Species dependence of mitochondrial calcium transients during excitation-contraction coupling in isolated cardiomyocytes.

Whether mitochondrial Ca(2+) transport is rapid enough to respond to changes in cytosolic [Ca(2+)] ([Ca(2+)](c)) which occur during excitation-contraction coupling in the heart is controversial; different results wereobtained with different techniques and different species. In this study mitochondrial [Ca(2+)] ([Ca(2+)](m)) was measured in indo-1/AM-loaded myocytes from rat and guinea-pig hearts where the cytosolic indo-1 had been removed by extended incubation of cells at 37 degrees C ("heat treatment"). The mitochondrial origin of the remaining fluorescence was confirmed by sensitivity of the indo-1 signal to ruthenium red. In resting rat myocytes, [Ca(2+)](m) was lower than [Ca(2+)](c), whereas in guinea-pig cells [Ca(2+)](m) was higher than [Ca(2+)](c). Upon electrical stimulation of cells, no change occurred in [Ca(2+)](m) in rat myocytes. However, in guinea-pig cells mitochondrial Ca(2+) transients were clearly visible with a mean indo-1 ratio amplitude of 0.153 +/- 0.2 (n = 20), compared with 0.306 +/- 0.02 (n = 25), p < 0.001, prior to heat treatment. These observations suggest significant differences in mitochondrial Ca(2+) transport in cardiomyocytes from different species.     

Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium

During development fast-contracting atrial and ventricular chambers develop from a peristaltic-contracting heart tube. This study addresses the question of whether chamber formation is paralleled by a matching expression of the sarcoplasmic reticulum (SR) Ca(2+) pump. We studied indo-1 Ca(2+) transients elicited by field stimulation of linear heart tube stages and of explants from atria and outflow tracts of the prototypical preseptational E13 rat heart. Ca(2+) transients of H/H 11+ chicken hearts, which constitute the prototypic linear heart tube stage, were sensitive to verapamil only, indicating a minor contribution of Ca(2+)-triggered SR Ca(2+) release. Outflow tract transients displayed sensitivity to the inhibitors similar to that of the linear heart tube stages. Atrial Ca(2+) transients disappeared upon addition of ryanodine, tetracaine, or verapamil, indicating the presence of Ca(2+)-triggered SR Ca(2+) release. Quantitative radioactive in situ hybridization on sections of E13 rat hearts showed approximately 10-fold higher SERCA2a mRNA levels in the atria compared to nonmyocardial tissue and approximately 5-fold higher expression in compact ventricular myocardium. The myocardium of atrioventricular canal, outflow tract, inner curvature, and ventricular trabecules displayed weak expression. Immunohistochemistry on sections of rat and human embryos showed a similar pattern. The significance of these findings is threefold. (i) A functional SR is present long before birth. (ii) SR development is concomitant with cardiac chamber development, explaining regional differences in cardiac function. (iii) The pattern of SERCA2a expression underscores a manner of chamber development by differentiation at the outer curvature, rather than by segmentation of the linear heart tube.     

Effects of chronic administration of clenbuterol on function and metabolism of adult rat cardiac muscle

Clenbuterol (Clen), a beta(2)-agonist, is known to produce skeletal and myocardial hypertrophy. This compound has recently been used in combination with left ventricular assist devices for the treatment of end-stage heart failure to reverse or prevent the adverse effects of unloading-induced myocardial atrophy. However, the mechanisms of action of Clen on myocardial cells have not been fully elucidated. In an attempt to clarify this issue, we examined the effects of chronic administration of Clen on Ca(2+) handling and substrate preference in cardiac muscle. Rats were treated with either 2 mg x kg(-1) x day(-1) Clen or saline (Sal) for 4 wk with the use of osmotic minipumps. Ventricular myocytes were enzymatically dissociated. Cells were field stimulated at 0.5, 1, and 2 Hz, and cytoplasmic Ca(2+) transients were monitored with the use of the fluorescent indicator indo-1 acetoxymethyl ester. Two-dimensional surface area and action potentials in current clamp were also measured. We found that in the Clen group there was significant hypertrophy at the organ and cellular levels compared with Sal. In Clen myocytes, the amplitude of the indo-1 ratio transients was significantly increased. Sarcoplasmic reticulum Ca(2+) content, estimated by rapid application of 20 mM caffeine, was significantly increased in the Clen group. The action potential was prolonged in the Clen group compared with Sal. Carbohydrate contribution to the tricarboxylic cycle (Krebs cycle) flux was increased several times in the Clen group. This increase was associated with decreased expression of peroxisome proliferator-activated receptor-alpha. This study shows that chronic administration of Clen induces cellular hypertrophy and increases oxidative carbohydrate utilization together with an increase in sarcoplasmic reticulum Ca(2+) content, which results in increased amplitude of the Ca(2+) transients. These effects could be important when Clen is used in conjunction with left ventricular assist devices treatment.     

