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1.
Edward G LAKATTA 《中国科学:生命科学英文版》2004,47(1):31-37
Ca2+ sparks are the elementary events of intracellular Ca2+ release from the sar-coplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca2+ channel activation contributes to the genesis of spontaneous Ca2+ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca2+ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI2 which inhibits spontaneous L-type Ca2+ channel activation, the rate of occurrence of spontaneous Ca2+ sparks was halved from 4.20 to 2.04 events/(100 μm·s), with temporal and spatial properties of individual Ca2+ sparks unchanged. Analysis of the Cd2+-sensitive spark production revealed an open probability of-10-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca2+ channels in resting heart cells contribute significantly to the production of spontaneous Ca2+ sparks. 相似文献
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The patterning of cardiac myocytes on a micron scale ( approximately 5 microm) was achieved by microcontact printing of fibronectin onto a hydrophobically pretreated glass substrate. The patterned cardiac myocytes conjugated with each other by forming a gap junction, as judged from the synchronized Ca(2+) transition over the pattern, and thus simultaneously contracted. The dynamic change of the Ca(2+) concentration within the patterned tissue was analyzed quantitatively during successive contraction and relaxation using a Nipkow-type high-speed confocal microscope. 相似文献
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Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of fast and slow skeletal and cardiac muscles 总被引:3,自引:0,他引:3
Crystalline arrays of Ca2+ transport ATPase develop in sarcoplasmic reticulum membranes after treatment with Na3VO4 in a calcium-free medium [ Dux , L. and Martonosi , A. (1983) J. Biol. Chem. 258, 2599-2603]. The proportion of vesicles containing Ca2+-ATPase crystals in microsome preparations isolated from rat muscle of different fiber types (semimembranosus, levator ani, extensor digitorum longus, diaphragm, soleus, and heart) correlates well with the Ca2+-ATPase content and Ca2+-modulated ATPase activity. This implies that the concentration of Ca2+-ATPase in sarcoplasmic reticulum membranes of fast and slow skeletal or cardiac muscles differs only slightly, and the low Ca2+ transport activity of 'sarcoplasmic reticulum' preparations isolated from slow-twitch skeletal and cardiac muscles is due to the presence of large amount of non-sarcoplasmic-reticulum membrane elements. This is in accord with the relatively small differences in the density of 8.5-nm intramembranous particles seen by freeze-etch electron microscopy in sarcoplasmic reticulum of red and white muscles. The dimensions of the Ca2+-ATPase crystal lattice are similar in sarcoplasmic reticulum membranes of different fiber types; therefore if structural differences exist between 'isoenzymes' of Ca2+-ATPase, these are not reflected in the crystal-lattice. 相似文献
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Snopko RM Aromolaran AS Karko KL Ramos-Franco J Blatter LA Mejía-Alvarez R 《Cell calcium》2007,41(1):13-25
In heart, the excitation-contraction coupling (ECC) mechanism changes during development. Primary cell culture has been used to study Ca(2+) signaling in newborn (NB) rat heart. In this work, the effects of cell culture on the action potential (AP) and ECC Ca(2+) signaling during development were investigated. Specifically, AP, Ca(2+) currents (I(Ca)), and ryanodine receptor (RyR) properties (i.e. density, distribution, and contribution to Ca(2+) transients and Ca(2+) sparks) were defined in cultured myocytes (CM) from 0-day-old NB rat at different times in culture (1-4 days). Compared with acutely dissociated myocytes (ADM) from NB of equivalent ages (1-4 days), CM showed lower RyR density (50% at 1 day, 25% at 4 days), but larger RyR contribution to the Ca(2+) transient (25% at 1 day, 57% at 4 days). Additionally, Ca(2+) sparks were larger, longer, wider, and more frequent in CM than in ADM. RyR cellular distribution also showed different arrangement. While in CM, RyRs were located peripherally, in ADM of equivalent ages a sarcomeric arrangement was predominant. Finally, CM showed a two-fold increase in sarcolemmal Ca(2+) entry during the AP. These results indicated that primary culture is a feasible model to study Ca(2+) signaling in heart; however, it does not precisely reproduce what occurs in ECC during development. 相似文献
