Model of intracellular calcium cycling in ventricular myocytes.

We present a mathematical model of calcium cycling that takes into account the spatially localized nature of release events that correspond to experimentally observed calcium sparks. This model naturally incorporates graded release by making the rate at which calcium sparks are recruited proportional to the whole cell L-type calcium current, with the total release of calcium from the sarcoplasmic reticulum (SR) being just the sum of local releases. The dynamics of calcium cycling is studied by pacing the model with a clamped action potential waveform. Experimentally observed calcium alternans are obtained at high pacing rates. The results show that the underlying mechanism for this phenomenon is a steep nonlinear dependence of the calcium released from the SR on the diastolic SR calcium concentration (SR load) and/or the diastolic calcium level in the cytosol, where the dependence on diastolic calcium is due to calcium-induced inactivation of the L-type calcium current. In addition, the results reveal that the calcium dynamics can become chaotic even though the voltage pacing is periodic. We reduce the equations of the model to a two-dimensional discrete map that relates the SR and cytosolic concentrations at one beat and the previous beat. From this map, we obtain a condition for the onset of calcium alternans in terms of the slopes of the release-versus-SR load and release-versus-diastolic-calcium curves. From an analysis of this map, we also obtain an understanding of the origin of chaotic dynamics.

Gap junctional hemichannels in the heart.

Upon contacting each other, cells form gap junctions, in which each cell contributes half of the channel linking their cytoplasms, enabling them to share their metabolome up to a molecular weight of 1000. Each hemichannel (or connexon) is randomly inserted into the plasma membrane and then migrates to the site of cell-to-cell contact before pairing with the neighbouring cell's hemichannel to form a communicating conduit. This review summarizes the evidence for hemichannels in heart ventricular myocytes. Morphological findings are summarized describing how hemichannels are inserted into the plasma membrane. Once in the plasma membrane, hemichannels can be functionally detected electrophysiologically or by dye uptake assays. Each technique reveals specific aspects of hemichannel function. Using dye uptake studies, it is possible to investigate the biological regulation of hemichannels in vivo. Evidence is summarized which indicates that hemichannels are normally kept closed in the presence of normal extracellular Ca because they are phosphorylated at residues in the C-terminus regulated by the MAPK signalling pathway. When hemichannels are dephosphorylated, the channels open and allow dye uptake into the cells, as well as potentially deleterious ion exchange. Biological stresses, such as hyperosmolarity and metabolic inhibition, open hemichannels by this mechanism through activating phosphatases. The resulting ion fluxes may have important roles in heart physiology and pathophysiology.

Injection of tissue plasminogen activator into a branch retinal vein in eyes with central retinal vein occlusion.

PURPOSE: Central retinal vein occlusion (CRVO) often produces significant and permanent loss of vision in the affected eye. The purpose of this study was to determine if patients with vision loss secondary to CRVO treated with retinal vein cannulation and infusion of tissue plasminogen activator (t-PA) experienced recovery of visual acuity. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Thirty eyes of 30 consecutive patients with CRVO underwent the procedure, but two were subsequently excluded. The remaining 28 eyes of 28 patients with CRVO for an average of 4.9 months before intervention (range, 0.25-30 months) and best-corrected visual acuity 20/63 or worse were included in the study. INTERVENTION: All patients underwent pars plana vitrectomy with cannulation and infusion of t-PA into a branch retinal vein. MAIN OUTCOME MEASURES: Change in visual acuity and the development of complications such as vitreous hemorrhage and neovascular glaucoma were monitored. RESULTS: Twenty-two of 28 patients (79%) experienced at least one line of visual improvement during the follow-up period (average, 11.8 months; range, 3-24 months), and the same number had this level of improvement at the last follow-up examination. Fifteen patients (54%) gained 3 or more lines of acuity within 6 months after the procedure, and 14 (50%) had acuity at last follow-up at least 3 lines better than baseline acuity (average, 6.8 lines). Seven patients had postoperative vitreous hemorrhages ranging from 1 week to 11 months after the procedure; two cleared spontaneously. One patient had a postoperative retinal detachment from a peripheral retinal break that was repaired successfully with pneumatic retinopexy. No other serious intraoperative or early postoperative complications were noted. CONCLUSIONS: Vitrectomy with retinal vein cannulation and infusion of t-PA is a relatively safe procedure that may improve vision in eyes with CRVO.

