel molecular mechanism of toxicity for the environmental pollutant B[a]P and introduce the notion that chronic exposure of human population to B[a]P and possibly other polycyclic aromatic hydrocarbons could have an impact on metabolic disorders, such as obesity.     

One-way blocks in cardiac tissue: A mechanism for propagation failure in purkinje fibres

The concept of a one-way block, arising from a region of depressed tissue, has remained central to theories for cardiac arrhythmias. We show that both the geometry of a depressed region and spatial heterogeneities in depression are key factors for inducing such a block. By using an asymptotic approximation, known as the eikonal equation, to model qualitatively the movement of a depolarization wave-front down a Purkinje fibre bundle, we show how a one-way block in conduction may result from asymmetric constriction in the width of a depressed bundle. We demonstrate that this theory is valid for biologically relevant parameters and simulate a one-way block by numerically solving the eikonal approximation. We consider the case of non-uniform depression, where the planar travelling wave speed is spatially dependent. Here, numerical simulations indicate that such a spatial dependency may, in itself, be sufficient to produce a one-way block.     

Pulsed low-energy stimulation initiates electric turbulence in cardiac tissue

Interruptions in nonlinear wave propagation, commonly referred to as wave breaks, are typical of many complex excitable systems. In the heart they lead to lethal rhythm disorders, the so-called arrhythmias, which are one of the main causes of sudden death in the industrialized world. Progress in the treatment and therapy of cardiac arrhythmias requires a detailed understanding of the triggers and dynamics of these wave breaks. In particular, two very important questions are: 1) What determines the potential of a wave break to initiate re-entry? and 2) How do these breaks evolve such that the system is able to maintain spatiotemporally chaotic electrical activity? Here we approach these questions numerically using optogenetics in an in silico model of human atrial tissue that has undergone chronic atrial fibrillation (cAF) remodelling. In the lesser studied sub-threshold illumination régime, we discover a new mechanism of wave break initiation in cardiac tissue that occurs for gentle slopes of the restitution characteristics. This mechanism involves the creation of conduction blocks through a combination of wavefront-waveback interaction, reshaping of the wave profile and heterogeneous recovery from the excitation of the spatially extended medium, leading to the creation of re-excitable windows for sustained re-entry. This finding is an important contribution to cardiac arrhythmia research as it identifies scenarios in which low-energy perturbations to cardiac rhythm can be potentially life-threatening.     

Artificial cardiac stimulation: a current view of physiologic pacemakers.

Artificial pacing of the heart has evolved rapidly over the last 20 years; the physician can now implant "physiologic" pacemakers that preserve the natural order of atrial and ventricular systole. The commonly used pacemakers that pace only the ventricle can induce dizziness, fatigue and syncope and increase congestive heart failure. Physiologic pacemakers can eliminate many of these side effects, but they are more expensive, can be less durable and may induce arrhythmias. Physiologic pacing can provide the greatest benefit and cost-effectiveness when the particular functions of the device are matched to the specific needs of the patient.     

Modification of a cylindrical bidomain model for cardiac tissue.

Previous models based on a cylindrical bidomain assumed either that the ratio of intracellular and interstitial conductivities in the principal directions were the same or that there was no radial variation in potential (i.e., a planar front, delta Vm/delta rho = 0). This paper presents a formulation and the expressions for the intracellular, interstitial, extracellular, and transmembrane potentials arising from nonplanar propagation along a cylindrical bundle of cardiac tissue represented as a bidomain with arbitrary anisotropy. For unequal anisotropy, the transmembrane current depends not only on the local change of the transmembrane potential but also on the nature of the transmembrane potential throughout the volume.     

