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 (I _Ca) and corresponding whole-cell [Ca²⁺]_i transients in hESC-CMs and hFVMs, and the amplitude of both I _Ca 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 I _Ca during EC coupling develops early in human cardiomyocytes.     

Dilated Cardiomyopathy with Increased SR Ca2+ Loading Preceded by a Hypercontractile State and Diastolic Failure in the α1CTG Mouse

Mice over-expressing the α₁₋subunit (pore) of the L-type Ca²⁺ channel (α_1CTG) by 4months (mo) of age exhibit an enlarged heart, hypertrophied myocytes, increased Ca²⁺ current and Ca²⁺ transient amplitude, but a normal SR Ca²⁺ load. With advancing age (8–11 mo), some mice demonstrate advanced hypertrophy but are not in congestive heart failure (NFTG), while others evolve to frank dilated congestive heart failure (FTG). We demonstrate that older NFTG myocytes exhibit a hypercontractile state over a wide range of stimulation frequencies, but maintain a normal SR Ca²⁺ load compared to age matched non-transgenic (NTG) myocytes. However, at high stimulation rates (2–4 Hz) signs of diastolic contractile failure appear in NFTG cells. The evolution of frank congestive failure in FTG is accompanied by a further increase in heart mass and myocyte size, and phospholamban and ryanodine receptor protein levels and phosphorylation become reduced. In FTG, the SR Ca²⁺ load increases and Ca²⁺ release following excitation, increases further. An enhanced NCX function in FTG, as reflected by an accelerated relaxation of the caffeine-induced Ca²⁺ transient, is insufficient to maintain a normal diastolic Ca²⁺ during high rates of stimulation. Although a high SR Ca²⁺ release following excitation is maintained, the hypercontractile state is not maintained at high rates of stimulation, and signs of both systolic and diastolic contractile failure appear. Thus, the dilated cardiomyopathy that evolves in this mouse model exhibits signs of both systolic and diastolic failure, but not a deficient SR Ca²⁺ loading or release, as occurs in some other cardiomyopathic models.     

Defective sarcoplasmic reticulum–mitochondria calcium exchange in aged mouse myocardium

