Disialoganglioside GD3 synthase expression recruits membrane transglutaminase 2 during erythroid differentiation of the human chronic myelogenous leukemia K562 cells

By employing proteomics analysis tool, we examined the effects of GD3 synthase expression on the differentiation properties of chronic myelogenous leukemia (CML)-derived leukemia cells K562. Forced expression of GD3 synthase induced erythroid differentiation as determined by an increase in glycophorin A expression and synthesis of hemoglobins. The proteomic analysis revealed that 15 proteins were increased by GD3 synthase. In contrast, we observed three protein gel spots decreased in contents in the cell membranes of GD3 synthase-transfected K562 cells. Among the increased proteins, membrane transglutaminase 2 (TG2) was specifically increased in the cell membrane of GD3 synthase-transfected K562 cells. Then, we generated the GD3 synthase-transfected cells in the K562 cells. Interestingly, the TG2 level was increased in GD3 synthase-transfected cells compared with vector- and plasma membrane-associated ganglioside sialidase (Neu3)-transfected cells. In addition, its ability to be photoaffinity-labeled with [alpha-(32)P]GTP was also increased in the GD3 synthase- and TG2-transfected cells. Moreover, small interfering RNA (siRNA) analysis for the GD3 synthase showed the decrease or abolishment of the membrane TG2. Finally, GD3 synthase-transfected cells accelerated the erythroid differentiation. Therefore, we propose that the recruitment of TG2 into membranes by GD3 might play an important role in the erythroid differentiation in K562 cells.     

Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli

Using biological iron chelators to control specifically iron availability to Escherichia coli K-12 in conjunction with radioactive pulse-labels, we examined the biosynthesis of six iron-regulated membrane proteins. Iron deprivation induced the synthesis of five proteins, which had molecular weights of 83,000 (83K), 81K (Fep), 78K (TonA), 74K (Cir), and 25K. The kinetics of induction were the same in entA and entA⁺ strains, but were affected by the initial iron availability in the media. Iron-poor cells induced rapidly (half-time, 10 min), whereas iron-rich cells began induction after a lag and showed a slower induction half-time (30 min). Within this general pattern of induction after iron deprivation, several different kinetic patterns were apparent. The 83K, 81K, and 74K proteins were coordinately controlled under all of the conditions examined. The 78K and 25K proteins were regulated differently. The synthesis of a previously unrecognized 90K inner membrane protein was inhibited by iron deprivation and stimulated by iron repletion. Both ferrichrome and ferric enterobactin completely repressed 81K and 74K synthesis when the siderophores were supplied at concentrations of 5 μM in vivo (half-time, 2.5 min). At concentrations less than 5 μM, however, both siderophores repressed synthesis only temporarily; the duration of repression was proportional to the amount of ferric siderophore added. The half-lives of the 81K and 74K mRNAs, as measured by rifampin treatment, were 1.2 and 1.6 min, respectively. The results of this study suggest that enteric bacteria are capable of instantaneously detecting and reacting to fluctuations in the extracellular iron concentration and that they store iron during periods of iron repletion for utilization during periods of iron stress. Neither iron storage nor iron regulation of envelope protein synthesis is dependent on the ability of the bacteria to form heme.     

Differing expression of genes involved in non-transferrin iron transport across plasma membrane in various cell types under iron deficiency and excess

We studied the effect of iron deficiency, i.e., 24-h preincubation in iron-free medium, and the effect of high level of non-transferrin iron, i.e., the preincubation in ferric citrate medium containing 500 muM ferric citrate, on the expression of DMT1, Dcytb, ferroportin, hephaestin, and ceruloplasmin in various functional types of human cells. The expression of these proteins potentially involved in non-transferrin iron transport across cell membranes was tested on mRNA level by quantitative real-time PCR as well as on protein level by western blot analysis in Caco-2 (colorectal carcinoma), K562 (erythroleukemia), and HEP-G2 (hepatocellular carcinoma) cells. We found that changes in non-transferrin iron availability, i.e., iron deficiency and high level of non-transferrin iron, affect the expression of tested proteins in a cell type-specific manner. We also demonstrated that changes in the expression on mRNA level do not often correlate with relevant changes on protein level.     

