A condition for setting off ectopic waves in computational models of excitable cells

The purpose of this paper is to study the stability of steady state solutions of the Monodomain model equipped with Luo-Rudy I kinetics. It is well established that re-entrant arrhythmias can be created in computational models of excitable cells. Such arrhythmias can be initiated by applying an external stimulus that interacts with a partially refractory region, and spawn breaking waves that can eventually generate extremely complex wave patterns commonly referred to as fibrillation. An ectopic wave is one possible stimulus that may initiate fibrillation. Physiologically, it is well known that ectopic waves exist, but the mechanism for initiating ectopic waves in a large collection of cells is poorly understood. In the present paper we consider computational models of collections of excitable cells in one and two spatial dimensions. The cells are modeled by Luo-Rudy I kinetics, and we assume that the spatial dynamics is governed by the Monodomain model. The mathematical analysis is carried out for a reduced model that is known to provide good approximations of the initial phase of solutions of the Luo-Rudy I model. A further simplification is also introduced to motivate and explain the results for the more complicated models. In the analysis the cells are divided into two regions; one region (N) consists of normal cells as model by the standard Luo-Rudy I model, and another region (A) where the cells are automatic in the sense that they would act as pacemaker cells if they where isolated from their surroundings. We let delta denote the spatial diffusion and a denote a characteristic length of the automatic region. It has previously been shown that reducing diffusion or increasing the automatic region enhances ectopic activity. Here we derive a condition for the transition from stable resting state to ectopic wave spread. Under suitable assumptions on the model we provide mathematical and computational arguments indicating that there is a constant eta such that a steady state solution of this system is stable whenever delta approximately > etaa(2), and unstable whenever delta approximately < etaa(2).     

Development and application of human virtual excitable tissues and organs: from premature birth to sudden cardiac death

The electrical activity of cardiac and uterine tissues has been reconstructed by detailed computer models in the form of virtual tissues. Virtual tissues are biophysically and anatomically detailed, and represent quantitatively predictive models of the physiological and pathophysiological behaviours of tissue within an isolated organ. The cell excitation properties are quantitatively reproduced by equations that describe the kinetics of a few dozen proteins. These equations are derived from experimental measurements of membrane potentials, ionic currents, fluxes, and concentrations. Some of the measurements were taken from human cells and human ion channel proteins expressed in non-human cells, but they were mostly taken from cells of other animal species. Data on tissue geometry and architecture are obtained from the diffusion tensor magnetic resonance imaging of ex vivo or post mortem tissue, and are used to compute the spread of current in the tissue. Cardiac virtual tissues are well established and reproduce normal and pathological patterns of cardiac excitation within the atria or ventricles of the human heart. They have been applied to increase the understanding of normal cardiac electrophysiology, to evaluate the candidate mechanisms for re-entrant arrhythmias that lead to sudden cardiac death, and to predict the tissue level effects of mutant or pharmacologically-modified ion channels. The human full-term virtual uterus is still in development. This virtual tissue reproduces the in vitro behaviour of uterine tissue biopsies, and provides possible mechanisms for premature labour.     

IGF-1和动态微环境对脂肪干细胞向心肌细胞分化作用的研究

体外组织工程模型中,生物化学和机械信号对心肌再生起着很重要的促进作用,对人胰岛素样生长因子(IGF-1)和三维动态微环境对脂肪干细胞向心肌细胞分化过程中的促进作用进行了研究．带有IGF-1基因的质粒整合到胶原-壳聚糖支架中,脂肪干细胞接种到整合质粒的支架内,未整合质粒的支架作为对照组,心肌细胞培养基作为分化培养基,转瓶生物反应器提供动态微环境．经2周分化培养后,检测质粒在支架内释放及表达情况、细胞在支架内的活性以及心肌功能性蛋白和基因的表达．结果表明：动态微环境能促进质粒DNA的释放和转染;IGF-1可促进脂肪干细胞在胶原-壳聚糖支架内增殖以及向心肌细胞分化;动态微环境可加强IGF-1的促增殖分化作用．因此,IGF-1和动态微环境能独立或相互促进脂肪干细胞在胶原-壳聚糖支架内活性,动态微环境还可强化IGF-1对脂肪干细胞的促分化作用．对体外构建工程化心肌组织进行心肌再生研究有着重要的指导意义．     