Mitochondrial alterations are critically involved in increased vulnerability to disease during aging. We investigated the contribution of mitochondria–sarcoplasmic reticulum (SR) communication in cardiomyocyte functional alterations during aging. Heart function (echocardiography) and ATP/phosphocreatine (NMR spectroscopy) were preserved in hearts from old mice (>20 months) with respect to young mice (5–6 months). Mitochondrial membrane potential and resting O₂ consumption were similar in mitochondria from young and old hearts. However, maximal ADP-stimulated O₂ consumption was specifically reduced in interfibrillar mitochondria from aged hearts. Second generation proteomics disclosed an increased mitochondrial protein oxidation in advanced age. Because energy production and oxidative status are regulated by mitochondrial Ca²⁺, we investigated the effect of age on mitochondrial Ca²⁺ uptake. Although no age-dependent differences were found in Ca²⁺ uptake kinetics in isolated mitochondria, mitochondrial Ca²⁺ uptake secondary to SR Ca²⁺ release was significantly reduced in cardiomyocytes from old hearts, and this effect was associated with decreased NAD(P)H regeneration and increased mitochondrial ROS upon increased contractile activity. Immunofluorescence and proximity ligation assay identified the defective communication between mitochondrial voltage-dependent anion channel and SR ryanodine receptor (RyR) in cardiomyocytes from aged hearts associated with altered Ca²⁺ handling. Age-dependent alterations in SR Ca²⁺ transfer to mitochondria and in Ca²⁺ handling could be reproduced in cardiomyoctes from young hearts after interorganelle disruption with colchicine, at concentrations that had no effect in aged cardiomyocytes or isolated mitochondria. Thus, defective SR–mitochondria communication underlies inefficient interorganelle Ca²⁺ exchange that contributes to energy demand/supply mistmach and oxidative stress in the aged heart.Age is the main independent risk factor for cardiovascular morbidity and mortality.¹ It increases heart vulnerability to cardiac diseases as well as the severity of their clinical manifestations, and reduces the efficacy of cardioprotective interventions.² At the cellular level, some of the structural and functional age-dependent changes resemble those of failing cardiac myocytes.^{3, 4} Specifically, disturbed Ca²⁺ homeostasis and excitation–contraction coupling,⁵ as well as deficient mitochondrial energetics⁶ and excessive ROS production,⁷ have been consistently reported in senescent cardiomyocytes. These subcellular alterations likely contribute to the reduced adaptive capacity to stress (exercise, β-adrenergic stimulation) and increased vulnerability to disease of the aged hearts.In cardiac cells, electrochemical coupling and metabolic adaptations are based upon the coordination between sarcoplasmic reticulum (SR) and mitochondria tightly interconnected forming an interface to support local ionic exchange and signal transduction in a beat-to-beat basis.⁸ This privileged interorganelle communication facilitates mitochondrial ATP transport for SR Ca²⁺ cycling and ensures energy replenishment by reciprocal Ca²⁺ and ADP exchange. Ca²⁺ is taken up by mitochondria using a low-affinity uniporter whose activity is driven by the elevated Ca²⁺ concentration in the microenvironment present around ryanodine receptors (RyR).⁹ Indeed, the kinetics of mitochondrial Ca²⁺ uptake is more dependent on the concentration of Ca²⁺ at the SR–mitochondria contact points than on bulk cytosolic Ca²⁺ concentration.⁸ Mitochondrial Ca²⁺ uptake allows energy supply–demand matching through the activation of Krebs cycle dehydrogenases and electron transport chain activity, and at the same time it regulates the regeneration of Krebs-coupled antioxidative defenses (NAD(P)H).¹⁰Defective SR–mitochondria cross talk has been causally linked to the abnormal mitochondrial Ca²⁺ uptake in failing hearts and may underlie their increased oxidative stress.¹¹ Also, in diabetic cardiomyopathy, intracellular Ca²⁺ overload and depletion of energy stores appear to develop as a consequence of sequential SR–mitochondria dysfunction.¹² Atrial fibrillation has been associated with an increased fusion of mitochondria and a subsequent increased colocalization of giant mitochondria with SR, a subcellular remodeling process that contributes to the perpetuation of the arrhythmia.¹³ Because mitochondria are highly dynamic structures, some molecular links have been proposed to provide a stable physical interorganelle bridge^{14, 15} while others appear to facilitate direct tunneling of Ca²⁺ and other signaling mediators.¹⁶ In the present study, we hypothesized that aging may negatively impact on mitochondria–SR communication by mechanisms involving defective Ca²⁺ transmission, and we identified reduced physical interaction between RyR and mitochondrial voltage-dependent anion channel (VDAC) as the main responsible of this effect.     

Excitation-contraction coupling of the mouse embryonic cardiomyocyte

In the mammalian embryo, the primitive tubular heart starts beating during the first trimester of gestation. These early heartbeats originate from calcium-induced contractions of the developing heart muscle cells. To explain the initiation of this activity, two ideas have been presented. One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca²⁺ release from intracellular stores initiating spontaneous intracellular calcium oscillations. We show with experiments that both of these mechanisms coexist and operate in mouse cardiomyocytes during embryonic days 9–11. Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats. This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.     

Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization

This study presents an investigation of pacemaker mechanisms underlying lymphatic vasomotion. We tested the hypothesis that active inositol 1,4,5-trisphosphate receptor (IP₃R)-operated Ca²⁺ stores interact as coupled oscillators to produce near-synchronous Ca²⁺ release events and associated pacemaker potentials, this driving action potentials and constrictions of lymphatic smooth muscle. Application of endothelin 1 (ET-1), an agonist known to enhance synthesis of IP₃, to quiescent lymphatic smooth muscle syncytia first enhanced spontaneous Ca²⁺ transients and/or intracellular Ca²⁺ waves. Larger near-synchronous Ca²⁺ transients then occurred leading to global synchronous Ca²⁺ transients associated with action potentials and resultant vasomotion. In contrast, blockade of L-type Ca²⁺ channels with nifedipine prevented ET-1 from inducing near-synchronous Ca²⁺ transients and resultant action potentials, leaving only asynchronous Ca²⁺ transients and local Ca²⁺ waves. These data were well simulated by a model of lymphatic smooth muscle with: 1), oscillatory Ca²⁺ release from IP₃R-operated Ca²⁺ stores, which causes depolarization; 2), L-type Ca²⁺ channels; and 3), gap junctions between cells. Stimulation of the stores caused global pacemaker activity through coupled oscillator-based entrainment of the stores. Membrane potential changes and positive feedback by L-type Ca²⁺ channels to produce more store activity were fundamental to this process providing long-range electrochemical coupling between the Ca²⁺ store oscillators. We conclude that lymphatic pacemaking is mediated by coupled oscillator-based interactions between active Ca²⁺ stores. These are weakly coupled by inter- and intracellular diffusion of store activators and strongly coupled by membrane potential. Ca²⁺ store-based pacemaking is predicted for cellular systems where: 1), oscillatory Ca²⁺ release induces depolarization; 2), membrane depolarization provides positive feedback to induce further store Ca²⁺ release; and 3), cells are interconnected. These conditions are met in a surprisingly large number of cellular systems including gastrointestinal, lymphatic, urethral, and vascular tissues, and in heart pacemaker cells.     

Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP?/? Mice

Background

The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.

Principal Findings

We used intact olfactory epithelium obtained from WT and OMP^−/− mice to monitor the Ca²⁺ dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca²⁺ channels, or Ca²⁺ stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca²⁺-homeostasis in these neurons by influencing the activity of the plasma membrane Na⁺/Ca²⁺-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca²⁺ elevation by stimulating the reverse mode of NCX in both WT and OMP^−/− mice. The resulting Ca²⁺ responses indicate that OMP facilitates NCX activity and allows rapid Ca²⁺ extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).

Conclusions

Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions.     

Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle

Activation of store-operated Ca²⁺ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca²⁺ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca²⁺ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca²⁺ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca²⁺ into the sarcoplasmic reticulum (SR). A novel Ca²⁺-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca²⁺. Our data suggest that cytosolic Ca²⁺ can facilitate SOCE whereas SR luminal Ca²⁺ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca²⁺ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca²⁺ entry in response to physiological demand. Such Ca²⁺ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.     