Regulation of cloned ATP-sensitive K channels by adenine nucleotides and sulfonylureas: interactions between SUR1 and positively charged domains on Kir6.2.

K(ATP) channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K(ATP) channel. In contrast, the R301C and R314C mutants, which are also "PIP2-insensitive," remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the K(ATP) channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since K(ATP) channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.

Patterns of wave break during ventricular fibrillation in isolated swine right ventricle.

Several different patterns of wave break have been described by mapping of the tissue surface during fibrillation. However, it is not clear whether these surface patterns are caused by multiple distinct mechanisms or by a single mechanism. To determine the mechanism by which wave breaks are generated during ventricular fibrillation, we conducted optical mapping studies and single cell transmembrane potential recording in six isolated swine right ventricles (RV). Among 763 episodes of wave break (0.75 times x s(-1) x cm(-2)), optical maps showed three patterns: 80% due to a wave front encountering the refractory wave back of another wave, 11.5% due to wave fronts passing perpendicular to each other, and 8.5% due to a new (target) wave arising just beyond the refractory tail of a previous wave. Computer simulations of scroll waves in three-dimensional tissue showed that these surface patterns could be attributed to two fundamental mechanisms: head-tail interactions and filament break. We conclude that during sustained ventricular fibrillation in swine RV, surface patterns of wave break are produced by two fundamental mechanisms: head-tail interaction between waves and filament break.

Effects of diacetyl monoxime and cytochalasin D on ventricular fibrillation in swine right ventricles.

Whether or not the excitation-contraction (E-C) uncoupler diacetyl monoxime (DAM) and cytochalacin D (Cyto D) alter the ventricular fibrillation (VF) activation patterns is unclear. We recorded single cell action potentials and performed optical mapping in isolated perfused swine right ventricles (RV) at different concentrations of DAM and Cyto D. Increasing the concentration of DAM results in progressively shortened action potential duration (APD) measured to 90% repolarization, reduced the slope of the APD restitition curve, decreased Kolmogorov-Sinai entropy, and reduced the number of VF wave fronts. In all RVs, 15-20 mmol/l DAM converted VF to ventricular tachycardia (VT). The VF could be reinduced after the DAM was washed out. In comparison, Cyto D (10-40 micromol/l) has no effects on APD restitution curve or the dynamics of VF. The effects of DAM on VF are associated with a reduced number of wave fronts and dynamic complexities in VF. These results are compatible with the restitution hypothesis of VF and suggest that DAM may be unsuitable as an E-C uncoupler for optical mapping studies of VF in the swine RVs.

Coexistence of multiple spiral waves with independent frequencies in a heterogeneous excitable medium.

We studied the interactions and coexistence of stable spiral waves with independent frequencies in a heterogeneous excitable medium, using numerical simulations of a spatial system based on the FitzHugh-Nagumo cell model. When the heterogeneity of the medium exceeded a critical value, a transition took place from a single dominant spiral wave to a coexistence of multiple spiral waves with independent frequencies and n:n-1 wave conduction blocks. In this case, multiple spiral waves could coexist because they are "insulated" from each other by chaotic regions.

Phenylarsine oxide induces mitochondrial permeability transition, hypercontracture, and cardiac cell death.

