Local Control of Excitation-Contraction Coupling in Human Embryonic Stem Cell-Derived Cardiomyocytes

We investigated the mechanisms of excitation-contraction (EC) coupling in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and fetal ventricular myocytes (hFVMs) using patch-clamp electrophysiology and confocal microscopy. We tested the hypothesis that Ca2+ influx via voltage-gated L-type Ca2+ channels activates Ca2+ release from the sarcoplasmic reticulum (SR) via a local control mechanism in hESC-CMs and hFVMs. Field-stimulated, whole-cell [Ca2+]i transients in hESC-CMs required Ca2+ entry through L-type Ca2+ channels, as evidenced by the elimination of such transients by either removal of extracellular Ca2+ or treatment with diltiazem, an L-type channel inhibitor. Ca2+ release from the SR also contributes to the [Ca2+]i transient in these cells, as evidenced by studies with drugs interfering with either SR Ca2+ release (i.e. ryanodine and caffeine) or reuptake (i.e. thapsigargin and cyclopiazonic acid). As in adult ventricular myocytes, membrane depolarization evoked large L-type Ca2+ currents (ICa) and corresponding whole-cell [Ca2+]i transients in hESC-CMs and hFVMs, and the amplitude of both ICa and the [Ca2+]i transients were finely graded by the magnitude of the depolarization. hESC-CMs exhibit a decreasing EC coupling gain with depolarization to more positive test potentials, “tail” [Ca2+]i transients upon repolarization from extremely positive test potentials, and co-localized ryanodine and sarcolemmal L-type Ca2+ channels, all findings that are consistent with the local control hypothesis. Finally, we recorded Ca2+ sparks in hESC-CMs and hFVMs. Collectively, these data support a model in which tight, local control of SR Ca2+ release by the ICa during EC coupling develops early in human cardiomyocytes

Calcium channel diversity in the cardiovascular system

The flux of calcium ions (Ca2+) into the cytosol, where they serve as intracellular messengers, is regulated by two distinct families of Ca2+ channel proteins. These are the intracellular Ca2+ release channels, which allow Ca2+ to enter the cytosol from intracellular stores, and the plasma membrane Ca2+ channels, which control Ca2+ entry from the extracellular space. Each of these two families of channel proteins contains several subgroups. The intracellular channels include the large Ca2+ channels (“ryanodine receptors”) that participate in cardiac and skeletal muscle excitation-contraction coupling, and smaller inositol trisphosphate (InsP3)—activated Ca2+ channels. The latter serve several functions, including the pharmacomechanical coupling that activates smooth muscle contraction, and possibly regulation of diastolic tone in the heart. The InsP3-activated Ca2+ channels may also participate in signal transduction systems that regulate cell growth. The family of plasma membrane Ca2+ channels includes L-type channels, which respond to membrane depolarization by generating a signal that opens the intracellular Ca2+ release channels. Calcium ion entry through L-type Ca2+ channels in the sinoatrial (SA) node contributes to pacemaker activity, whereas L-type Ca2+ channels in the atrioventricular (AV) node are essential for AV conduction. The T-type Ca2+ channels, another member of the family of plasma membrane Ca2+ channels, participate in pharmacomechanical coupling in smooth muscle. Opening of these channels in response to membrane depolarization participates in SA node pacemaker currents, but their role in the working cells of the atria and ventricle is less clear. Like the InsP3-activated intracellular Ca2+ release channels, T-type plasma membrane channels may regulate cell growth. Because most of the familiar Ca2+ channel blocking agents currently used in cardiology, such as nifedipine, verapamil and diltiazem, are selective for L-type Ca2+ channels, the recent development of drugs that selectively block T-type Ca2+ channels offers promise of new approaches to cardiovascular therapy

2014 Annual town report town of Randolph, New Hampshire January 1st through December 31st 2014.

This is an annual report containing vital statistics for a town/city in the state of New Hampshire