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

Glycogen synthase kinase-3β (GSK3β) is a multifunctional kinase whose inhibition is known to limit myocardial ischemia–reperfusion injury. However, the mechanism mediating this beneficial effect still remains unclear. Mitochondria and sarco/endoplasmic reticulum (SR/ER) are key players in cell death signaling. Their involvement in myocardial ischemia–reperfusion injury has gained recognition recently, but the underlying mechanisms are not yet well understood. We questioned here whether GSK3β might have a role in the Ca²⁺ transfer from SR/ER to mitochondria at reperfusion. We showed that a fraction of GSK3β protein is localized to the SR/ER and mitochondria-associated ER membranes (MAMs) in the heart, and that GSK3β specifically interacted with the inositol 1,4,5-trisphosphate receptors (IP₃Rs) Ca²⁺ channeling complex in MAMs. We demonstrated that both pharmacological and genetic inhibition of GSK3β decreased protein interaction of IP₃R with the Ca²⁺ channeling complex, impaired SR/ER Ca²⁺ release and reduced the histamine-stimulated Ca²⁺ exchange between SR/ER and mitochondria in cardiomyocytes. During hypoxia reoxygenation, cell death is associated with an increase of GSK3β activity and IP₃R phosphorylation, which leads to enhanced transfer of Ca²⁺ from SR/ER to mitochondria. Inhibition of GSK3β at reperfusion reduced both IP₃R phosphorylation and SR/ER Ca²⁺ release, which consequently diminished both cytosolic and mitochondrial Ca²⁺ concentrations, as well as sensitivity to apoptosis. We conclude that inhibition of GSK3β at reperfusion diminishes Ca²⁺ leak from IP₃R at MAMs in the heart, which limits both cytosolic and mitochondrial Ca²⁺ overload and subsequent cell death.Glycogen synthase kinase-3 (GSK3) was originally identified as a phosphorylating kinase for glycogen synthase.^{1, 2} It has two isoforms, α and β, that possess strong homology in their kinase domains with, however, distinct functions.³ GSK3 is constitutively active but it can be inhibited by phosphorylation on serine 21 (Ser21) for GSK3α and Ser9 for GSK3β.⁴ In the heart, GSK3β has several important roles in cardiac hypertrophy⁵ and ischemia–reperfusion (IR) injury.⁶ Accumulating evidence indicates that phospho-Ser9-GSK3β-mediated cytoprotection is achieved by an increased threshold for permeability transition pore (PTP) opening.^{6, 7, 8, 9} The mechanism by which GSK3β delays PTP opening still remains unclear. It has been reported that GSK3β could interact with ANT at the inner mitochondrial membrane in the heart⁹ and/or to phosphorylate voltage-dependent anion channel (VDAC) and cyclophilin D (CypD) in cancer cells.^{10, 11} GSK3β also has other proposed mechanisms of action, including a poorly characterized role in calcium (Ca²⁺) homeostasis regulation¹² and protein–protein interactions,⁹ as well as functions in different subcellular fractions such as the nucleus, cytosol and mitochondria.¹³Reperfusion is the most powerful intervention to salvage ischemic myocardium. However, it can also paradoxically lead to cardiomyocyte injury and death.¹⁴ One of the main actors of this lethal reperfusion injury is cellular Ca²⁺ overload,¹⁵ which results in part from excessive sarco/endoplasmic reticulum (SR/ER) Ca²⁺ release and Ca²⁺ influx through the plasma membrane (e.g. through L-type Ca²⁺channel and NCX (sodium-calcium exchanger)).¹⁶ Although ryanodine receptors (RyRs) are the major cardiac SR/ER Ca²⁺-release channels involved in excitation–contraction coupling (ECC)¹⁷ and ischemia–reperfusion (IR) injury,¹⁸ recent studies reported an increasing role for inositol 1,4,5-trisphosphate receptors (IP₃Rs) Ca²⁺-release channels in the modulation of ECC and cell death.^{19, 20} Ca²⁺-handling proteins of ER and mitochondria are highly concentrated at mitochondria-associated ER membranes (MAMs), providing a direct and proper mitochondrial Ca²⁺ signaling, including VDAC, Grp75 and IP₃R1.^{20, 21, 22}Here, we provide evidence that, following IR, a fraction of cellular GSK3β is localized at the SR/ER and MAMs. At the MAMs interface, GSK3β can specifically interact and regulate the protein composition of the IP₃R Ca²⁺ channeling complex and modulate Ca²⁺ transfer between SR/ER and mitochondria. These findings support a novel mechanism of action of GSK3β in cell death process during reperfusion injury.     

Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl⁻ ion channels that are activated by intracellular Ca²⁺. At present, the structures and mechanisms responsible for Ca²⁺ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca²⁺ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca²⁺ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca²⁺ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca²⁺. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca²⁺ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca²⁺-binding loop (312–323) reduced the apparent Ca²⁺ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca²⁺-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca²⁺ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca²⁺ and suggest a model in which Ca²⁺ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ~100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca²⁺ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca²⁺.     