Erythroid 5‐aminolevulinate synthase mediates the upregulation of membrane band 3 protein expression by iron

Iron deficiency leads to abnormal expression and function of band 3 protein in erythrocytes, but the underlying mechanisms remain elusive. The mRNA of erythroid‐specific 5‐aminolevulinate synthase (eALAS) contains an iron response element and the eALAS protein is an important mediator of iron utilization by erythrocytes. In this study, we investigated the effect of short hairpin RNA (shRNA) mediated silencing of eALAS on the expression of band 3 protein induced by iron. By real‐time RT‐PCR and Western blot we showed that at mRNA and protein level iron‐induced expression of band 3 protein was lower in eALAS‐shRNA transfected K562 cells than in control cells. Of note, the lowest expression was detected in K562 cells cultured in iron deficiency condition (p < 0.01). Thus either iron deficiency or depletion of eALAS could suppress the expression of erythroid band 3 protein. These results demonstrated for the first time that iron and the iron‐regulatory system regulate the expression of the erythrocyte membrane proteins. Copyright © 2010 John Wiley & Sons, Ltd.     

Humanin delays apoptosis in K562 cells by downregulation of P38 MAP kinase

Humanin (HN) is a newly identified neuroprotective peptide. In this study, we investigated its antiapoptotic effect and the potential mechanisms in K562 cells. Upon serum deprivation, expression of HN in K562 cells decreased and its intracellular distribution changed from cytoplasm to cell membrane. In HN stably transfected K562 cells, apoptosis was delayed compared with control vector transfected cells as measured by flow cytometry. Furthermore, analysis of different mitogen-activated protein (MAP) kinases activity revealed that extracellular signal-regulated kinase (ERK) pathway was inhibited while p38 signaling was activated following serum deprivation in K562 cells. And in HN transfected K562 cells, ERK downregulation was not affected, but p38 activation was suppressed, which may responsible for the delayed apoptosis in these cells. Activation of the ERK signaling pathway by phorbol myristate 13-acetate (PMA) and sorbitol protected K562 cells from serum deprivation induced apoptosis. Additionally, overexpression of HN reduced megakaryocytic differentiation of K562 cells. The present data outline the role of ERK and p38 MAP kinases in serum deprivation induced apoptosis in K562 cells and figure out p38 signaling pathway as molecular target for HN delaying apoptosis in K562 cells.     

A novel tubulin polymerization inhibitor,MPT0B206, downregulates Bcr-Abl expression and induces apoptosis in imatinib-sensitive and imatinib-resistant CML cells

Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.     

Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells

K562 erythroleukemia cells import non-transferrin-bound iron (NTBI) by an incompletely understood process that requires initial iron reduction. The mechanism of NTBI ferrireduction remains unknown but probably involves transplasma membrane electron transport. We here provide evidence for a novel mechanism of NTBI reduction and uptake by K562 cells that utilizes transplasma membrane ascorbate cycling. Incubation of cells with dehydroascorbic acid, but not ascorbate, resulted in (i) accumulation of intracellular ascorbate that was blocked by the glucose transporter inhibitor, cytochalasin B, and (ii) subsequent release of micromolar concentrations of ascorbate into the external medium via a route that was sensitive to the anion channel inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Ascorbate-deficient control cells demonstrated low levels of ferric citrate reduction. However, incubation of the cells with dehydroascorbic acid resulted in a dose-dependent stimulation of both iron reduction and uptake from radiolabeled [(55)Fe]ferric citrate. This stimulation was abrogated by ascorbate oxidase treatment, suggesting dependence on direct chemical reduction by ascorbate. These results support a novel model of NTBI reduction and uptake by K562 cells in which uptake is preceded by reduction of iron by extracellular ascorbate, the latter of which is subsequently regenerated by transplasma membrane ascorbate cycling.     

Overexpression of sorcin in multidrug resistant human leukemia cells and its role in regulating cell apoptosis

In an attempt to identify novel proteins involved in the emergence of multidrug resistance (MDR) in leukemia cells, we adopted a proteomics approach to analyze protein expression patterns in leukemia cell lines, K562, and its MDR counterpart, K562/A02. Combining high-resolution two-dimensional gel electrophoresis and mass spectrometry, we compared the protein expression profiles between K562 and K562/A02. A total number of 22 protein spots with altered abundances of more than 2-fold were detected and 14 proteins were successfully identified. Consistent with our previous observations by cDNA microarray, sorcin, a 22-kDa calcium-binding protein, was also identified by this proteomic approach with a 10.4-fold up-regulation in K562/A02 cells. Overexpression of sorcin protein in K562 cells by gene transfection led to significantly reduced cytosolic calcium level and increased resistance to cell apoptosis. Further, leukemia cell lines over-expressing sorcin also showed up-regulation of Bcl-2, along with decreased level of Bax. Taken together, our results suggest that sorcin plays an important role in the emergence of MDR in leukemia cells via regulating cell apoptosis pathways, thus may represent both a new MDR marker for prognosis and a good target for anti-MDR drug development.     