心肌细胞钙瞬变和细胞收缩的激光共聚焦成像研究

目的：游离钙离子参与机体的多种重要生理功能。本研究着重探讨如何利用激光共聚焦显微镜线扫描成像技术同时记录正常情况下心肌细胞的钙瞬变以及由此引起的细胞收缩过程。方法与结果：本研究以分离的心室肌细胞为对象,通过局部场刺激诱发细胞的钙瞬变和收缩,同时配合使用激光共聚焦显微镜成像系统,以线扫描方式记录实验结果。结果表明,钙瞬变先于细胞收缩发生(约早31ms),而收缩最大处远落后于钙瞬变峰值发生处(约慢346ms)。结论：激光共聚焦显微镜线扫描成像技术具有较好的时问分辨率和空间分辨率,其实验结果直观、明确、可靠,是较理想的研究钙瞬变和细胞收缩的光学记录方法。     

Morphological and functional changes in cardiac myocytes isolated from mice overexpressing TNF-alpha

Transgenic (TG) TNF1.6 mice, which cardiac specifically overexpress tumor necrosis factor-alpha (TNF-alpha), exhibit heart failure (HF) and increased mortality, which is markedly higher in young (<20 wk) males (TG-M) than females (TG-F). HF in this model may be partly caused by remodeling of the extracellular matrix and/or structure/function alterations at the single myocyte level. We studied left ventricular (LV) structure and function using echocardiography and LV myocyte morphometry, contractile function, and intracellular Ca(2+) (Ca(i)(2+)) handling using cell edge detection and fura 2 fluorescence, respectively, in 12-wk-old TG-M and TG-F mice and their wild-type (WT) littermates. TG-F mice showed LV hypertrophy without dilatation and only a small reduction of basal fractional shortening (FS) and response to isoproterenol (Iso). TG-M mice showed a large LV dilatation, higher mRNA levels of beta-myosin heavy chain and atrial natriuretic factor versus TG-F mice, reduced FS relative to both WT and TG-F mice, and minimal response to Iso. TG-F and TG-M myocytes were similarly elongated (by approximately 20%). The amplitude of Ca(i)(2+) transients and contractions and the response to Iso were comparable in WT and TG-F myocytes, whereas the time to 50% decline (TD(50%)) of the Ca(i)(2+) transient, an index of the rate of sarcoplasmic reticulum Ca(2+) uptake, was prolonged in TG-F myocytes. In TG-M myocytes, the amplitudes of Ca(i)(2+) transients and contractions were reduced, TD(50%) of the Ca(i)(2+) transient was prolonged, and the inotropic effect of Iso on Ca(i)(2+) transients was reduced approximately twofold versus WT myocytes. Protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and phospholamban was unaltered in TG versus WT hearts, suggesting functional origins of impaired Ca(2+) handling in the former. These results indicate that cardiac-specific overexpression of TNF-alpha induces myocyte hypertrophy and gender-dependent alterations in Ca(i)(2+) handling and contractile function, which may at least partly account for changes in LV geometry and in vivo cardiac function in this model.     