Myosin light chain replacement in the heart

Myosin-actin cross-bridge kinetics are an important determinant for cardiac systolic and diastolic function. We compared the effects of myosin light chain substitutions on the ability of the fibers to contract in response to calcium and in their ability to produce power. Transgenesis was used to effect essentially complete replacement of the target contractile protein isoform specifically in the heart. Atrial and ventricular fibers derived from the various transgenic (TG) lines were skinned, and the force-velocity relationships, unloaded shortening velocities, and Ca(2+)-stimulated Mg(2+)-ATPase activities were determined. Replacement with an ectopic isoform resulted in significant changes in cross-bridge cycling kinetics but without any overt effects on morbidity or mortality. To confirm that this result was not light chain specific, a modified alpha-myosin heavy chain isoform that resulted in significant changes in force development was also engineered. The animals appeared healthy and have normal lifespans, and the changes in force development did not result in significant remodeling or overt hypertrophy. We conclude that myosin light chains can control aspects of cross-bridge cycling and alter force development. The myosin heavy chain data also show that changes in the kinetics of force development and power output do not necessarily lead to activation of the hypertrophic response or significant cardiac remodeling.     

Are astroglial cells involved in morphine tolerance?

Morphine gives rise to a cascade of events in the nervous system affecting, among others, neurotransmitter metabolism. Tolerance develops for various effects shortly after administration of the drug. Also, physical dependence develops and can be demonstrated by precipitation of withdrawal reactions. Biochemical events in nervous tissue have been extensively studied during morphine treatment. This overview will focus upon brain protein metabolism since macromolecular events might be of importance for development of long-term effects, such as tolerance and physical dependence. Both dose-and time-dependent changes in brain protein synthesis and the syntheses of specific proteins have been demonstrated after morphine treatment, although methodological considerations are important. Different experimental models (animal and tissue culture models) are presented. It might be interesting to note that astroglial protein synthesis and the secretion of proteins to the extracellular medium are both changed after morphine treatment, these having been evaluated in astroglial enriched primary cultures and in brain tissue slices. The possibility is suggested that proteins released from astroglial cells participate in the communication with other cells, including via synaptic regions, and that such communication might be of significance in modifying the synaptic membranes during morphine intoxication.     

d4eBP acts downstream of both dTOR and dFoxo to modulate cardiac functional aging in Drosophila

dTOR (target of rapamycin) and dFoxo respond to changes in the nutritional environment to induce a broad range of responses in multiple tissue types. Both dTOR and dFoxo have been demonstrated to control the rate of age-related decline in cardiac function. Here, we show that the Eif4e-binding protein (d4eBP) is sufficient to protect long-term cardiac function against age-related decline and that up-regulation of dEif4e is sufficient to recapitulate the effects of high dTOR or insulin signaling. We also provide evidence that d4eBP acts tissue-autonomously and downstream of dTOR and dFoxo in the myocardium, where it enhances cardiac stress resistance and maintains normal heart rate and myogenic rhythm. Another effector of dTOR and insulin signaling, dS6K , may influence cardiac aging nonautonomously through its activity in the insulin-producing cells, possibly by regulating dilp2 expression. Thus, elevating d4eBP activity in cardiac tissue represents an effective organ-specific means for slowing or reversing cardiac functional changes brought about by normal aging.     

Cell–cell junction remodeling in the heart: Possible role in cardiac conduction system function and arrhythmias?

Anchoring cell-cell junctions (desmosomes, fascia adherens) play crucial roles in maintaining mechanical integrity of cardiac muscle cells and tissue. Genetic mutations and/or loss of critical components in these macromolecular structures are increasingly being associated with arrhythmogenic cardiomyopathies; however, their specific roles have been primarily attributed to effects within the working (ventricular) cardiac muscle. Growing evidence also points to a key role for anchoring cell-cell junction components in cardiac muscle cells of the cardiac conduction system. This is not only evidenced by the molecular and ultra-structural presence of anchoring cell junctions in specific compartments/structures of the cardiac conduction system (sinoatrial node, atrioventricular node, His-Purkinje system), but also because conduction system-related arrhythmias can be found in humans and mouse models of cardiomyopathies harboring defects and/or mutations in key anchoring cell-cell junction proteins. These studies emphasize the clinical need to understand the molecular and cellular role(s) for anchoring cell-cell junctions in cardiac conduction system function and arrhythmias. This review will focus on (i) experimental findings that underline an important role for anchoring cell-cell junctions in the cardiac conduction system, (ii) insights regarding involvement of these structures in age-related cardiac remodeling of the conduction system, (iii) summarizing available genetic mouse models that can target cardiac conduction system structures and (iv) implications of these findings on future therapies for arrhythmogenic heart diseases.     

Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression

The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin.     

Sensing scaffolds to monitor cellular activity using impedance measurements

Scaffolds are cell adhesive matrices for the realisation of tissue constructs. Here we describe how scaffolds for tissue engineering can also be used as sensors for monitoring cellular activity such as adhesion and spreading. Carbon nanotube polymer composites were fabricated into membranes and scaffolds with electro-conductive properties. Impedance techniques were used to measure the effects of media and cell cultures on composite membranes and the results were analysed using lumped parameter models. We show that protein adhesion can be distinguished from cell adhesion as the impedance changes are much smaller for the latter (5%). In the presence of cells, impedance changes are of the order of 40% and can be correlated with adhesion, spreading and changes in cell density.     

Platelet-activating factor and cardiac diseases: therapeutic potential for PAF inhibitors

Platelet-activating factor (PAF) is a potent phospholipid mediator released from inflammatory cells in response to diverse immunologic and non-immunologic stimuli. Animal studies have implicated PAF as a major mediator involved in coronary artery constriction, modulation of myocardial contractility and the generation of arrhythmias which may bear on cardiac disorders such as ischemia, infarction and sudden cardiac death. PAF effects are induced by direct actions of PAF on cardiac tissue to modify chronotropic and inotropic activity, or indirectly via the release of eicosanoids such as thromboxane A₂ (TXA,), leukotrienes (LT) or cytokines (TNFx). The development of selective, high affinity PAF receptor antagonists has permitted investigations on the role of PAF in experimental animal models of cardiac injury. In vivo and in vitro studies strongly suggest that PAF receptor antagonists might convey therapeutic benefits in ischemic conditions and certain arrhythmias. In addition, PAF antagonists might have a cardiac allograft-preservation effect. Although clinical studies with PAF receptor antagonists in patients with cardiac diseases have not yet been reported, the experimental results to date suggest that PAF receptor antagonist might be useful in some specific cardiac disorders in humans.     

Caffeine: cardiorespiratory effects and tissue protection in animal models

The aim of this review is to analyze the cardiorespiratory and tissue-protective effects of caffeine in animal models. Peer-reviewed literature published between 1975 and 2021 was retrieved from CAB Abstracts, PubMed, ISI Web of Knowledge, and Scopus. Extracted data were analyzed to address the mechanism of action of caffeine on cardiorespiratory parameters (heart rate and rhythm), vasopressor effects, and some indices of respiratory function; we close this review by discussing the current debate on the research carried out on the effects of caffeine on tissue protection. Adenosine acts through specific receptors and is a negative inotropic and chronotropic agent. Blockage of its cardiac receptors can cause tachycardia (with arrhythmogenic potential) due to the intense activity of β1 receptors. In terms of tissue protection, caffeine inhibits hyperoxia-induced pulmonary inflammation by decreasing proinflammatory cytokine expression in animal models. The protection that caffeine provides to tissues is not limited to the CNS, as studies have demonstrated that it generates attenuation of inflammatory effects in pulmonary tissue. It inhibits the effects of some pro-inflammatory cytokines and prevents functional and structural changes.     

Bacillus Calmette-Guérin and TLR4 agonist prevent cardiovascular hypertrophy and fibrosis by regulating immune microenvironment

Hypertension-induced cardiovascular hypertrophy and fibrosis are critical in the development of heart failure. The activity of TLRs has been found to be involved in the development of pressure overload-induced myocardial hypertrophy and cardiac fibrosis. We wondered whether vaccine bacillus Calmette-Guérin (BCG), which activated TLR4 to elicit immune responses, modulated the pressure overload-stimulated cardiovascular hypertrophy and cardiac fibrosis in the murine models of abdominal aortic constriction (AAC)-induced hypertension. Before or after AAC, animals received BCG, TLR4 agonist, IFN-gamma, or TLR4 antagonist i.p. BCG and TLR4 agonist significantly prevented AAC-induced cardiovascular hypertrophy and reactive cardiac fibrosis with no changes in hemodynamics. Moreover, TLR4 antagonist reversed the BCG- and TLR4 agonist-induced actions of anti-cardiovascular hypertrophy and cardiac fibrosis. BCG decreased the expression of TLR2 or TLR4 on the heart tissue but TLR4 agonist increased the expression of TLR2 or TLR4 on the immune cells that infiltrate into the heart tissue. This led to an increased expression ratio of IFN-gamma/TGF-beta in the heart. The cardiac protective effects of BCG and TLR4 agonist are related to their regulation of ERK-Akt and p38-NF-kappaB signal pathways in the heart. In conclusion, the activity of TLR4 plays a critical role in the mediation of pressure overload-induced myocardial hypertrophy and fibrosis. The regulation of immune responses by BCG and TLR4 agonist has a great potential for the prevention and treatment of hypertension-induced myocardial hypertrophy and cardiac fibrosis.     