Lactoferrin activates plasma membrane oxidase and Na+/H+ antiport activity.

Lactoferrin is a growth stimulant. The basis for this effect is not clear since it is not thought to be involved in iron uptake through endocytosis. Ferric lactoferrin supports external ferrous chelate formation by K562 and HeLa cells, and ferric lactoferrin stimulates the reduction of external ferric iron by cells. Ferric lactoferrin also stimulates NADH oxidase activity in isolated rat liver plasma membranes and stimulates amiloride sensitive proton release from K562 cells. The evidence that ferric lactoferrin can participate in oxidoreduction reactions at the plasma membrane leading to activation of Na+/H+ exchange provides an alternative explanation for the proliferative effect.     

Regulation of hexokinase binding to VDAC

Hexokinase isoforms I and II bind to mitochondrial outer membranes in large part by interacting with the outer membrane voltage-dependent anion channel (VDAC). This interaction results in a shift in the susceptibility of mitochondria to pro-apoptotic signals that are mediated through Bcl2-family proteins. The upregulation of hexokinase II expression in tumor cells is thought to provide both a metabolic benefit and an apoptosis suppressive capacity that gives the cell a growth advantage and increases its resistance to chemotherapy. However, the mechanisms responsible for the anti-apoptotic effect of hexokinase binding and its regulation remain poorly understood. We hypothesize that hexokinase competes with Bcl2 family proteins for binding to VDAC to influence the balance of pro-and anti-apoptotic proteins that control outer membrane permeabilization. Hexokinase binding to VDAC is regulated by protein kinases, notably glycogen synthase kinase (GSK)-3β and protein kinase C (PKC)-ɛ. In addition, there is evidence that the cholesterol content of the mitochondrial membranes may contribute to the regulation of hexokinase binding. At the same time, VDAC associated proteins are critically involved in the regulation of cholesterol uptake. A better characterization of these regulatory processes is required to elucidate the role of hexokinases in normal tissue function and to apply these insights for optimizing cancer treatment.     

Ginsenoside Rg1 protects H9c2 cells against nutritional stress-induced injury via aldolase /AMPK/PINK1 signalling

Insufficient nutrients supply will greatly affect the function of cardiac myocytes. The adaptive responses of cardiac myocytes to nutritional stress are not fully known. Ginsenoside Rg1 is one of the most pharmacologically active components in Panax Ginseng and possesses protective effects on cardiomyocyte. Here, we investigate the effects of ginsenoside Rg1 on H9c2 cells which were subjected to nutritional stress. Nutritional stress-induced by glucose deprivation strongly induced cell death and this response was inhibited by ginsenoside Rg1. Importantly, glucose deprivation decreased intracellular ATP levels and mitochondrial membrane potential. Ginsenoside Rg1 rescued ATP levels and mitochondrial membrane potential in nutrient-starved cells. For molecular mechanisms, ginsenoside Rg1 increased the expressions of PTEN-induced kinase 1 (PINK1) and p-AMPK in glucose deprivation treated H9c2 cells. Reducing the expression of aldolase in H9c2 cells inhibited ginsenoside Rg1′s actions on PINK1 and p-AMPK. Further, the nutritional stress mice were used to verify the mechanisms obtained in vitro. Ginsenoside Rg1 increased the expressions of aldolase, p-AMPK, and PINK1 in starved mice heart. Taken together, our results reveal that ginsenoside Rg1 limits nutritional stress-induced H9c2 cells injury by regulating the aldolase /AMP-activated protein kinase/PINK1 pathway.     

Iron deprivation induces apoptosis independently of p53 in human and murine tumour cells