Voltage-independent changes in L-type Ca(2+) current uncoupled from SR Ca(2+) release in cardiac myocytes

To determine the effect of voltage-independent alterations of L-type Ca(2+) current (I(Ca)) on the sarcoplasmic reticular (SR) Ca(2+) release in cardiac myocytes, we measured I(Ca) and cytosolic Ca(2+) transients (Ca(i)(2+); intracellular Ca(2+) concentration) in voltage-clamped rat ventricular myocytes during 1) an abrupt increase of extracellular [Ca(2+)] (Ca(o)(2+)) or 2) application of 1 microM FPL-64176, a Ca(2+) channel agonist, to selectively alter I(Ca) in the absence of changes in SR Ca(2+) loading. On the first depolarization in higher Ca(o)(2+), peak I(Ca) was increased by 46 +/- 6% (P < 0.001), but the increases in the maximal rate of rise of Ca(i)(2+) (dCa(i)(2+)/dt(max), where t is time; an index of SR Ca(2+) release flux) and the Ca(i)(2+) transient amplitude were not significant. Rapid exposure to FPL-64176 greatly slowed inactivation of I(Ca), increasing its time integral by 117 +/- 8% (P < 0.001) without significantly increasing peak I(Ca), dCa(i)(2+)/dt(max), or amplitude of the corresponding Ca(i)(2+) transient. Prolongation of exposure to higher Ca(o)(2+) or FPL-64176 did not further increase peak I(Ca) but greatly increased dCa(i)(2+)/dt(max), Ca(i)(2+) transient amplitude, and the gain of Ca(2+) release (dCa(i)(2+)/dt(max)/I(Ca)), evidently due to augmentation of the SR Ca(2+) loading. Also, the time to peak dCa(i)(2+)/dt(max) was significantly increased in the continuous presence of higher Ca(o)(2+) (by 37 +/- 5%, P < 0.001) or FPL-64176 (by 63 +/- 5%, P < 0.002). Our experiments provide the first evidence of a marked disparity between an increased peak I(Ca) and the corresponding SR Ca(2+) release. We attribute this to saturation of the SR Ca(2+) release flux as predicted by local control theory. Prolongation of the SR Ca(2+) release flux, caused by combined actions of a larger I(Ca) and maximally augmented SR Ca(2+) loading, might reflect additional Ca(2+) release from corbular SR.     

Intracellular calibration of the calcium indicator indo-1 in isolated fibers of Xenopus muscle.

Estimates of the free myoplasmic [Ca2+] ([Ca2+]i) with fluorescent dyes are complicated by the fact that some properties of these dyes are altered in the intracellular environment. In the present study indo-1 was used to measure [Ca2+]i in isolated muscle fibers from Xenopus frogs. Fluorescent ratio signals obtained from indo-1 were converted into [Ca2+]i by means of an intracellular calibration method, which involved microinjection of 0.5 M EGTA and 1 M CaCl2 to get the ratio at very low (Rmin) and high (Rmax) [Ca2+], respectively; ratios at intermediate [Ca2+] were obtained by injection of solutions with different EGTA/Ca(2+)-EGTA proportions. This calibration gave an intracellular Ca2+ dissociation constant of indo-1 of 311 nM and a [Ca2+]i at rest of 52 +/- 4 nM (mean +/- SE; n = 15). Indo-1 records during twitches were compared with records obtained with the much faster indicator mag-indo-1. This analysis suggests a Ca2+ dissociation rate of indo-1 of 52 s-1 (22 degrees C). This makes indo-1 less suitable for measurements of [Ca2+]i during twitches, whereas it is fast enough to follow most aspects of [Ca2+]i during tetani, including the relaxation phase.     

Temporal and spatial properties of cellular Ca2+ flux in trout ventricular myocytes