Regulation of tissue plasminogen activator activity by cells. Domains responsible for binding and mechanism of stimulation

A number of cell types have previously been shown to bind tissue plasminogen activator (tPA), which in some cases can remain active on the cell surface resulting in enhanced plasminogen activation kinetics. We have investigated several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and shown that they bind both tPA and plasminogen and are able to act as promoters of plasminogen activation in kinetic assays. To understand what structural features of tPA are involved in cell surface interactions, we performed kinetic assays with a range of tPA domain deletion mutants consisting of full-length glycosylated and nonglycosylated tPA (F-G-K1-K2-P), DeltaFtPA (G-K1-K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P). Deletion variants were made in Escherichia coli and were nonglycosylated. Plasminogen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum levels of stimulation up to 80-fold with full-length, glycosylated tPA. Stimulation for nonglycosylated full-length tPA dropped to 45-60% of this value. Loss of N-terminal domains as in DeltaFtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value. The protease domain alone was stimulated at very low levels of up to 2-fold. Thus, a number of different sites are involved in cell interactions especially within finger and kringle domains, which is similar to the regulation of tPA activity by fibrin. A model was developed to explain the mechanism of stimulation and compared with actual data collected with varying cell, plasminogen, or tPA concentrations and different tPA variants. Experimental data and model predictions were generally in good agreement and suggest that stimulation is well explained by the concentration of reactants by cells.     

X chromosome monosomy: a common mechanism for autoimmune diseases

The majority of human autoimmune diseases are characterized by female predominance. Although sex hormone influences have been suggested to explain this phenomenon, the mechanism remains unclear. In contrast to the role of hormones, it has been suggested, based on pilot data in primary biliary cirrhosis, that there is an elevation of monosomy X in autoimmune disease. Using peripheral white blood cells from women with systemic sclerosis (SSc), autoimmune thyroid disease (AITD), or healthy age-matched control women, we studied the presence of monosomy X rates using fluorescence in situ hybridization. We also performed dual-color fluorescence in situ hybridization analysis with a chromosome Y alpha-satellite probe to determine the presence of the Y chromosome in the monosomic cells. In subsets of patients and controls, we determined X monosomy rates in white blood cell subpopulations. The rates of monosomy X increased with age in all three populations. However, the rate of monosomy X was significantly higher in patients with SSc and AITD when compared with healthy women (6.2 +/- 0.3% and 4.3 +/- 0.3%, respectively, vs 2.9 +/- 0.2% in healthy women, p < 0.0001 in both comparisons). Importantly, X monosomy rate was more frequent in peripheral T and B lymphocytes than in the other blood cell populations, and there was no evidence for the presence of male fetal microchimerism. These data highlight the thesis that chromosome instability is common to women with SSc and AITD and that haploinsufficiency for X-linked genes may be a critical factor for the female predominance of autoimmune diseases.     

Action potential propagation in inhomogeneous cardiac tissue: safety factor considerations and ionic mechanism

Heterogeneity of myocardial structure and membrane excitability is accentuated by pathology and remodeling. In this study, a detailed model of the ventricular myocyte in a multicellular fiber was used to compute a location-dependent quantitative measure of conduction (safety factor, SF) and to determine the kinetics and contribution of sodium current (I(Na)) and L-type calcium current [I(Ca(L))] during conduction. We obtained the following results. 1) SF decreases sharply for propagation into regions of increased electrical load (tissue expansion, increased gap junction coupling, reduced excitability, hyperkalemia); it can be <1 locally (a value indicating conduction failure) and can recover beyond the transition region to resume propagation. 2) SF and propagation across inhomogeneities involve major contribution from I(Ca(L)). 3) Modulating I(Na) or I(Ca(L)) (by blocking agents or calcium overload) can cause unidirectional block in the inhomogeneous region. 4) Structural inhomogeneity causes local augmentation of I(Ca(L)) and suppression of I(Na) in a feedback fashion. 5) Propagation across regions of suppressed I(Na) is achieved via a I(Ca(L))-dependent mechanism. 6) Reduced intercellular coupling can effectively compensate for reduced SF caused by tissue expansion but not by reduced membrane excitability.     