The mitochondrial permeability transition (MPT) is implicated in cardiac reperfusion/reoxygenation injury. In isolated ventricular myocytes, the sulfhydryl (SH) group modifier and MPT inducer phenylarsine oxide (PAO) caused MPT, severe hypercontracture, and irreversible membrane injury associated with increased cytoplasmic free [Ca(2+)]. Removal of extracellular Ca(2+) or depletion of nonmitochondrial Ca(2+) pools did not prevent these effects, whereas the MPT inhibitor cyclosporin A was partially protective and the SH-reducing agent dithiothreitol fully protective. In permeabilized myocytes, PAO caused hypercontracture at much lower free [Ca(2+)] than in its absence. Thus PAO induced hypercontracture by both increasing myofibrillar Ca(2+) sensitivity and promoting mitochondrial Ca(2+) efflux during MPT. Hypercontracture did not directly cause irreversible membrane injury because lactate dehydrogenase (LDH) release was not prevented by abolishing hypercontracture with 2,3-butanedione monoxime. However, loading myocytes with the membrane-permeable Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) prevented PAO-induced LDH release, thus implicating the PAO-induced rise in cytoplasmic [Ca(2+)] as obligatory for irreversible membrane injury. In conclusion, PAO induces MPT and enhanced susceptibility to hypercontracture in isolated cardiac myocytes, both key features also implicated in cardiac reperfusion and reoxygenation injury.

Effects of simulated ischemia on spiral wave stability.

Regional hyperkalemia during acute myocardial ischemia is a major factor promoting electrophysiological abnormalities leading to ventricular fibrillation (VF). However, steep action potential duration restitution, recently proposed to be a major determinant of VF, is typically decreased rather than increased by hyperkalemia and acute ischemia. To investigate this apparent contradiction, we simulated the effects of regional hyperkalemia and other ischemic components (anoxia and acidosis) on the stability of spiral wave reentry in simulated two-dimensional cardiac tissue by use of the Luo-Rudy ventricular action potential model. We found that the hyperkalemic "ischemic" area promotes wavebreak in the surrounding normal tissue by accelerating the rate of spiral wave reentry, even after the depolarized ischemic area itself has become unexcitable. Furthermore, wavebreak and fibrillation can be prevented if the dynamical instability of the normal tissue is reduced significantly by targeting electrical restitution properties, suggesting a novel therapeutic approach.

Adenosine enhances neuroexcitability by inhibiting a slow postspike afterhyperpolarization in rabbit vagal afferent neurons.

BACKGROUND: Electrophysiological mechanisms by which adenosine may activate cardiac afferent neurons are unknown. Slow afterhyperpolarizations (AHPs) follow action potentials in a subset of vagal C afferents, rendering them inexcitable. The purpose of this study was to test the hypothesis that adenosine increases vagal neuronal excitability by blocking slow AHPs and to determine the adenosine receptor subtype mediating these effects. METHODS AND RESULTS: Using the perforated patch-clamp technique, we identified cultured adult rabbit nodose ganglion cells with slow AHPs in current-clamp mode. Trains of 100 current pulses at 20% above threshold were injected, with an interspike interval of 100 ms, and the number of action potentials triggered were counted and reported as the action potential response rate. During adenosine (10 micromol/L), slow AHPs were suppressed and action potential response rate was augmented from 3.8+/-0.5% at baseline to 28+/-7% after adenosine (P:=0.0009). The selective A(2)-adenosine receptor agonist NECA but not the A(1)-adenosine agonist CCPA replicated the adenosine effect. The selective A(2A)-adenosine antagonist ZM 241385 (10 nmol/L) but not the A(1) adenosine antagonist DPCPX (5 micromol/L) abolished the adenosine effect. We considered two alternative hypotheses: (1) A(2)-receptor-mediated suppression of I(Ca) leading to smaller increases in intracellular Ca during stimulation, resulting in less activation of I(K(Ca)) and consequent suppression of slow AHPs, or (2) A(2)-receptor-mediated elevation of cAMP directly suppressing slow AHPs. Under voltage-clamp conditions, adenosine did not significantly inhibit I(Ca), making the latter hypothesis more likely. CONCLUSIONS: Adenosine inhibits slow AHPs in vagal afferent neurons. This effect is most likely caused by A(2A)-receptor-mediated stimulation of cAMP production.

Electrophysiological heterogeneity and stability of reentry in simulated cardiac tissue.