Iron deprivation induces apoptosis in some sensitive cultured tumour cells, while other cells are resistant. In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we studied the expression of p53 and the expression of selected p53-regulated genes. To discriminate between changes coupled only with iron deprivation and changes involved in apoptosis induction by iron deprivation, we compared the expression of the genes in sensitive (human Raji, mouse 38C13) versus resistant (human HeLa, mouse EL4) cells under iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. The level of p53 mRNA decreased significantly under iron deprivation in sensitive cells, but it did not change in resistant cells. On the contrary, the level of the p53 protein under iron deprivation was slightly increased in sensitive cells while it was not changed in resistant cells. The activity of p53 was assessed by the expression of selected p53-regulated targets, i.e. p21(WAF1/CIP1) gene, mdm2, bcl-2 and bax. We did not detect any relevant change in mRNA levels as well as in protein levels of these genes under iron deprivation with the exception of p21(WAF1/CIP1). We detected a significant increase in the level of p21 mRNA in both (sensitive and resistant) mouse cell lines tested, however, we did not find any change in both (sensitive and resistant) human cell lines. Moreover, the p21(WAF1/CIP1) protein was accumulated in mouse-sensitive 38C13 cells under iron deprivation while all other cell lines tested, including human-sensitive cell line Raji, did not show any accumulation of p21(WAF1/CIP1) protein. It seems that the p21(WAF1/CIP1) mRNA, as well as protein accumulation, is not specifically coupled with apoptosis induction by iron deprivation and that it is rather cell-line specific. Taken together, we suggest that iron deprivation induces apoptosis at least in some cell types independently of the p53 pathway.     

The outcome of poliovirus infections in K562 cells is cytolytic rather than persistent after hemin-induced differentiation.

K562-Mu erythroleukemia cells readily establish a long-term persistent poliovirus infection characterized by continuous virus production in the absence of complete p220 cleavage and host translation shutoff (R. E. Lloyd and M. Bovee, Virology 194:200-209, 1993). The mechanism of resistance appears to be modulated at the intracellular level and to be related to decreased virus-mediated cytopathic effects (P. A. Benton, J. W. Murphy, and R. E. Lloyd Virology 213:7-18, 1995). It is well documented that hemin induces the differentiation of K562 cells and alters the expression of several host proteins. We report here that growth of K562 cells in hemin prior to poliovirus infection results in a dose-dependent increase in virus-induced cell lysis and thereby alters the normally persistent outcome of infection to a more lytic phenotype. K562 cells infected after hemin treatment displayed increased host translation shutoff, p220 cleavage, viral protein synthesis, and viral RNA accumulation compared with nontreated cells. Since hemin treatment of K562 cells also induced the increased expression of several heat shock proteins (Hsp70, Hsc70, Hsp90, and cohort p60), we tested the hypothesis that their increased expression may play a role in altering poliovirus infection in hemin-treated K562 cells. However, neither heat stress nor oxidative stress, inducers of heat shock protein synthesis, altered the outcome (of virus infections. In addition, we report the novel finding that subunits of two translation initiation factors, p220 (eIF-4G) and eIF-2alpha, are cleaved as a result of hemin treatment of K562 cells. It is proposed that hemin alters the expression of specific host proteins in K562 cells, probably other than heat shock proteins, which changes the initial response to poliovirus infections from persistent to lytic.     

DMT1 (IRE) expression in intestinal and erythroid cells is regulated by peripheral benzodiazepine receptor-associated protein 7

The divalent metal transporter 1 (DMT1) is essential for cellular uptake of iron, mediating iron absorption across the duodenal brush border membrane. We have previously shown that with iron feeding DMT1 in the brush border membrane undergoes endocytosis into the subapical compartment of enterocytes. To understand the mechanisms of iron-induced endocytosis of DMT1, we used the yeast two-hybrid system to find proteins that interact with DMT1 and isolated from a rat duodenal cDNA library a protein that interacts specifically with the IRE containing isoform of DMT1 {DMT1 [iron-responsive element (IRE)]}. The protein (Genbank AY336075) is 97.5% identical with peripheral benzodiazepine receptor-associated protein 7 (PAP7), a protein that interacts with the peripheral benzodiazepine receptor. PAP7 is ubiquitously expressed in the rat and in multiple cell lines with consensus sequences including a nuclear localization signal and a Golgi dynamic domain. PAP7, expressed on the brush border of rat duodenum, copurified with DMT1 in brush border membrane vesicles, and following iron feeding, was internalized in parallel with the internalization of DMT1. To determine if PAP7 plays a role in cellular iron metabolism, we downregulated PAP7 expression in K562 cells with small interfering RNA. Following the decrease in PAP7 protein, DMT1 (IRE) protein but not mRNA was significantly downregulated but without effect on DMT1 (non-IRE), transferin (Tf)R1, or ferritin expression. Lowered levels of PAP7 resulted also in decreased cell proliferation and G(1) cell cycle arrest. These data are consistent with PAP7 interacting with DMT1 (IRE) and regulating DMT1 (IRE) expression in K562 cells by modulating expression of DMT1 (IRE) protein.     

Demonstration of the specific binding of bovine transferrin to the human transferrin receptor in K562 cells: evidence for interspecies transferrin internalization

Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000-fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves.     