Luminal Mg2+, a key factor controlling RYR2-mediated Ca2+ release: cytoplasmic and luminal regulation modeled in a tetrameric channel

In cardiac muscle, intracellular Ca²⁺ and Mg²⁺ are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca²⁺] in the SR ([Ca²⁺]_L) stimulates the Ca²⁺ release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg²⁺, which has not been regarded as an important regulator of Ca²⁺ release.     

Imaging Single Cardiac Ryanodine Receptor Ca2+ Fluxes in Lipid Bilayers

In this and an accompanying report we describe two steps, single-channel imaging and channel immobilization, necessary for using optical imaging to analyze the function of ryanodine receptor (RyR) channels reconstituted in lipid bilayers. An optical bilayer system capable of laser scanning confocal imaging of fluo-3 fluorescence due to Ca²⁺ flux through single RyR2 channels and simultaneous recording of single channel currents was developed. A voltage command protocol was devised in which the amplitude, time course, shape, and hence the quantity of Ca²⁺ flux through a single RyR2 channel is controlled solely by the voltage imposed across the bilayer. Using this system, the voltage command protocol, and concentrations of Ca²⁺ (25–50 mM) that result in saturating RyR2 Ca²⁺ currents, proportional fluo-3 fluorescence was recorded simultaneously with Ca²⁺ currents having amplitudes of 0.25–14 pA. Ca²⁺ sparks, similar to those obtained with conventional microscope-based laser scanning confocal systems, were imaged in mouse ventricular cardiomyocytes using the optical bilayer system. The utility of the optical bilayer for systematic investigation of how cellular factors extrinsic to the RyR2 channel, such as Ca²⁺ buffers and diffusion, alter fluo-3 fluorescent responses to RyR2 Ca²⁺ currents, and for addressing other current research questions is discussed.     

Rem uncouples excitation–contraction coupling in adult skeletal muscle fibers

In skeletal muscle, excitation–contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca²⁺ channel (Ca_V1.1) to be communicated to the type 1 ryanodine-sensitive Ca²⁺ release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein–protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca_V1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α_1S subunit of Ca_V1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary β_1a subunit of Ca_V1.1 is a conduit for this intermolecular communication. However, a direct role for β_1a has been difficult to test because β_1a serves two other functions that are prerequisite for conformational coupling between Ca_V1.1 and RYR1. Specifically, β_1a promotes efficient membrane expression of Ca_V1.1 and facilitates the tetradic ultrastructural arrangement of Ca_V1.1 channels within plasma membrane–SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established β subunit–interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca²⁺ transients without greatly affecting Ca_V1.1 targeting, intramembrane gating charge movement, or releasable SR Ca²⁺ store content. In contrast, a β_1a-binding–deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca²⁺ release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca_V1.1 from RYR1-mediated SR Ca²⁺ release via its ability to interact with β_1a. Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the β_1a subunit in skeletal-type EC coupling.     

Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes

Store-operated Ca²⁺ entry (SOCE), which is Ca²⁺ entry triggered by the depletion of intracellular Ca²⁺ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca²⁺ loading (load_SR) that was not altered by inhibition of L-type Ca²⁺ channels, reverse mode Na⁺/Ca²⁺ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca²⁺] to 0 mM or adding either 0.5 mM Zn²⁺ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 µM) inhibited load_SR at rest. Interestingly, inhibition of forward mode NCX with 30 µM KB-R7943 stimulated SOCE significantly and resulted in enhanced load_SR. In addition, manipulation of the extracellular and intracellular Na⁺ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca²⁺ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca²⁺ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca²⁺ overload and arrhythmogenesis. cardiac ontogeny; cardiac excitation-contraction coupling; calcium homeostasis     

Voltage-Activated Calcium Signals in Myotubes Loaded with High Concentrations of EGTA