Myosin heavy chain expression in embryonic cardiac cell cultures

Chick embryonic heart cell isolates and monolayer cultures were prepared from atria and ventricles at selected stages of cardiac development. The cardiac myocytes were assayed for myosin heavy chain (MHC) content using monoclonal antibodies (McAbs) specific in the heart for atrial (B-1), ventricular (ALD-19), or conductive system (ALD-58) isoforms. Using immunofluorescence microscopy or radioimmunoassay, MHC accumulation was measured before plating and at 48 hr or 7 days in culture. Reproducible changes in MHC antigenicity were observed by 7 days in both atrial and ventricular cultures. The changes were stage dependent and tissue specific but generally resulted in a decreased reactivity with the tissue specific MHC McAbs. In addition, the isoform recognized by ALD-58, characteristic of the conductive system cells in vivo, was never present in cultured myocytes. These results indicate that MHC isoforms produced in vivo may be replaced in monolayer cultures by an isoform(s) not recognized by our tissue specific MHC McAbs. This suggests that the intrinsic program of cardiac myogenesis, within cardiac myocytes, may not be sufficient to establish and maintain differential expression of tissue specific MHC in monolayer cell culture.     

Redox regulation of calcium release in skeletal and cardiac muscle

In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity. In this work, we studied the effects of Ca2+ (endogenous agonist) and Mg2+ (endogenous inhibitor) on the kinetics of Ca2+ release from sarcoplasmic reticulum vesicles isolated from skeletal or cardiac mammalian muscle. Native skeletal vesicles exhibited maximal stimulation of release kinetics by 10-20 microM [Ca2+], whereas in native cardiac vesicles, maximal stimulation of release required only 1 microM [Ca2+]. In 10 microM [Ca2+], free [Mg2+] < 0.1 mM produced marked inhibition of release from skeletal vesicles but free [Mg2+] < or = 0.8 mM did not affect release from cardiac vesicles. Incubation of skeletal or cardiac vesicles with the oxidant thimerosal increased their susceptibility to stimulation by Ca2+ and decreased the inhibitory effect of Mg2+ in skeletal vesicles. Sulfhydryl-reducing agents fully reversed the effects of thimerosal. The endogenous redox species, glutathione disulfide and S-nitrosoglutathione, also stimulated release from skeletal sarcoplasmic reticulum vesicles. In 10 microM [Ca2+], 35S-nitrosoglutathione labeled a protein fraction enriched in release channels through S-glutathiolation. Free [Mg2+] 1 mM or decreasing free [Ca2+] to the nM range prevented this reaction. Possible physiological and pathological consequences of redox modification of release channels on Ca2+ signaling in heart and muscle cells are discussed.     

Regulation of alternative splicing by SRrp86 through coactivation and repression of specific SR proteins

SRrp86 is an 86-kDa member of the SR protein superfamily that is unique in that it can alter splice site selection by regulating the activity of other SR proteins. To study the function of SRrp86, inducible cell lines were created in which the concentration of SRrp86 could be varied and its effects on alternative splicing determined. Here, we show that SRrp86 can activate SRp20 and repress SC35 in a dose-dependent manner both in vitro and in vivo. These effects are apparently mediated through direct protein-protein interaction, as pull-down assays showed that SRrp86 interacts with both SRp20 and SC35. Consistent with the hypothesis that relatively modest changes in the concentration or activity of one or more splicing factors can combinatorially regulate overall splicing, protein expression patterns of SRrp86, SRp20, and SC35 reveal that each tissue maintains a unique ratio of these factors. Regulation of SR protein activity, coupled with regulated protein expression, suggest that SRrp86 may play a crucial role in determining tissue specific patterns of alternative splicing.     