铁剥夺在TPA 诱导K562 细胞分化中对细胞 生长分化及EGR1mRNA 表达的影

目的：通过,IPA诱导K562细胞分化过程中干预细胞铁代谢探讨白血病细胞铁与细胞分化的关系及对EGR1mRNA表达的影响。方法：应用体外细胞培养技术通过细胞形态,细胞化学染色观察细胞生长分化情况;用FCM、RT—PCR等技术检细胞周期、细胞表面分化抗原CD33、CD14及EGR1mRNA的表达。结果：在,IPA诱导K562细胞分化过程中铁剥夺可明显抑制K562细胞生长,并可阻止,IPA诱导K562细胞分化,使K562细胞停止在S期。铁剥夺可降低,TPA诱导K562细胞分化过程中EGR1mRNA的表达。讨论：铁剥夺明显抑制K562细胞生长、阻止TPA诱导K562细胞分化,故铁剥夺剂（DFO）可能作为一种辅助抗癌药用于白血病的化疗,但由于它能阻止白血病细胞的分化,故不宜用于白血病的诱导分化治疗。铁剥夺使K562细胞分化过程中E—GR1mRNA表达降低可能参与了阻止TPA诱导K562细胞的分化过程。     

Hypoxia reduces the expression of heme oxygenase-2 in various types of human cell lines. A possible strategy for the maintenance of intracellular heme level

Heme oxygenase consists of two structurally related isozymes, heme oxygenase-1 and and heme oxygenase-2, each of which cleaves heme to form biliverdin, iron and carbon monoxide. Expression of heme oxygenase-1 is increased or decreased depending on cellular microenvironments, whereas little is known about the regulation of heme oxygenase-2 expression. Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxygenase-2 mRNA and protein after 48 h of incubation in human cell lines, including Jurkat T-lymphocytes, YN-1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses. In contrast, the expression level of heme oxygenase-1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN-1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells. Moreover, heme oxygenase-1 protein was decreased in YN-1 cells under the conditions used, despite the induction of heme oxygenase-1 mRNA under hypoxia. The heme oxygenase activity was significantly decreased in YN-1, K562 and HepG2 cells after 48 h of hypoxia. To explore the mechanism for the hypoxia-mediated reduction of heme oxygenase-2 expression, we showed that hypoxia shortened the half-life of heme oxygenase-2 mRNA (from 12 h to 6 h) in YN-1 cells, without affecting the half-life of heme oxygenase-1 mRNA (9.5 h). Importantly, the heme contents were increased in YN-1, HepG2 and HeLa cells after 48 h of incubation under hypoxia. Thus, the reduced expression of heme oxygenase-2 may represent an important adaptation to hypoxia in certain cell types, which may contribute to the maintenance of the intracellular heme level.     

Voltage-dependent anion channel 2 modulates resting Ca²+ sparks, but not action potential-induced Ca²+ signaling in cardiac myocytes

Voltage-dependent anion channels (VDACs) are pore forming proteins predominantly found in the outer mitochondrial membrane and are thought to transport Ca(2+). In this study, we have investigated the possible role of type 2 VDAC (VDAC2) in cardiac Ca(2+) signaling and Ca(2+) sparks using a lentiviral knock-down (KD) technique and two-dimensional confocal Ca(2+) imaging in immortalized autorhythmic adult atrial cells, HL-1. We confirmed high expression of VDAC2 protein in ventricular, atrial, and HL-1 cells using Western blot analysis. Infection of HL-1 cells with VDAC2-targeting lentivirus reduced the level of VDAC2 protein to ～10%. Comparisons of autorhythmic Ca(2+) transients between wild-type (WT) and VDAC2 KD cells showed no significant change in the magnitude, decay, and beating rate of the Ca(2+) transients. Caffeine (10mM)-induced Ca(2+) release, which indicates sarcoplasmic reticulum (SR) Ca(2+) content, was not altered by VDAC2 KD. Interestingly, however, the intensity, width, and duration of the individual Ca(2+) sparks were significantly increased by VDAC2 KD in resting conditions, with no change in the frequency of sparks. VDAC2 KD significantly delayed mitochondrial Ca(2+) uptake during artificial Ca(2+) pulses in permeabilized HL-1 cells. These results suggest that VDAC2 may facilitate mitochondrial Ca(2+) uptake and restrict Ca(2+) spark expansion without regulating activations of sparks under resting conditions, thereby providing evidence on the functional role of VDAC2 in cardiac local Ca(2+) signaling.