Single-channel gating and regulation of human L-type calcium channels in cardiomyocytes of transgenic mice

Overexpression of human cardiac L-type Ca(2+) channel pores (hCa(v)1.2) in mice causes heart failure. Earlier studies showed Ca(v)1.2-mRNA increase by 2.8-fold, but whole-cell current density enhancement by 相似文献   

Calcium-transporting system of erythrocytes in psoriasis

The activity of Ca-ATPase and permeability of erythrocyte membrane for calcium in patients with psoriasis were studied with the aim to reveal disturbances in the calcium membrane transport under psoriasis. In the presence of endogenic activators the mean values of the maximal Ca-ATPase activity of erythrocyte membranes in patients with psoriasis and in healthy people have no essential differences and make up 264 +/- 12 and 244 +/- 10 mumol P/1 cells per 1 min, respectively. The rate of 45Ca accumulation in erythrocytes under inhibition of Ca-ATPase in patients suffering from psoriasis is by 64% higher than in healthy people. The data obtained along with the previously revealed changes in the calcium metabolism in patients with psoriasis make it possible to suppose the presence of the system disturbance of the calcium membrane transport, in particular an increase in the plasma membrane permeability for cells of different types. Such a disturbance may distort a regulatory (messenger) function of calcium ions in the processes of proliferation, differentiation, functional activity and death of different cell types.     

Measuring fast calcium fluxes in cardiomyocytes

Cardiomyocytes have multiple Ca(2+) fluxes of varying duration that work together to optimize function (1,2). Changes in Ca(2+) activity in response to extracellular agents is predominantly regulated by the phospholipase Cβ- Gα(q;) pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine (3,4). We have recently found that plasma membrane protein domains called caveolae(5,6) can entrap activated Gα(q;)(7). This entrapment has the effect of stabilizing the activated state of Gα(q;) and resulting in prolonged Ca(2+) signals in cardiomyocytes and other cell types(8). We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca(2+) indicator. In our studies, we used Ca(2+) Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca(2+) responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca(2+) waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca(2+) waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca(2+)fluxes in the cells, and to determine their impact on various cellular events.     

Voltage-gated calcium channel blockers deregulate macroautophagy in cardiomyocytes

Voltage-gated calcium channel blockers are widely used for the management of cardiovascular diseases, however little is known about their effects on cardiac cells in vitro.We challenged neonatal ventricular cardiomyocytes (CMs) with therapeutic L-type and T-type Ca²⁺ channel blockers (nifedipine and mibefradil, respectively), and measured their effects on cell stress and survival, using fluorescent microscopy, Q-PCR and Western blot. Both nifedipine and mibefradil induced a low-level and partially transient up-regulation of three key mediators of the Unfolded Protein Response (UPR), indicative of endoplasmic (ER) reticulum stress. Furthermore, nifedipine triggered the activation of macroautophagy, as evidenced by increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), decreased levels of polyubiquitin-binding protein p62/SQSTM1 and ubiquitinated protein aggregates, that was followed by cell death. In contrast, mibefradil inhibited CMs constitutive macroautophagy and did not promote cell death. The siRNA-mediated gene silencing approach confirmed the pharmacological findings for T-type channels.We conclude that L-type and T-type Ca²⁺ channel blockers induce ER stress, which is divergently transduced into macroautophagy induction and inhibition, respectively, with relevance for cell viability. Our work identifies VGCCs as novel regulators of autophagy in the heart muscle and provides new insights into the effects of VGCC blockers on CMs homeostasis, that may underlie both noxious and cardioprotective effects.     

Epigenetic regulation of neonatal cardiomyocytes differentiation

The relationship between DNA methylation, histone modifications and terminal differentiation in cardiomyocytes was investigated in this study. The upregulation of methylation-related proteins, including DNA methyltransferase (DNMT) 1, methyl-CpG binding domain proteins 1, 2 and 3, and the increase in global methylation during rat neonatal heart development were observed. Moreover, an increase in DNA synthesis and a delay in differentiation were found in 5-azacytidine (5-azaC)-treated cardiomyocytes. Increase in acetylation of H3-K9, H4-K5, H4-K8 and methylation of H3-K4 suggested a more accessible chromatin structure in 5-azaC-treated cells. Furthermore, methyl-CpG-binding protein 2 was found to be upregulated in differentiated cardiomyocytes. Overexpression of this protein resulted in an increase of global methylation levels. Therefore, we suggest that a hypermethylated genome and a more compact chromatin structure are formed during terminal differentiation of cardiomyocytes.     