Generation of wave break is a characteristic feature of cardiac fibrillation. In this study, we investigated how dynamic factors and fixed electrophysiological heterogeneity interact to promote wave break in simulated two-dimensional cardiac tissue, by using the Luo-Rudy (LR1) ventricular action potential model. The degree of dynamic instability of the action potential model was controlled by varying the maximal amplitude of the slow inward Ca(2+) current to produce spiral waves in homogeneous tissue that were either nearly stable, meandering, hypermeandering, or in breakup regimes. Fixed electrophysiological heterogeneity was modeled by randomly varying action potential duration over different spatial scales to create dispersion of refractoriness. We found that the degree of dispersion of refractoriness required to induce wave break decreased markedly as dynamic instability of the cardiac model increased. These findings suggest that reducing the dynamic instability of cardiac cells by interventions, such as decreasing the steepness of action potential duration restitution, may still have merit as an antifibrillatory strategy.

Regulation of the mitochondrial permeability transition by matrix Ca(2+) and voltage during anoxia/reoxygenation.

We studied the interplay between matrix Ca(2+) concentration ([Ca(2+)]) and mitochondrial membrane potential (Deltapsi) in regulation of the mitochondrial permeability transition (MPT) during anoxia and reoxygenation. Without Ca(2+) loading, anoxia caused near-synchronous Deltapsi dissipation, mitochondrial Ca(2+) efflux, and matrix volume shrinkage when a critically low PO(2) was reached, which was rapidly reversible upon reoxygenation. These changes were related to electron transport inhibition, not MPT. Cyclosporin A-sensitive MPT did occur when extramitochondrial [Ca(2+)] was increased to promote significant Ca(2+) uptake during anoxia, depending on the Ca(2+) load size and ability to maintain Deltapsi. However, when [Ca(2+)] was increased after complete Deltapsi dissipation, MPT did not occur until reoxygenation, at which time reactivation of electron transport led to partial Deltapsi regeneration. In the setting of elevated extramitochondrial Ca(2+), this enhanced matrix Ca(2+) uptake while promoting MPT because of less than full recovery of Deltapsi. The interplay between Deltapsi and matrix [Ca(2+)] in accelerating or inhibiting MPT during anoxia/reoxygenation has implications for preventing reoxygenation injury associated with MPT.

Metabolic inhibition activates a non-selective current through connexin hemichannels in isolated ventricular myocytes.

Intracellular Na(+)accumulation and K(+)loss play important roles in the pathogenesis of arrhythmias and injury in the ischemic heart. We investigated the role of metabolically sensitive connexin hemichannels as a potential route for Na(+)influx and K(+)efflux during ischemia, using dye uptake and electrophysiological measurements to assay hemichannel activity in isolated rabbit ventricular myocytes. Consistent with the known size selectivity of connexin hemichannels,;50% of myocytes exposed to either low extracellular Ca(2+)(an established method for opening connexin hemichannels) or to metabolic inhibitors (a recently described method for opening hemichannels) accumulated fluorescent dyes with <1000 MW (propidium iodide and calcein), but excluded a larger dye with 1500-3000 MW (dextran-rhodamine). Using the whole cell patch clamp technique, we found that metabolic inhibitors activated a non-selective current permeant to both small and large cations, and blocked by La(3+), similar to the properties of connexin 43 when overexpressed in human embryonic kidney (HEK) cells. These findings indicate that isolated cardiac myocytes endogenously express metabolically-sensitive connexin hemichannels. If activated during ischemia, these hemichannels could contribute significantly to altered ionic fluxes promoting arrhythmias and myocardial injury.

From local to global spatiotemporal chaos in a cardiac tissue model.

Two kinds of chaos can occur in cardiac tissue, chaotic meander of a single intact spiral wave and chaotic spiral wave breakup. We studied these behaviors in a model of two-dimensional cardiac tissue based on the Luo-Rudy I action potential model. In the chaotic meander regime, chaos is spatially localized to the core of the spiral wave. When persistent spiral wave breakup occurs, there is a transition from local to global spatiotemporal chaos.