Confocal microscopy was used to investigate the temporal and spatial properties of Ca(2+) transients and Ca(2+) sparks in ventricular myocytes of the rainbow trout (Oncorhynchus mykiss). Confocal imaging confirmed the absence of T tubules and the long ( approximately 160 microm), thin ( approximately 8 microm) morphology of trout myocytes. Line scan imaging of Ca(2+) transients evoked by electrical stimulation in cells loaded with fluo 4 revealed spatial inhomogeneities in the temporal properties of Ca(2+) transients across the width of the myocytes. The Ca(2+) wavefront initiated faster, rose faster, and reached larger peak amplitudes in the periphery of the myocyte compared with the center. These differences were exacerbated by stimulation with the L-type Ca(2+) channel agonist (-)BAY K 8644 or by sarcoplasmic reticulum (SR) inhibition with ryanodine and thapsigargin. Results reveal that the shape of the trout myocyte allows for rapid diffusion of Ca(2+) from the cell periphery to the cell center, with SR Ca(2+) release contributing to the cytosolic Ca(2+) rise in a time-dependent manner. Spontaneous Ca(2+) sparks were exceedingly rare in trout myocytes under control conditions (1 sparking cell from 238 cells examined). This is in marked contrast to the rat where a total of 56 spontaneous Ca(2+) sparks were observed in 9 of 11 myocytes examined. Ca(2+) sparklike events were observed in a very small number of trout myocytes (15 sparks from 9 of 378 cells examined) after stimulation with either (-)BAY K 8644 or high Ca(2+) (6 mM). Reducing temperature to 15 degrees C in intact myocytes or permeabilizing myocytes to adjust intracellular conditions to favor Ca(2+) spark detection was without significant effects. Possible reasons for the rarity of Ca(2+) sparks in a cardiac myocyte with an active SR are discussed.     

Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution

Recent developments in microscopy and fluorescent indicators now make it possible to monitor the activity and localization of membrane ion channels by imaging Ca(2+) flux through individual channels. Such optical approaches have advantages over electrophysiological single-channel techniques in that they are less invasive, provide spatial information and can simultaneously and independently monitor hundreds of channels. However, their kinetic resolution does not yet approach that of patch-clamp recordings. To help understand the processes that determine the temporal resolution and noise level of single-channel Ca(2+) fluorescence signals (SCCaFTs), we simulated the microdomains of Ca(2+) ions and Ca(2+)-bound indicator dye that exist around the mouth of an open channel. Further, as an aid to development of improved optical techniques, we modeled the dependence of the amplitude and kinetics of SCCaFTs on parameters such as the imaging volume, the indicator concentration, affinity and mobility, and the presence of endogenous and exogenous Ca(2+) buffers. The results indicate that under optimal conditions, including the use of confocal or total-internal reflection microscopy to image from sub-femtolitre volumes, SCCaFTs should resolve channel openings as brief as 1ms with a signal-to-noise ratio >10.     

Profound cardioprotection with timolol in a female rat model of aging-related altered left ventricular function

Increasing evidence shows a marked beneficial effect with β-blockers in heart dysfunction via scavenging reactive oxygen species. Previously we showed that chronic treatment with either timolol or propranolol possessed similar beneficial effects for heart function in male rats as age increased, whereas only timolol exerted similar benefits in female rats. Therefore, in this study, we aimed first to examine the cellular bases for age-related alterations in excitation-contraction coupling in ventricular myocytes from female rats and, second, to investigate the hypothesis that age-related changes in [Ca(2+)](i) homeostasis and receptor-mediated system can be prevented with chronic timolol treatment. Chronic timolol treatment of 3-month-old female rats abolished age-related decrease in left ventricular developed pressure and the attenuated responses to β-adrenoreceptor stimulation. It also normalized the altered parameters of [Ca(2+)](i) transients, decreased Ca(2+) loading of sarcoplasmic reticulum and increased basal [Ca(2+)](i), and decreased L-type Ca(2+) currents in 12-month-old female rats compared with the 3-month-old group. Adenylyl cyclase activity, β-adrenoreceptor affinity to its agonist, and β-adrenoreceptor density of the 12-month-old group are normalized to those of the 3-month-old group. Moreover, timolol treatment prevented dysfunction of the antioxidant system, including increased lipid peroxidation, decreased ratio of reduced glutathione to oxidized glutathione, and decreased activities of thioredoxin reductase and glucose-6-phosphate dehydrogenase, in the left ventricle of hearts from the 12-month-old group. Our data confirmed that aging-related early myocardial impairment is primarily related to a dysfunctional antioxidant system and impairment of Ca(2+) homeostasis, which can be prevented with chronic timolol treatment.     