ATP regulation in adult rat cardiomyocytes: time-resolved decoding of rapid mitochondrial calcium spiking imaged with targeted photoproteins

The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand have not been fully elucidated. Here we used an adenoviral system to express the photoproteins luciferase and aequorin, targeted to the mitochondria or cytosol of adult cardiomyocytes, to investigate the interrelationship between ATP and Ca(2+) in these compartments. In neither compartment were changes in free [ATP] observed upon increased workload (addition of isoproterenol) in myocytes that were already beating. However, when myocytes were stimulated to beat rapidly from rest, in the presence of isoproterenol, a significant but transient drop in mitochondrial [ATP] ([ATP](m)) occurred (on average to 10% of the initial signal). Corresponding changes in cytosolic [ATP] ([ATP](c)) were much smaller (<5%), indicating that [ATP](c) was effectively buffered in this compartment. Although mitochondrial [Ca(2+)] ([Ca(2+)](m)) is an important regulator of respiratory chain activity and ATP production in other cells, the kinetics of mitochondrial Ca(2+) transport are controversial. Parallel experiments in cells expressing mitochondrial aequorin showed that the drop in [ATP](m) occurred over the same time scale as average [Ca(2+)](m) was increasing. Conversely, in the absence or presence of isoproterenol, clear beat-to-beat peaks in [Ca(2+)](m) were observed at 0.9 or 1.3 mum, respectively, concentrations similar to those observed in the cytosol. These results suggest that mitochondrial Ca(2+) transients occur during the contractile cycle and are translated into a time-averaged increase in mitochondrial ATP production that keeps pace with increased cytosolic demand.     

Growth hormone secretagogues protect mouse cardiomyocytes from in vitro ischemia/reperfusion injury through regulation of intracellular calcium

Background

Ischemic heart disease is a leading cause of mortality. To study this disease, ischemia/reperfusion (I/R) models are widely used to mimic the process of transient blockage and subsequent recovery of cardiac coronary blood supply. We aimed to determine whether the presence of the growth hormone secretagogues, ghrelin and hexarelin, would protect/improve the function of heart from I/R injury and to examine the underlying mechanisms.

Methodology/Principal Findings

Isolated hearts from adult male mice underwent 20 min global ischemia and 30 min reperfusion using a Langendorff apparatus. Ghrelin (10 nM) or hexarelin (1 nM) was introduced into the perfusion system either 10 min before or after ischemia, termed pre- and post-treatments. In freshly isolated cardiomyocytes from these hearts, single cell shortening, intracellular calcium ([Ca²⁺]_i) transients and caffeine-releasable sarcoplasmic reticulum (SR) Ca²⁺ were measured. In addition, RT-PCR and Western blots were used to examine the expression level of GHS receptor type 1a (GHS-R1a), and phosphorylated phospholamban (p-PLB), respectively. Ghrelin and hexarelin pre- or post-treatments prevented the significant reduction in the cell shortening, [Ca²⁺]_i transient amplitude and caffeine-releasable SR Ca²⁺ content after I/R through recovery of p-PLB. GHS-R1a antagonists, [D-Lys3]-GHRP-6 (200 nM) and BIM28163 (100 nM), completely blocked the effects of GHS on both cell shortening and [Ca²⁺]_i transients.

Conclusion/Significance

Through activation of GHS-R1a, ghrelin and hexarelin produced a positive inotropic effect on ischemic cardiomyocytes and protected them from I/R injury probably by protecting or recovering p-PLB (and therefore SR Ca²⁺ content) to allow the maintenance or recovery of normal cardiac contractility. These observations provide supporting evidence for the potential therapeutic application of ghrelin and hexarelin in patients with cardiac I/R injury.     

Store-operated calcium entry is present in HL-1 cardiomyocytes and contributes to resting calcium

Store-operated Ca(2+) entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca(2+) during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells expressed stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE was tightly coupled to sarcoplasmic reticulum (SR)-Ca(2+) release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca(2+) channels (L-type and T-type channels) or reverse mode Na(+)/Ca(2+) exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca(2+) and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca(2+) homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart.     