In the present study we describe the analysis of optically recorded whole cell Ca²⁺ transients elicited by depolarization in cultured skeletal myotubes. Myotubes were obtained from the mouse muscle-derived cell line C2C12 and from mouse satellite cells. The cells were voltage-clamped and perfused with an artificial intracellular solution containing 15 mM EGTA to ensure that the bulk of the Ca²⁺ mobilized by depolarization is bound to this extrinsic buffer. The apparent on- and off-rate constants of EGTA and the dissociation rate constant of fura-2 in the cell were estimated by investigating the Ca²⁺-dependence of kinetic components of the fluorescence decay after repolarization. These parameters were used to calculate the time course of the total voltage-controlled flux of Ca²⁺ to the myoplasmic space (Ca²⁺ input flux). The validity of the procedure was confirmed by model simulations using artificial Ca²⁺ input fluxes. Both C2C12 and primary-cultured myotubes showed a very similar phasic-tonic time course of the Ca²⁺ input flux. In most measurements, the input flux was considerably larger and showed a different time course than the estimated Ca²⁺ flux carried by the L-type Ca²⁺ channels, indicating that it consists mainly of voltage-controlled Ca²⁺ release from the sarcoplasmic reticulum. In cells with extremely small fluorescence transients, the calculated input fluxes matched the kinetic characteristics of the Ca²⁺ inward current, indicating that Ca²⁺ release was absent. These measurements served as a control for the fidelity of the fluorimetric flux analysis. The procedures promise a deeper insight into alterations of Ca²⁺ release gating in studies employing myotube expression systems for mutant or chimeric protein components of excitation-contraction coupling.     

Measurements of the BKCa channel's high-affinity Ca2+ binding constants: effects of membrane voltage

It has been established that the large conductance Ca²⁺-activated K⁺ channel contains two types of high-affinity Ca²⁺ binding sites, termed the Ca²⁺ bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance–voltage relations determined over a series of Ca²⁺ concentrations with models of channel gating that include both voltage sensing and Ca²⁺ binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca²⁺ dose–response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca²⁺ binding site) and for channels that have had one or the other Ca²⁺ binding site disabled via mutation. To accurately determine these dose–response curves we have used a series of 22 Ca²⁺ concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at −80 mV the Ca²⁺ bowl has higher affinity for Ca²⁺ than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca²⁺ to the RCK1 site is voltage dependent, whereas at the Ca²⁺ bowl it is not.     

The Skeletal L-type Ca2+ Current Is a Major Contributor to Excitation-coupled Ca2+ entry

The term excitation-coupled Ca²⁺ entry (ECCE) designates the entry of extracellular Ca²⁺ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca²⁺ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn²⁺. Here, we have examined the effects of these agents on the L-type Ca²⁺ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La³⁺, and Gd³⁺) and dantrolene all inhibited the L-type Ca²⁺ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca²⁺ channel displays permeability to Mn²⁺ in the absence of external Ca²⁺ and produces a Ca²⁺ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca²⁺ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca²⁺; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α_1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca²⁺-free solution and were unaffected by Ca²⁺ removal; and (3) after block of SR Ca²⁺ release by 200 μM ryanodine, normal myotubes still displayed a large Ca²⁺ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca²⁺ entry attributed to ECCE.     

Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

Background

Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of “Ca²⁺ spikes” (i.e., [Ca²⁺]_c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca²⁺ spikes are related to the slow circadian rhythms.

Methodology/Principal Findings

We addressed this issue based on a Ca²⁺ indicator dye (fluo-4) and a protein Ca²⁺ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca²⁺ spikes in 18% of rat SCN cells in acute brain slices, but the Ca²⁺ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca²⁺ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca²⁺ spikes was increased to 13∼14%.

Conclusions/Significance

Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca²⁺ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca²⁺ spiking activity is caused by the Ca²⁺ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca²⁺]_c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca²⁺ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca²⁺ spikes in the function of SCN.     