Glutamate pretreatment affects Ca2+ signaling in processes of astrocyte pairs

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca(2+) imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) in the processes of cultured rat cortical astrocytes existing as pairs. Transient Ca(2+) changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, Ca(2+) transients were recorded following pretreatment of cells with glutamate (500 microM for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl(4-carboxy-phenyl) glycine (MCPG). Estimation of local Ca(2+) diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the Ca(2+) transients in the heparin-loaded astrocyte but attenuated the [Ca(2+)](i) responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate (IP(3)) may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the [Ca(2+)](i) responses in both the control and glutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamate-induced alterations in Ca(2+) signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), IP(3), internal Ca(2+) stores, VGCC and gap junction channels appear to play an important role.     

Abnormalities in gap junctions and Ca2+ dynamics in cardiomyocytes at the border zone of myocardial infarcts

Abnormalities in gap junction function and Ca2+ dynamics are believed to be important factors in arrhythmogenesis after myocardial infarction. To elucidate the relationship between changes in Ca2+ dynamics and gap junctions, we analyzed by real-time in situ Ca2+ imaging of fluo-3 loaded whole hearts the spatiotemporal occurrence of Ca2+ waves and the localization of connexin43 (Cx43) at the border zone of myocardial infarcts induced in the rat by coronary ligation. At early time points (2-4 hours postligation), different regions of the left ventricle showed distinct changes in cytosolic free Ca2+ concentrations [Ca2+]i. While some cardiomyocytes of infarcted regions exhibited high levels of resting fluo-3 fluorescence, at border zones frequent Ca2+ waves were observed. Some of the waves were abolished by spontaneous Ca2+ transients and others were not. Intact myocardium apart from infarcted regions exhibited homogenous Ca2+ transients. Confocal imaging of Cx43 and actin filaments in the rat heart fixed 2 hours after coronary ligation revealed that Cx43 was markedly decreased in the area of myocyte necrosis with contraction bands and in the neighboring myocardium. These results suggest that abnormal expression and function of gap junctions could be associated with Ca2+ waves at the border zone of myocardial infarcts, possibly through Ca2+ overload.     

Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques

The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).     

Neuroendocrine properties of intrinsic cardiac adrenergic cells in fetal rat heart

Intrinsic cardiac adrenergic (ICA) cells in developing rat heart constitute a novel adrenergic signaling system involved in cardiac regulation. Regulatory mechanisms of ICA cells remain to be defined. Immunohistochemical study of fetal rat hearts demonstrated ICA cells with catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). The mRNA of TH and PNMP was also detected in fetal rat hearts before sympathetic innervation. Immunoreactivity of norepinephrine transporter (NET) was localized to ICA cells in rat heart tissue and primary cell culture. For the functional study, the activity of intracellular Ca2+ concentration ([Ca2+]i) transients was quantified by a ratio fluorescent spectrometer in cultured ICA cells and myocytes. ICA cells generated spontaneous [Ca2+]i transients that were eliminated by tetrodotoxin or Ca(2+)-free solutions and showed greatly reduced amplitude with the addition of L-type Ca2+ channel blocker nifedipine. [3H]norepinephrine studies demonstrate release and uptake of norepinephrine. Functional interaction between catecholamines produced by the ICA cells and cocultured myocytes was evident by the effect of the beta-adrenergic blocker atenolol eliciting a dose-dependent reduction in the amplitude and frequency of [Ca2+]i transients of beating myocytes. Hypoxia inhibited [Ca2+]i transient activity of ICA cells, which subsequently produced a reoxygenation-mediated rebound augmentation of [Ca2+]i transients. We conclude that ICA cells are capable of catecholamine synthesis, release, and uptake. They generate spontaneous [Ca2+]i transient activity that can be regulated by oxygen tension. ICA cells may provide an alternative adrenergic supply to maintain cardiac contractile and pacemaker function at rest and during stress in the absence of sympathetic innervation.     