154 Protein folding and binding funnels: a common driving force and a common mechanism

Under the free energy landscape theory, both the protein-folding and protein–ligand binding processes are driven by the decrease in total Gibbs free energy of the protein-solvent or protein–ligand-solvent system, which involves the non-complementary changes between the entropy and enthalpy, ultimately leading to a global free energy minimization of these thermodynamic systems (Ji & Liu, 2011; Liu et al., 2012; Yang, Ji & Liu, 2012). In the case of protein folding, the lowering of the system free energy coupled with the gradual reduction in conformational degree of freedom of the folding intermediates determines that the shape of the free energy landscape for protein folding must be funnel-like (Dill & Chan, 1997), rather than non-funneled shapes (Ben-Naim, 2012). In the funnel-like free energy landscape, protein folding can be viewed as going down the hill via multiple parallel routes from a vast majority of individual non-native states on surface outside the funnel to the native states located around the bottom of the funnel. The first stage of folding, i.e. the rapid hydrophobic collapse process, is driven by the solvent entropy maximization. Concretely, the water molecules squeeze and sequestrate the hydrophobic amino acid side chains within the interior of the folding intermediates while exposing the polar and electrostatically charged side chains on the intermediate surface so as to minimize the solvent-accessible surface area of the solute and thus, the minimal contacts between the folding intermediates and the water molecules. This will maximize the entropy of the solvent, thus contributing substantially to lowering of the system free energy due to an absolute advantage of the solvent in both quantity and mass (Yang, Ji & Liu, 2012). The resulting molten globule states (Ohgushi & Wada, 1983), within which a few transient secondary structural components and tertiary contacts have been formed but many native contacts or close residue–residue interactions has yet to form, need to be further sculptured into the native states. This is a relatively slow “bottleneck” process because the competitive interactions between protein residues within the folding intermediates and between residues and water molecules may repeat many rounds to accumulate a large enough number of stable noncovalent bonds capable of counteracting the conformational entropy loss of the intermediates, thus putting this bottleneck stage under the enthalpy control (i.e. negative enthalpy change), contributing further to the lowering of the system free energy. Although the protein–ligand association occurs around the rugged bottom of the free energy landscape, the exclusion of water from the binding interfaces and the formation of noncovalent bonds between the two partners can still lower the system free energy. In conjunction with the loss of the rotational and translational degrees of freedom of the two partners as well as the loss of the conformational entropy of the protein, these processes could merge, downwards expand, and further narrow the free energy wells within which the protein–ligand binding process takes place, thereby making them look like a funnel, which we term the binding funnel. In this funnel, the free energy downhill process follows a similar paradigm to the protein-folding process. For example, if the initial collisions/contacts occur between the properly complementary interfaces of the protein and ligand, a large amount of water molecules (which usually form a water network around the solute surface) will be displaced to suit the need for maximizing the solvent entropy. This process is similar to that of the hydrophobic collapse during protein folding, resulting in a loosely associated protein–ligand complex that needs also to be further adapted into a tight complex, i.e. the second step which is mainly driven by the negative enthalpy change through intermolecular competitive interactions to gradually accumulate the noncovalent bonds and ultimately, to stabilize the complex at a tightly bound state. Taken together, we conclude that whether in the protein-folding or in the protein–ligand binding process, both the entropy-driven first step and the enthalpy-driven second step contribute to the lowering of the system free energy, resulting in the funnel-like folding or binding free energy landscape.     

Early afterdepolarizations in cardiac myocytes: mechanism and rate dependence.

A model of the cardiac ventricular action potential that accounts for dynamic changes in ionic concentrations was used to study the mechanism, characteristics, and rate dependence of early after depolarizations (EADs). A simulation approach to the study of the effects of pharmacological agents on cellular processes was introduced. The simulation results are qualitatively consistent with experimental observations and help resolve contradictory conclusions in the literature regarding the mechanism of EADs. Our results demonstrate that: 1) the L-type calcium current, ICa, is necessary as a depolarizing charge carrier during an EAD; 2) recovery and reactivation of ICa is the mechanism of EAD formation, independent of the intervention used to induce the EADs (cesium, Bay K 8644, or isoproterenol were used in our simulations, following similar published experimental protocols); 3) high [Ca2+]i is not required for EADs to develop and calcium release by the sarcoplasmic reticulum does not occur during the EAD; 4) although the primary mechanism of EAD formation is recovery of ICa, other plateau currents can modulate EAD formation by affecting the balance of currents during a conditional phase before the EAD take-off; and 5) EADs are present at drive cycle lengths longer than 1000 ms. Because of the very long activation time constant of the delayed rectifier potassium current, IK, the activation gate of IK does not deactivate completely between consecutive stimuli at fast rates (drive cycle length < 1000 ms). As a result, IK plays a key role in determining the rate dependence of EADs.