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca²⁺ transient. The global elevation of the intracellular Ca²⁺ concentration arises from the spatial and temporal summation of elementary Ca²⁺ release events, Ca²⁺ sparks. Ca²⁺ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca²⁺, Mg²⁺, and ATP). Here, we examined by which mechanism the free intracellular Mg²⁺ ([Mg²⁺]_free) affects various Ca²⁺ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg²⁺ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg²⁺ from the possible activation by ATP and Mg²⁺-ATP via the adenine binding site of the channel. Changes in [Mg²⁺]_free generally led to biphasic alterations of the Ca²⁺ spark frequency. For example, lowering [Mg²⁺]_free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca²⁺ depletion. Fitting the Ca²⁺ spark inhibition by [Mg²⁺]_free with a Hill equation revealed a K_i of 0.1 mM. In conclusion, our results support the notion that local Ca²⁺ release and Ca²⁺ sparks are modulated by Mg²⁺ in the intracellular environment. This seems to occur predominantly by hindering Ca²⁺-dependent activation of the RYRs through competitive Mg²⁺ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg²⁺ and ATP can change.     

Reconciling depressed Ca2+ sparks occurrence with enhanced RyR2 activity in failing mice cardiomyocytes

Abnormalities in cardiomyocyte Ca²⁺ handling contribute to impaired contractile function in heart failure (HF). Experiments on single ryanodine receptors (RyRs) incorporated into lipid bilayers have indicated that RyRs from failing hearts are more active than those from healthy hearts. Here, we analyzed spontaneous Ca²⁺ sparks (brief, localized increased in [Ca²⁺]_i) to evaluate RyR cluster activity in situ in a mouse post-myocardial infarction (PMI) model of HF. The cardiac ejection fraction of PMI mice was reduced to ∼30% of that of sham-operated (sham) mice, and their cardiomyocytes were hypertrophied. The [Ca²⁺]_i transient amplitude and sarcoplasmic reticulum (SR) Ca²⁺ load were decreased in intact PMI cardiomyocytes compared with those from sham mice, and spontaneous Ca²⁺ sparks were less frequent, whereas the fractional release and the frequency of Ca²⁺ waves were both increased, suggesting higher RyR activity. In permeabilized cardiomyocytes, in which the internal solution can be controlled, Ca²⁺ sparks were more frequent in PMI cells (under conditions of similar SR Ca²⁺ load), confirming the enhanced RyR activity. However, in intact cells from PMI mice, the Ca²⁺ sparks frequency normalized by the SR Ca²⁺ load in that cell were reduced compared with those in sham mice, indicating that the cytosolic environment in intact cells contributes to the decrease in Ca²⁺ spark frequency. Indeed, using an internal “failing solution” with less ATP (as found in HF), we observed a dramatic decrease in Ca²⁺ spark frequency in permeabilized PMI and sham myocytes. In conclusion, our data show that, even if isolated RyR channels show more activity in HF, concomitant alterations in intracellular media composition and SR Ca²⁺ load may mask these effects at the Ca²⁺ spark level in intact cells. Nonetheless, in this scenario, the probability of arrhythmogenic Ca²⁺ waves is enhanced, and they play a potential role in the increase in arrhythmia events in HF patients.     

Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale

In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).

Objective

To characterize RyR functional properties in relation to TT proximity, at baseline and after MI.

Methods

Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca²⁺ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca²⁺ release, F>F₅₀ within 20 ms) or their absence (delayed areas). Spontaneous Ca²⁺ release events during diastole, Ca²⁺ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.

Results

In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na⁺/Ca²⁺ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca²⁺ influx via Ca²⁺ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca²⁺ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca²⁺ removal by NCX at the membrane was significantly lower in MI.

Conclusion

TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca²⁺ loss and raise SR Ca²⁺ content, but may promote Ca²⁺ waves.