Mechanisms of ATP release and signalling in the blood vessel wall

The nucleotide adenosine 5′-triphosphate (ATP) has classically been considered the cell's primary energy currency. Importantly, a novel role for ATP as an extracellular autocrine and/or paracrine signalling molecule has evolved over the past century and extensive work has been conducted to characterize the ATP-sensitive purinergic receptors expressed on almost all cell types in the body. Extracellular ATP elicits potent effects on vascular cells to regulate blood vessel tone but can also be involved in vascular pathologies such as atherosclerosis. While the effects of purinergic signalling in the vasculature have been well documented, the mechanism(s) mediating the regulated release of ATP from cells in the blood vessel wall and circulation are now a key target of investigation. The aim of this review is to examine the current proposed mechanisms of ATP release from vascular cells, with a special emphasis on the transporters and channels involved in ATP release from vascular smooth muscle cells, endothelial cells, circulating red blood cells, and perivascular sympathetic nerves, including vesicular exocytosis, plasma membrane F1/F0-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channel

Modeling CICR in rat ventricular myocytes: voltage clamp studies

<p>Abstract</p> <p>Background</p> <p>The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect <it>Ca</it>²⁺loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR <it>Ca</it>²⁺release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic <it>Ca</it>²⁺concentration ([<it>Ca</it>²⁺]<it>_myo</it>).</p> <p>Methods</p> <p>The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed <it>Ca</it>²⁺channels (trigger-channel and release-channel). It releases <it>Ca</it>²⁺flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[<it>Ca</it>²⁺]<it>_myo</it>.</p> <p>Results</p> <p>Our model reproduces measured VC data published by several laboratories, and generates graded <it>Ca</it>²⁺release at high <it>Ca</it>²⁺gain in a homeostatically-controlled environment where [<it>Ca</it>²⁺]<it>_myo</it>is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR <it>Ca</it>²⁺release, its activation by trigger <it>Ca</it>²⁺, and its refractory characteristics mediated by the luminal SR <it>Ca</it>²⁺sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence <it>Ca</it>²⁺homeostasis.</p> <p>Conclusions</p> <p>We examine the role of the above <it>Ca</it>²⁺regulating mechanisms in handling various types of induced disturbances in <it>Ca</it>²⁺levels by quantifying cellular <it>Ca</it>²⁺balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses.</p

Functional Ca2+-couplings among the mitochondrion, endoplasmic reticulum and plasmalemma in thermogenic brown adipocytes : Possible roles in energy consumption

Obesity is the result of excess energy intake and/or decreased energy consumption. Energy is consumed by basal metabolic activity, muscle activity and thermogenesis. Adrenergic activations of lipolysis and uncoupling proteins in brown adipocytes lead to heat production without oxidative phosphorylation. The energy dissipated by this process is the H+ electrochemical potential across the mitochondrial membrane generated by H+ pumps, respiratory chains, that require NADH and FADH2 produced by Ca2+-dependent dehydrogenases in TCA cycle. Thus, this process is strongly affected by the level of intracellular free Ca2+ ([Ca2+]i), which are regulated by Ca2+ binding to Ca2+ binding proteins, Ca2+ entry and extrusion at the plasma membrane, Ca2+ release and uptake into, and from, mitochondria and the endoplasmic reticulum (ER). We review here how these organelles and the plasmalemma communicate with each other in regulating [Ca2+]i and discuss how these coupling are involved in thermogenesis in rat brown adipocytes. Our recent observations suggest the new mechanisms of [Ca2+]i regulation in brown adipocytes: 1) uncoupling of oxidative phosphorylation and the subsequent recoupling activate Ca2+ entry at the plasmalemma that depends on restoration of H+ electrochemical potential or ATP synthesis, 2) mitochondrial Ca2+ release induces Ca2+ release from the ER, 3) Ca2+ depletion in the ER via mitochondria-induced Ca2+ release activates store-operated Ca2+ entry (SOC) in a fraction of cells and 4) Ca2+ depletion in the ER activates Ca2+ release from mitochondria. Since these mechanisms are activated by the a- and b-actions of noradrenaline, they likely play important roles in thermogenesis

Calcium Homeostasis in a Local/Global Whole Cell Model of Permeabilized Ventricular Myocytes with a Langevin Description of Stochastic Calcium Release