Effects of Na(+)/Ca(2+) exchanger downregulation on contractility and [Ca(2+)](i) transients in adult rat myocytes

Postmyocardial infarction (MI) rat myocytes demonstrated depressed Na(+)/Ca(2+) exchange (NCX1) activity, altered contractility, and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients. We investigated whether NCX1 downregulation in normal myocytes resulted in contractility changes observed in MI myocytes. Myocytes infected with adenovirus expressing antisense (AS) oligonucleotides to NCX1 had 30% less NCX1 at 3 days and 66% less NCX1 at 6 days. The half-time of relaxation from caffeine-induced contracture was twice as long in ASNCX1 myocytes. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase abundance, SR Ca(2+) uptake, resting membrane potential, action potential amplitude and duration, L-type Ca(2+) current density and cell size were not affected by ASNCX1 treatment. At extracellular Ca(2+) concentration ([Ca(2+)](o)) of 5 mM, ASNCX1 myocytes had significantly lower contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents than control myocytes. At 0.6 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents were significantly higher in ASNCX1 myocytes. At 1.8 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes were not different between control and ASNCX1 myocytes. This pattern of contractile and [Ca(2+)](i) transient abnormalities in ASNCX1 myocytes mimics that observed in rat MI myocytes. We conclude that downregulation of NCX1 in adult rat myocytes resulted in decreases in both Ca(2+) influx and efflux during a twitch. We suggest that depressed NCX1 activity may partly account for the contractile abnormalities after MI.     

Calcium dynamics associated with a single action potential in a CNS presynaptic terminal.

Calcium dynamics associated with a single action potential were studied quantitatively in the calyx of Held, a large presynaptic terminal in the rat brainstem. Terminals were loaded with different concentrations of high- or low-affinity Ca2+ indicators via patch pipettes. Spatially averaged Ca2+ signals were measured fluorometrically and analyzed on the basis of a single compartment model. A single action potential led to a total Ca2+ influx of 0.8-1 pC. The accessible volume of the terminal was about 0.4 pl; thus the total calcium concentration increased by 10-13 microM. The Ca(2+)-binding ratio of the endogenous buffer was about 40, as estimated from the competition with Fura-2, indicating that 2.5% of the total calcium remained free. This is consistent with the peak increase in free calcium concentration of about 400 nM, which was measured directly with MagFura-2. The decay of the [Ca2+]i transients was fast, with time constants of 100 ms at 23 degrees C and 45 ms at 35 degrees C, indicating Ca2+ extrusion rates of 400 and 900 s-1, respectively. The combination of the relatively low endogenous Ca(2+)-binding ratio and the high rate of Ca2+ extrusion provides an efficient mechanism for rapidly removing the large Ca2+ load of the terminal evoked by an action potential.     

A ryanodine receptor-dependent Ca(i)(2+) asymmetry at Hensen's node mediates avian lateral identity

In mouse, the establishment of left-right (LR) asymmetry requires intracellular calcium (Ca(i)(2+)) enrichment on the left of the node. The use of Ca(i)(2+) asymmetry by other vertebrates, and its origins and relationship to other laterality effectors are largely unknown. Additionally, the architecture of Hensen's node raises doubts as to whether Ca(i)(2+) asymmetry is a broadly conserved mechanism to achieve laterality. We report here that the avian embryo uses a left-side enriched Ca(i)(2+) asymmetry across Hensen's node to govern its lateral identity. Elevated Ca(i)(2+) was first detected along the anterior node at early HH4, and its emergence and left-side enrichment by HH5 required both ryanodine receptor (RyR) activity and extracellular calcium, implicating calcium-induced calcium release (CICR) as the novel source of the Ca(i)(2+). Targeted manipulation of node Ca(i)(2+) randomized heart laterality and affected nodal expression. Bifurcation of the Ca(i)(2+) field by the emerging prechordal plate may permit the independent regulation of LR Ca(i)(2+) levels. To the left of the node, RyR/CICR and H(+)V-ATPase activity sustained elevated Ca(i)(2+). On the right, Ca(i)(2+) levels were actively repressed through the activities of H(+)K(+) ATPase and serotonin-dependent signaling, thus identifying a novel mechanism for the known effects of serotonin on laterality. Vitamin A-deficient quail have a high incidence of situs inversus hearts and had a reversed calcium asymmetry. Thus, Ca(i)(2+) asymmetry across the node represents a more broadly conserved mechanism for laterality among amniotes than had been previously believed.     

Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes.