Adult rat cardiomyocytes exhibit capacitative calcium entry

Capacitative Ca(2+) entry (CCE) refers to the influx of Ca(2+) through plasma membrane channels activated on depletion of endoplasmic-sarcoplasmic reticulum Ca(2+) stores. We utilized two Ca(2+)-sensitive dyes (one monitoring cytoplasmic free Ca(2+) and the other free Ca(2+) within the sarcoplasmic reticulum) to determine whether adult rat ventricular myocytes exhibit CCE. Treatments with inhibitors of the sarcoplasmic endoplasmic reticulum Ca(2+)-ATPases were not efficient in releasing Ca(2+) from stores. However, when these inhibitors were coupled with either Ca(2+) ionophores or angiotensin II (an agonist generating inositol 1,4,5 trisphosphate), depletion of stores was observed. This depletion was accompanied by a significant influx of extracellular Ca(2+) characteristic of CCE. CCE was also observed when stores were depleted with caffeine. This influx of Ca(2+) was sensitive to four inhibitors of CCE (glucosamine, lanthanum, gadolinium, and SKF-96365) but not to inhibitors of L-type channels or the Na(+)/Ca(2+) exchanger. In the whole cell configuration, an inward current of approximately 0.7 pA/pF at -90 mV was activated when a Ca(2+) chelator or inositol (1,4,5)-trisphosphate was included in the pipette or when Ca(2+) stores were depleted with a Ca(2+)-ATPase inhibitor and ionophore. The current was maximal at hyperpolarizing voltages and inwardly rectified. The channel was relatively permeant to Ca(2+) and Ba(2+) but only poorly to Mg(2+) or Mn(2+). Taken together, these data support the existence of CCE in adult cardiomyocytes, a finding with likely implications to physiological responses to phospholipase C-generating agonists.     

牛磺酸对心肌细胞钙离子的调节作用

牛磺酸占心肌游离氨基酸的５０％以上,它对心肌细胞许多依赖于Ｃａ＾２＋的生理两全是现象具有明显的调节作用。牛磺酸能增加高亲和力Ｃａ＾２＋转驼系统转运Ｃａ＾２＋的量和速度,对要和力Ｃａ＾２＋转运系统的调节作用依胞外Ｃａ＾２＋浓度的不同而不同,具有稳态调节细胞内的Ｃａ＾２＋作用;另外,牛磺酸尚能抑制各种原因经起的胞内Ｃａ＾２＋超负荷,显示其具有细胞保护作用。     

Expression and regulation of chloride channels in neonatal rat cardiomyocytes

Using an ¹²⁵I^– efflux assay, we have studied the expression of various types of chloride channels in isolated neonatal rat cardiomyocytes. Three different classes of anion conductances were distinguished: (1) a Cal²⁺-sensitive Cl^– conductance, triggered upon stimulation of the cells with endothelin-1 or Ca²⁺-ionophore; (2) a CAMP/protein kinase A-operated Cl^– conductance, activated by addition of forskolin. This anion channel could be identified as the Cystic Fibrosis Transmembrane conductance Regulator (CFTR-CI^– channel) by Western blotting as well as by its enhanced activity in cultures pretreated with the tyrosine kinase inhibitor genistein; (3) a distinct class of cell volume-regulated Cl^– channels, potentiated in the presence of endothelin-1 or the phosphotyrosine phosphatase inhibitor pervanadate. The potential role of each class of Cl^– channels in the generation and/or modulation of action potentials as well as in maintaining cell volume is discussed.     

Taurine prevents intracellular calcium overload during calcium paradox of cultured cardiomyocytes

Summary The effect of taurine on the cellular distribution of [Ca²⁺]_i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca²⁺ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca²⁺-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca²⁺]; rapidly fell to a level of 3–6 × 10^–8M. Subsequent restoration of medium calcium increased [Ca²⁺]_i to level 4–7 times normal. Large increases in [Ca²⁺]_i were observed in most cells and were associated with the development of contracture and bleb formation.Taurine pretreatment (20mM) caused no significant effect on [Ca²⁺]_i during Ca²⁺ depletion. However, it inhibited excessive accumulation of [Ca²⁺]_i during the Ca²⁺ repletion. Moreover, taurine treated cells recovered their Ca²⁺-transients and beating pattern earlier than non-treated cells. Finally morphological abnormalities commonly associated with calcium overload were attenuated by taurine treatment.     