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The localization of S100A1 in rat soleus (SOL) and extensor digitorum longus (EDL) muscles was studied immunocytochemically at different sarcomere lengths (stretched, relaxed and contracted) at the ultrastructural level. The muscle fibres were contracted by application of 15 mmol/l caffeine. Following aldehyde fixation, dehydration and embedding in Lowicryl HM20 (-35 degrees C) ultrathin sections were incubated with rabbit polyclonal antiserum against S100A1. Goat antirabbit secondary antibodies conjugated with 10 nm gold particles were used to visualize antigen sites. Relative areas of Z-lines, A- and I-bands were estimated from longitudinal sections by the point counting method. The highest densities of the particles were found at the Z-lines. A higher incidence of S100A1 antigen sites in I-bands than in A-bands and a higher density of S100A1 in lateral parts of A-bands (with actin and myosin filaments overlapping) compared with the central area of A-bands are consistent with an interaction of S100A1 with F-actin in skeletal muscles. Antigen sites were also present at M-lines and at distinct locations of the sarcoplasmic reticulum. 相似文献
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Cardiac muscle excitation-contraction coupling is controlled by the Ca(2+)-induced Ca2+ release mechanism. The present study examines the effects of a calmodulin antagonist W-7 on Ca2+ current (ICa)-induced Ca2+ release in whole cell-clamped rat ventricular myocytes. Exposure of cells to W-7 suppressed ICa, but the intracellular Ca(2+)-transients showed a lesser degree of reduction, suggesting possible enhancement of Ca(2+)-induced Ca2+ release. The effects of W-7 on the efficacy of Ca2+ release were most prominent at negative potentials. At test potentials of -30 mV, 20 microM W-7 almost completely blocked ICa, but significant Ca(2+)-transients remained, thus causing a four to six-fold increase in the efficacy of Ca(2+)-induced Ca2+ release. The depolarization-dependent Ca(2+)-transients were eliminated in absence of extracellular Ca2+, blocked by Cd2+, and were absent when the sarcoplasmic reticulum was depleted of Ca2+, implicating dependency on Ca(2+)-signaling between the L-type channel and the ryanodine receptor. W-7 mediated increase in the efficacy of Ca(2+)-induced Ca2+ release was eliminated when myocytes were dialyzed with the internal solution containing gluathione (5 mM), suggesting the possible role of cellular redox state in the regulation of Ca2+ release by the calmodulin antagonist. 相似文献
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Examining calcium spark morphology and its relationship to the structure of the cardiac myocyte offers a direct means of understanding excitation-contraction coupling mechanisms. Traditional confocal line scanning achieves excellent temporal spark resolution but at the cost of spatial information in the perpendicular dimension. To address this, we developed a methodology to identify and analyze sparks obtained via two-dimensional confocal or charge-coupled device microscopy. The technique consists of nonlinearly subtracting the background fluorescence, thresholding the data on the basis of noise level, and then localizing the spark peaks via a generalized extrema test, while taking care to detect and separate adjacent peaks. In this article, we describe the algorithm, compare its performance to a previously validated spark detection algorithm, and demonstrate it by applying it to both a synthetic replica and an experimental preparation of a two-dimensional isotropic myocyte monolayer exhibiting sparks during a calcium transient. We find that our multidimensional algorithm provides better sensitivity than the conventional method under conditions of temporally heterogeneous background fluorescence, and the inclusion of peak segmentation reduces false negative rates when spark density is high. Our algorithm is robust and can be effectively used with different imaging modalities and allows spark identification and quantification in subcellular, cellular, and tissue preparations. 相似文献