Population density approaches to modeling local control of Ca2+ induced Ca2+ release in cardiac myocytes can be used to construct minimal whole cell models that accurately represent heterogeneous local Ca2+ signals. Unfortunately, the computational complexity of such local/global whole cell models scales with the number of Ca2+ release unit (CaRU) states, which is a rapidly increasing function of the number of ryanodine receptors (RyRs) per CaRU. Here we present an alternative approach based on a Langevin description of the collective gating of RyRs coupled by local Ca2+ concentration ([Ca2+]). The computational efficiency of this approach no longer depends on the number of RyRs per CaRU. When the RyR model is minimal, Langevin equations may be replaced by a single Fokker-Planck equation, yielding an extremely compact and efficient local/global whole cell model that reproduces and helps interpret recent experiments that investigate Ca2+ homeostasis in permeabilized ventricular myocytes. Our calculations show that elevated myoplasmic [Ca2+] promotes elevated network sarcoplasmic reticulum (SR) [Ca2+] via SR Ca2+ -ATPase-mediated Ca2+ uptake. However, elevated myoplasmic [Ca2+] may also activate RyRs and promote stochastic SR Ca2+ release, which can in turn decrease SR [Ca2+]. Increasing myoplasmic [Ca2+] results in an exponential increase in spark-mediated release and a linear increase in nonspark-mediated release, consistent with recent experiments. The model exhibits two steady-state release fluxes for the same network SR [Ca2+] depending on whether myoplasmic [Ca2+] is low or high. In the later case, spontaneous release decreases SR [Ca2+] in a manner that maintains robust Ca2+ sparks

The effect of modulating calcium-induced calcium release on the properties of spontaneous and systolic calcium release in rat ventricular myocytes.

The effects of modulation of CICR on spontaneous and systolic Ca2+ release were investigated in isolated rat ventricular myocytes. Spontaneous waves of Ca2 + release were initially abolished and then resumed at a lower frequency during exposure to 100 J.LMtetracaine. Both the duration of the initial quiescent period and oscillation frequency in tetracaine were dependent on the control oscillation frequency and the concentration of tetracaine applied. Electrophysiological quantification of the SR Ca2+ content of myocytes revealed a significant increase during exposure to tetracaine. The amplitude of spontaneous Ca2 + release was also increased such that despite decreased frequency, efflux per unit time activated by Ca2+ waves was not changed significantly. Using confocal microscopy, the spatial and temporal properties of Ca2+ waves were studied revealing that tetracaine inhibits the propagation of Ca2 + release. The increased SR Ca2+ content and the increased amplitude of Ca2+ release can reverse this effect. Application of 100 J.LMtetracaine to electrically stimulated cells transiently depressed systolic Ca2+ release and contraction but had no effect in the steady state. Removal of tetracaine was associated with potentiation of systolic Ca2+ release followed by gradual recove~. Quantification of the SR Ca2+ content revealed that in tetracaine the SR Ca + content was significantly increased in the steady state. This increase was accounted for by inhibition of systolic Ca2+ release activating less Ca2+ efflux in the presence of the same or increased Ca2+ influx on the L-type Ca2+ current. As the SR Ca2+ content increases, more efflux is activated until eventually efflux and influx balance in the steady state. The transient potentiation of contraction on removal of tetracaine is due to the increased SR Ca2 + content, which increases the gain of CICR in the absence of inhibition of Ca2 + release. The mechanism of post rest potentiation in rat cardiac tissue has not been conclusively elucidated by previous studies. This investigation provides evidence that changes in SR Ca2 + content and recovery of channels from inactivation could contribute to the potentiation of contraction observed in rat ventricle after a period of rest. Tetracaine enhances the degree of potentiation of contraction, which can only partially be attributed to its ability to enhance SR Ca2+ accumulation. During myocardial ischaemia dramatic changes in the substrate and metabolite levels in cells occur and a number of these changes are known to affect the RyR. However, the overall effects of metabolic blockade on the sensitivity CICR in intact cells have been overlooked. Experiments were carried out to investigate the effect of metabolic inhibition on spontaneous Ca2+ release and SR Ca2+ content in isolated rat ventricular myocytes. The results show that CICR is inhibited during metabolic inhibition. This could contribute to the degree of damaging and potentially fatal Ca2+ overload experienced on reperfusion of ischaemic tissue