Previous studies in adult myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated abnormal contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) homeostasis and decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) expression and activity, but sarcoplasmic reticulum Ca(2+) leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. Compared with sham-operated hearts, 3-wk MI hearts exhibited significantly higher left ventricular end-diastolic and end-systolic volumes but lower fractional shortening and ejection fraction, as measured by M-mode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3-wk MI myocytes did not affect Na(+)-Ca(2+) exchanger expression but restored the depressed SERCA2 levels toward those measured in sham myocytes. In addition, the reduced sarcoplasmic reticulum Ca(2+) uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca(2+) concentration of 5 mM, the subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6 mM extracellular Ca(2+) concentration, the supernormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca(2+)](i) transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca(2+) transport pathways, e.g., Na(+)-Ca(2+) exchanger, may also play an important role in contractile and [Ca(2+)](i) homeostatic abnormalities in MI myocytes.     

Altered spatial calcium regulation enhances electrical heterogeneity in the failing canine left ventricle: implications for electrical instability

Myocytes across the left ventricular (LV) wall of the mammalian heart are known to exhibit heterogeneity of electrophysiological properties; however, the transmural variation of cellular electrophysiology and Ca(2+) homeostasis in the failing LV is incompletely understood. We studied action potentials (APs), the L-type calcium (Ca(2+)) current (I(Ca,L)), and intracellular Ca(2+) transients ([Ca(2+)](i)) of subendocardial (Endo), midmyocardial (Mid), and subepicardial (Epi) tissue layers in the canine normal and tachycardia pacing-induced failing left ventricles. Heart failure (HF) was associated with significant prolongation of the AP duration in Mid myocytes. There were no differences in I(Ca,L) density in normal Endo, Mid, and Epi myocytes, whereas in the failing heart, I(Ca,L) density was downregulated by 45% and 26% (at +10 mV) in Endo and Mid myocytes, respectively. The rates of sarcoplasmic reticulum (SR) Ca(2+) release and decay of the [Ca(2+)](i) were slowed, and the amplitude of the [Ca(2+)](i) was depressed in Endo and Epi myocytes isolated from failing, compared with normal, hearts. Experiments in sodium (Na(+))-free solutions showed that Epi and Mid myocytes of the failing ventricle exhibit a greater reliance on the Na(+)-Ca(2+) exchanger to remove cytosolic Ca(2+) than myocytes isolated from normal hearts. Simulation studies in Endo, Mid, and Epi canine myocytes demonstrate the importance of L-type current density and SR Ca(2+) uptake in modulating the potentially arrhythmogenic repolarization in HF. In conclusion, these results demonstrate that spatially heterogeneous decreases in I(Ca,L) and defective cytosolic Ca(2+) removal contribute to the altered [Ca(2+)](i) and AP profiles across the canine failing LV. These distinct electrophysiological features in myocytes from a failing heart contribute to a characteristic electrogram arising from increased dispersion of refractoriness across the LV, which may result in significant arrhythmogenic sequellae.     

Adrenergic stimulation of rat resistance arteries affects Ca(2+) sparks, Ca(2+) waves, and Ca(2+) oscillations

Confocal laser scanning microscopy and fluo 4 were used to visualize local and whole cell Ca(2+) transients within individual smooth muscle cells (SMC) of intact, pressurized rat mesenteric small arteries during activation of alpha1-adrenoceptors. A method was developed to record the Ca(2+) transients within individual SMC during the changes in arterial diameter. Three distinct types of "Ca(2+) signals" were influenced by adrenergic activation (agonist: phenylephrine). First, asynchronous Ca(2+) transients were elicited by low levels of adrenergic stimulation. These propagated from a point of origin and then filled the cell. Second, synchronous, spatially uniform Ca(2+) transients, not reported previously, occurred at higher levels of adrenergic stimulation and continued for long periods during oscillatory vasomotion. Finally, Ca(2+) sparks slowly decreased in frequency of occurrence during exposure to adrenergic agonists. Thus adrenergic activation causes a decrease in the frequency of Ca(2+) sparks and an increase in the frequency of asynchronous wavelike Ca(2+) transients, both of which should tend to decrease arterial diameter. Oscillatory vasomotion is associated with spatially uniform synchronous oscillations of cellular [Ca(2+)] and may have a different mechanism than the asynchronous, propagating Ca(2+) transients.