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This study investigates the contribution of Ca2+ entry via sarcolemmal (SL) Ca2+ channels to the Ca2+ transient and its relationship with sarcoplasmic reticulum (SR) Ca2+ content during steady-state contraction in guinea pig and rat ventricular myocytes. The action potential clamp technique was used to obtain physiologically relevant changes in membrane potential. A method is shown that allows calculation of Ca2+ entry through the SL Ca2+ channels by measuring Cd(2+)-sensitive current during the whole cardiac cycle. SR Ca2+ content was calculated from caffeine-induced transient inward current. In guinea pig cardiac myocytes stimulated at 0.5 Hz and 0.2 Hz, Ca2+ entry through SL Ca2+ channels during a cardiac cycle was approximately 30% and approximately 50%, respectively, of the SR Ca2+ content. In rat myocytes Ca2+ entry via SL Ca2+ channels at 0.5 Hz was approximately 3.5% of the SR Ca2+ content. In the presence of 500 nM thapsigargin Ca2+ entry via SL Ca2+ channels in guinea pig cardiac cells was 39% greater than in controls, suggesting a larger contribution of this mechanism to the Ca2+ transient when the SR is depleted of Ca2+. These results provide quantitative support to the understanding of the relationship between Ca2+ entry and the SR Ca2+ content and may help to explain differences in the Ca2+ handling observed in different species. 相似文献
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Giedrius Kanaporis 《Channels (Austin, Tex.)》2016,10(6):507-517
Cardiac alternans, defined beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias. We investigated mechanisms of cardiac alternans in single rabbit ventricular myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. A strong correlation between beat-to-beat alternations of AP morphology and CaT alternans was observed. During CaT alternans application of voltage clamp protocols in form of pre-recorded APs revealed a prominent Ca2+-dependent membrane current consisting of a large outward component coinciding with AP phases 1 and 2, followed by an inward current during AP repolarization. Approximately 85% of the initial outward current was blocked by Cl? channel blocker DIDS or lowering external Cl? concentration identifying it as a Ca2+-activated Cl? current (ICaCC). The data suggest that ICaCC plays a critical role in shaping beat-to-beat alternations in AP morphology during alternans. 相似文献
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Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators. 总被引:1,自引:1,他引:0
Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters. 相似文献
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Yatani A Irie K Otani T Abdellatif M Wei L 《American journal of physiology. Heart and circulatory physiology》2005,288(2):H650-H659
Regulation of ionic channels plays a pivotal role in controlling cardiac function. Here we show that the Rho family of small G proteins regulates L-type Ca2+ currents in ventricular cardiomyocytes. Ventricular myocytes isolated from transgenic (TG) mice that overexpress the specific GDP dissociation inhibitor Rho GDI-alpha exhibited significantly decreased basal L-type Ca2+ current density (approximately 40%) compared with myocytes from nontransgenic (NTG) mice. The Ca2+ channel agonist BAY K 8644 and the beta-adrenergic agonist isoproterenol increased Ca2+ currents in both NTG and TG myocytes to a similar maximal level, and no changes in mRNA or protein levels were observed in the Ca2+ channel alpha1-subunits. These results suggest that the channel activity but not the expression level was altered in TG myocytes. In addition, the densities of inward rectifier and transient outward K+ currents were unchanged in TG myocytes. The amplitudes and rates of basal twitches and Ca2+ transients were also similar between the two groups. When the protein was delivered directly into adult ventricular myocytes via TAT-mediated protein transduction, Rho GDI-alpha significantly decreased Ca2+ current density, which supports the idea that the defective Ca2+ channel activity in TG myocytes was a primary effect of the transgene. In addition, expression of a dominant-negative RhoA but not a dominant-negative Rac-1 or Cdc42 also significantly decreased Ca2+ current density, which indicates that inhibition of Ca2+ channel activity by overexpression of Rho GDI-alpha is mediated by inhibition of RhoA. This study points to the L-type Ca2+ channel activity as a novel downstream target of the RhoA signaling pathway. 相似文献
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Yamaoka K Yuki T Kawase K Munemori M Seyama I 《American journal of physiology. Heart and circulatory physiology》2002,282(3):H1092-H1101