Augmented O-GlcNAc signaling attenuates oxidative stress and calcium overload in cardiomyocytes

O-linked β-N-acetylglucosamine (O-GlcNAc) is an inducible, dynamically cycling and reversible post-translational modification of Ser/Thr residues of nucleocytoplasmic and mitochondrial proteins. We recently discovered that O-GlcNAcylation confers cytoprotection in the heart via attenuating the formation of mitochondrial permeability transition pore (mPTP) and the subsequent loss of mitochondrial membrane potential. Because Ca²⁺ overload and reactive oxygen species (ROS) generation are prominent features of post-ischemic injury and favor mPTP formation, we ascertained whether O-GlcNAcylation mitigates mPTP formation via its effects on Ca²⁺ overload and ROS generation. Subjecting neonatal rat cardiac myocytes (NRCMs, n ≥ 6 per group) to hypoxia, or mice (n ≥ 4 per group) to myocardial ischemia reduced O-GlcNAcylation, which later increased during reoxygenation/reperfusion. NRCMs (n ≥ 4 per group) infected with an adenovirus carrying nothing (control), adenoviral O-GlcNAc transferase (adds O-GlcNAc to proteins, AdOGT), adenoviral O-GlcNAcase (removes O-GlcNAc to proteins, AdOGA), vehicle or PUGNAc (blocks OGA; increases O-GlcNAc levels) were subjected to hypoxia–reoxygenation or H₂O₂, and changes in Ca²⁺ levels (via Fluo-4AM and Rhod-2AM), ROS (via DCF) and mPTP formation (via calcein-MitoTracker Red colocalization) were assessed using time-lapse fluorescence microscopy. Both OGT and OGA overexpression did not significantly (P > 0.05) alter baseline Ca²⁺ or ROS levels. However, AdOGT significantly (P < 0.05) attenuated both hypoxia and oxidative stress-induced Ca²⁺ overload and ROS generation. Additionally, OGA inhibition mitigated both H₂O₂-induced Ca²⁺ overload and ROS generation. Although AdOGA exacerbated both hypoxia and H₂O₂-induced ROS generation, it had no effect on H₂O₂-induced Ca²⁺ overload. We conclude that inhibition of Ca²⁺ overload and ROS generation (inducers of mPTP) might be one mechanism through which O-GlcNAcylation reduces ischemia/hypoxia-mediated mPTP formation.     

Endolysosomal calcium regulation and disease

Until recently, the mechanisms that regulate endolysosomal calcium homoeostasis were poorly understood. The discovery of the molecular target of NAADP (nicotinic acid-adenine dinucleotide phosphate) as the two-pore channels resident in the endolysosomal system has highlighted this compartment as an important calcium store. The recent findings that dysfunctional NAADP release leads to defective endocytic function which in turn results in secondary lipid accumulation in the lysosomal storage disease Niemann-Pick type?C, is the first evidence of a direct connection between a human disease and defective lysosomal calcium release. In the present review, we provide a summary of the current knowledge on mechanisms of calcium homoeostasis within the endolysosomal system and how these mechanisms may be affected in human metabolic disorders.     

Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes

Hyperamylinemia is a condition that accompanies obesity and precedes type II diabetes, and it is characterized by above-normal blood levels of amylin, the pancreas-derived peptide. Human amylin oligomerizes easily and can deposit in the pancreas [1], brain [2], and heart [3], where they have been associated with calcium dysregulation. In the heart, accumulating evidence suggests that human amylin oligomers form moderately cation-selective [[4], [5]] channels that embed in the cell sarcolemma (SL). The oligomers increase membrane conductance in a concentration-dependent manner [5], which is correlated with elevated cytosolic Ca²⁺. These findings motivate our core hypothesis that non-selective inward Ca²⁺ conduction afforded by human amylin oligomers increase cytosolic and sarcoplasmic reticulum (SR) Ca²⁺ load, which thereby magnifies intracellular Ca²⁺ transients. Questions remain however regarding the mechanism of amylin-induced Ca²⁺ dysregulation, including whether enhanced SL Ca²⁺ influx is sufficient to elevate cytosolic Ca²⁺ load [6], and if so, how might amplified Ca²⁺ transients perturb Ca²⁺-dependent cardiac pathways. To investigate these questions, we modified a computational model of cardiomyocytes Ca²⁺ signaling to reflect experimentally-measured changes in SL membrane permeation and decreased sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) function stemming from acute and transgenic human amylin peptide exposure. With this model, we confirmed the hypothesis that increasing SL permeation alone was sufficient to enhance Ca²⁺ transient amplitudes. Our model indicated that amplified cytosolic transients are driven by increased Ca²⁺ loading of the SR and that greater fractional release may contribute to the Ca²⁺-dependent activation of calmodulin, which could prime the activation of myocyte remodeling pathways. Importantly, elevated Ca²⁺ in the SR and dyadic space collectively drive greater fractional SR Ca²⁺ release for human amylin expressing rats (HIP) and acute amylin-exposed rats (+Amylin) mice, which contributes to the inotropic rise in cytosolic Ca²⁺ transients. These findings suggest that increased membrane permeation induced by oligomeratization of amylin peptide in cell sarcolemma contributes to Ca²⁺ dysregulation in pre-diabetes.