Qualitative modeling of mechanoelectrical feedback in a ventricular cell

Mechanical changes in the heart muscle can influence its electrical properties through a process called mechanoelectrical feedback (MEF). This feedback can operate via changes in calcium dynamics during the cross-bridge cycle or via mechanosensitive (stretch-activated) channels. We present a four-variable ordinary differential equation (ODE) system that caricatures the electrical and mechanical activity of a ventricular cell and their mutual interactions. A three-variable excitable system with restitution properties of the FitzHugh-Nagumo type is coupled to a fourth equation which describes changes in cell length during a lightly loaded contraction. The resulting four-variable system models MEF in a cell and can be incorporated into spatially distributed models for mechanoelectric behavior during wave propagation in the cardiac tissue.     

Carvedilol inhibits mitochondrial complex I and induces resistance to H2O2 -mediated oxidative insult in H9C2 myocardial cells

Carvedilol, a beta-adrenoreceptor antagonist with strong antioxidant activity, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Although growing evidences suggest specific effects on mitochondrial metabolism, how carvedilol would exert its overall activity has not been completely disclosed. In the present work we have investigated the impact of carvedilol-treatment on mitochondrial bioenergetic functions and ROS metabolism in H9C2 cells. This analysis has revealed a dose-dependent decrease in respiratory fluxes by NAD-dependent substrates associated with a consistent decline of mitochondrial complex I activity. These changes were associated with an increase in mitochondrial H(2)O(2) production, total glutathione and protein thiols content. To evaluate the antioxidant activity of carvedilol, the effect of the exposure of control and carvedilol-pretreated H9C2 cells to H(2)O(2) were investigated. The H(2)O(2)-mediated oxidative insult resulted in a significant decrease of mitochondrial respiration, glutathione and protein thiol content and in an increased level of GSSG. These changes were prevented by carvedilol-pretreatment. A similar protective effect on mitochondrial respiration could be obtained by pre-treatment of the cells with a sub-saturating amount of rotenone, a complex I inhibitor. We therefore suggest that carvedilol exerts its protective antioxidant action both by a direct antioxidant effect and by a preconditioning-like mechanism, via inhibition of mitochondrial complex I.     

Large animal models for cardiac stem cell therapies

Cardiovascular disease is the leading cause of death in developed countries and is one of the leading causes of disease burden in developing countries. Therapies have markedly increased survival in several categories of patients, nonetheless mortality still remains high. For this reason high hopes are associated with recent developments in stem cell biology and regenerative medicine that promise to replace damaged or lost cardiac muscle with healthy tissue, and thus to dramatically improve the quality of life and survival in patients with various cardiomyopathies.Much of our insight into the molecular and cellular basis of cardiovascular biology comes from small animal models, particularly mice. However, significant differences exist with regard to several cardiac characteristics when mice are compared with humans. For this reason, large animal models like dog, sheep and pig have a well established role in cardiac research. A distinct characteristic of cardiac stem cells is that they can either be endogenous or derive from outside the heart itself; they can originate as the natural course of their differentiation programme (e.g., embryonic stem cells) or can be the result of specific inductive conditions (e.g., mesenchymal stem cells). In this review we will summarize the current knowledge on the kind of heart-related stem cells currently available in large animal species and their relevance to human studies as pre-clinical models.     

Steroid regulation of monoamine oxidase activity in the adrenal medulla

Administration of different steroid hormones in vivo has distinct and specific effects on the MAO activity of the adrenal medulla. In an effort to reconstitute these effects in defined cells, we have isolated endothelial cells and chromaffin cells from the bovine adrenal medulla and tested each cell type for sensitivity to these steroids. As in the intact animal, we found that endothelial cell MAO activity was stimulated 1.5- 2.5-fold by 10 microM progesterone, hydrocortisone, and dexamethasone, inhibited by ca. 50% by 17-alpha-estradiol, but unaffected by testosterone. The type of MAO in the endothelial cells was found to be exclusively of the A type. The chromaffin cells had MAO B exclusively and were inert to treatment with dexamethasone. The mode of action of the various steroids on MAO A activity in endothelial cells seemed to be that of affecting the number of MAO molecules, as binding of [3H]pargyline, an MAO inhibitor, changed in proportion to changes in enzyme activity. Consistently, the kinetic parameters for MAO A showed changes in Vmax but not Km under all conditions. The specificity of steroid action on MAO A activity was also supported by the fact that steroid-induced changes in total cell division ([14C]thymidine incorporation) and total protein synthesis ([14C]leucine incorporation) were seen after changes in MAO A. We conclude that the differential effects of steroids on MAO activity in the intact adrenal medulla can be reproduced in cultured adrenal medullary endothelial cells but not in chromaffin cells. Therefore we suggest that the action of these steroid hormones on the intact adrenal medulla may be restricted to the endothelial cell component of this tissue.