We examined the concentration-dependent blocking effects of intracellular Mg2+ on L-type Ca2+ channels in cardiac myocytes using the whole cell patch-clamp technique. The increase of L-type Ca2+ channel current (I(Ca)) (due to relief of Mg2+ block) occurred in two temporal phases. The rapid phase (runup) transiently appeared early (<5 min) in dialysis of the low-Mg2+ solution; the slow phase began later in dialysis (>10 min). Runup was not blocked by intracellular GTP (GTP(i)). The late phase of the I(Ca) increase (late I(Ca)) was suppressed by GTP(i) (0.4 mM) and was observed in myocytes of the guinea pig or frog at higher (32 or 24 degrees C, respectively) rather than lower temperatures (24 or 17.5 degrees C, respectively). At pMg = 6.0, raising the temperature from 24 to 32 degrees C evoked late I(Ca) with a Q10 of 14.5. Restoring the temperature to 24 degrees C decreased I(Ca) with a Q10 of only 2.4. The marked difference in the Q10 values indicated that late I(Ca) (pMg = 5-6) is an irreversible phenomenon. Phosphorylation suppressed the intracellular [Mg2+] dependency of late I(Ca). This effect of phosphorylation together with the inhibitory action of GTP(i) on Mg2+-dependent blocking of I(Ca) are common properties of mammalian and amphibian cardiomyocytes. 相似文献
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Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes 总被引:1,自引:0,他引:1
Huang J van Breemen C Kuo KH Hove-Madsen L Tibbits GF 《American journal of physiology. Cell physiology》2006,290(6):C1572-C1582
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 (loadSR) 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 loadSR at rest. Interestingly, inhibition of forward mode NCX with 30 µM KB-R7943 stimulated SOCE significantly and resulted in enhanced loadSR. 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 相似文献
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1. We studied the effect of verapamil, nitrendipine, 3',4'-dichlorobenzamil (DCB) and Cd2+ on the increase in cytosolic free Ca2+ ([Ca2+]c) and the rate of O2-uptake induced by depolarization of isolated rat cardiac myocytes with veratridine. 2. The degree of inhibition by the several drugs tested on the increase in [Ca2+]c and respiration was dependent on extracellular Ca2+, pH and Na+. 3. Low verapamil and nitrendipine concentrations (2.5 microM) were fully effective in Ca2+ channel blockade, as indicated from experiments with isoproterenol and in a low-Na+ medium. 4. A complete inhibition of veratridine-induced increase in [Ca2+]c and O2-uptake was attained with higher Ca2+ blocker concentrations (25-30 microM), implying that these processes depend to a major extent on some other Ca2+ transport system, probably Na+/Ca2+ exchange. 相似文献
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Markevich NI Korystova AF Grichenko AS Lankina DA Kokoz YM 《Membrane & cell biology》2000,14(1):109-127
The regulation of L-type Ca2+ current in isolated rat cardiac cells was studied using the perforated patch-clamp technique. A dual effect of the cAMP-dependent phosphorylation activator, isoproterenol, at different holding potentials (V(h)) was shown. The currents increased at V(h) = -50 mV and decreased at V(h) = -30 mV. A dihydropyridine agonist, BAY K 8644, and isoproterenol had an additive effect on the activation of Ca2+ channels at holding potentials close to the resting potential. The additivity was disturbed at more positive V(h). The activating effect of BAY K 8644 did not virtually change in the presence of a protein kinase blocker, H8, and a phosphatase activator, acetylcholine. The results were interpreted within the framework of a two-site phosphorylation model with two independent pathways of Ca2+ current regulation. 相似文献
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The measurements of the sarcomere length in dissociated cardiac ventricular myocytes are discussed using mainly our own experimental data. The striation periodicity of these unloaded cells was found to be that which is to be expected of a myocyte free of the ultrastructural constraints imposed upon it by the normal syncytial matrix of the ventricular wall. The sarcomere length and [Ca(2+)] relationship was consistent as expected from the intact tissue, when it was measured soon after partial rupturing the cell membrane. Miniature fluctuations of individual sarcomere length were demonstrated during rest, which was augmented by the Ca(2+) overload. The [Ca(2+)] could be estimated from the measurements of sarcomere length during the positive staircase of contraction. The usefulness of the optical measurement of sarcomere pattern was indicated. 相似文献