Origins of spiral wave meander and breakup in a two-dimensional cardiac tissue model.

We studied the stability of spiral waves in homogeneous two-dimensional cardiac tissue using phase I of the Luo-Rudy ventricular action potential model. By changing the conductance and the relaxation time constants of the ion channels, various spiral wave phenotypes, including stable, quasiperiodically meandering, chaotically meandering, and breakup were observed. Stable and quasiperiodically meandering spiral waves occurred when the slope of action potential duration (APD) restitution was < 1 over all diastolic intervals visited during reentry; chaotic meander and spiral wave breakup occurred when the slope of APD restitution exceeded 1. Curvature of the wave changes both conduction velocity and APD, and their restitution properties, thereby modulating local stability in a spiral wave, resulting in distinct spiral wave phenotypes. In the LRI model, quasiperiodic meander is most sensitive to the Na+ current, whereas chaotic meander and breakup are more dependent on the Ca2+ and K+ currents.

Mechanisms of discordant alternans and induction of reentry in simulated cardiac tissue.

BACKGROUND: T-wave alternans, which is associated with the genesis of cardiac fibrillation, has recently been related to discordant action potential duration (APD) alternans. However, the cellular electrophysiological mechanisms responsible for discordant alternans are poorly understood. METHODS AND RESULTS: We simulated a 2D sheet of cardiac tissue using phase 1 of the Luo-Rudy cardiac action potential model. A steep (slope >1) APD restitution curve promoted concordant APD alternans and T-wave alternans without QRS alternans. When pacing was from a single site, discordant APD alternans occurred only when the pacing rate was fast enough to engage conduction velocity (CV) restitution, producing both QRS and T-wave alternans. Tissue heterogeneity was not required for this effect. Discordant alternans markedly increases dispersion of refractoriness and increases the ability of a premature stimulus to cause localized wavebreak and induce reentry. In the absence of steep APD restitution and of CV restitution, sustained discordant alternans did not occur, but reentry could be induced if there was marked electrophysiological heterogeneity. Both discordant APD alternans and preexisting APD heterogeneity facilitate reentry by causing the waveback to propagate slowly. CONCLUSION: Discordant alternans arises dynamically from APD and CV restitution properties and markedly increases dispersion of refractoriness. Preexisting and dynamically induced (via restitution) dispersion of refractoriness independently increase vulnerability to reentrant arrhythmias. Reduction of dynamically induced dispersion by appropriate alteration of electrical restitution has promise as an antiarrhythmic strategy.

Mechanisms of ventricular fibrillation induction by 60-Hz alternating current in isolated swine right ventricle.

BACKGROUND: The mechanisms by which 60-Hz alternating current (AC) can induce ventricular fibrillation (VF) are unknown. METHODS AND RESULTS: We studied 7 isolated perfused swine right ventricles in vitro. The action potential duration restitution curve was determined. Optical mapping techniques were used to determine the patterns of activation on the epicardium during 5-second 60-Hz AC stimulation (10 to 999 microA). AC captured the right ventricles at 100+/-65 microA, which is significantly lower than the direct current pacing threshold (0.77+/-0.45 mA, P:<0.05). AC induced ventricular tachycardia or VF at 477+/-266 microA, when the stimulated responses to AC had (1) short activation CLs (128+/-14 ms), (2) short diastolic intervals (16+/-9 ms), and (3) short diastolic intervals associated with a steep action potential duration restitution curve. Optical mapping studies showed that during rapid ventricular stimulation by AC, a wave front might encounter the refractory tail of an earlier wave front, resulting in the formation of a wave break and VF. Computer simulations reproduced these results. CONCLUSIONS: AC at strengths less than the regular pacing threshold can capture the ventricle at fast rates. Accidental AC leak to the ventricles could precipitate VF and sudden death if AC results in a fast ventricular rate coupled with a steep restitution curve and a nonuniform recovery of excitability of the myocardium.