Responsive to fluctuations in intracellular Ca2+, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as an enticing therapeutic target in the management of ischemic heart injury. CaMKII is activated early in ischemia and to a greater extent in the first few minutes of reperfusion, at a time when reperfusion arrhythmias are particularly prominent. CaMKII phosphorylates and upregulates many of the key proteins involved in intracellular Na+ and Ca2+ loading in ischemia and reperfusion. Experimentally, selective inhibition of CaMKII activity reduces cardiomyocyte death and arrhythmic incidence post-ischemia. New evidence is emerging that CaMKII actions in ischemia and reperfusion involve specific splice variant targeted actions, selective and localized post-translational modifications, and organelle-directed substrate interactions. A more complete mechanistic understanding of CaMKII mode of action in ischemia and reperfusion is required to optimize intervention opportunities. This review summarizes the current experimentally derived understanding of CaMKII participation in mediating the pathophysiology of the heart in ischemia and in reperfusion, and highlights priority future research directions.Ischemic heart disease is a leading cause of death, and there is considerable imperative to identify effective therapeutic interventions. Cardiomyocyte Ca Though considerable advances have been made in the understanding of the processes involved in the pathological consequences of an ischemic event, effective therapeutic management of this disease has been difficult to achieve. Ca+/H+ exchanger (NHE) to maintain physiological pH, which contributes to an accumulation of intracellular Na+. The rise in intracellular Na+ is exacerbated by a decrease in Na efflux, including a reduction of Na+/K+-ATPase (NKA) activity that may be related to lower ATP availability (+ levels reduce the capacity for Ca2+ efflux via the Na+/Ca2+ exchanger (NCX) and promote the Ca2+ influx “reverse” operating mode of this transporter. Re-establishing coronary flow (reperfusion) is essential to ultimately allow any salvage of the ischemic myocardium, but involves a significant acute cardiomyocyte jeopardy. Reperfusion rapidly restores the trans-sarcolemmal proton gradient, exacerbating cellular Na+ accumulation (concomitant with restoration of NKA activity) and eventually culminating in Ca2+ overload. High cytosolic Ca2+ levels can have profound negative effects on the cardiomyocyte in reperfusion  in reperfusion, a response associated with reactive oxygen species (ROS) generation and the initiation of pro-death pathways . Furthermore, reports that CaMKII is susceptible to oxidation (ox-CaMKII(Met281/2)), promoting autonomous activation (2+) and reperfusion  provides an optimal setting for rapid CaMKII activation – and highlights the potential for intervention strategies targeting CaMKII.A complex series of cardiomyocyte events occur during both ischemia and reperfusion to culminate in an intracellular milieu primed for activating CaMKII. Briefly, insufficient tissue perfusion and oxygen availability in ischemia induce a shift to increased reliance on anaerobic glycolysis for ATP generation, promoting a build-up of metabolic intermediates including lactate and protons. This ionic shift stimulates the Nalability . Elevateerfusion , inducinpathways . As CaMKtivation , suggestex vivo rodent hearts have mapped phospholamban (PLB) phosphorylation at the CaMKII-specific site (P-PLB(Thr17)) as a marker of CaMKII activation during acute ischemia and reperfusion (2+ release channel (RyR2) and titin  activating CaMKII in ischemia and the NCX in reperfusion  and RyR2 phosphorylation (P-RyR2(Ser2814)) throughout 120 min of subsequent reperfusion. The substantial activation of CaMKII, which occurs in ischemia and reperfusion, would be expected to be an important determinant of cardiomyocyte Ca2+ homeostasis and post-ischemic outcomes.CaMKII is also undoubtedly influenced by the intracellular acidosis prevalent in ischemia and early reperfusion. Both CaMKII autophosphorylation (P-CaMKII(Thr287)) and P-PLB(Thr17) rapidly increase in acidic conditions, contributing to the recovery of Canditions . Inhibition; see ). An incion; see , consisterfusion . Interes2+ correlates with the onset of irreversible injury in ischemia . These studies found that CaMKII promotes apoptosis in vivo  by exacerbating SR and/or mitochondrial Ca2+ loading , sugges loading .2+ uptake, as P-PLB(Thr17) was suppressed in early reperfusion. Parallel experiments were conducted on hearts from mutant mice expressing a PLB Thr to Ala mutation at amino acid residue 17 . These 2+ release channel, augmenting SR Ca2+ leak and elevating cytosolic Ca2+ levels ) by CaMKII increases the open probability of the SR Ca+ levels . A CaMKIerfusion , both of+ levels , there i+ levels . Hearts  current . This wo2+ homeostasis at the SR and mitochondria, and may extend to direct pro-inflammatory actions. Indeed, cardiac-specific deletion of CaMKIIδ reduces chronic post-ischemic reperfusion (24 h) inflammation and injury in vivo, diminishing IκBα degradation and NF-κB activation . There igure 2, . Studiesigure 2, . This suigure 2, . CaMKII-igure 2, . This ab2+ handling processes, and the myocardial response to ischemia and reperfusion  in the myocyte, including PP1 and PP2a. Downstream Ca handling protein targets of CaMKII are regulated by these phosphatases. Indeed, PP1 is reported to form part of a multimeric complex in the regulation of PLB , which erfusion . Limitederfusion , and thaerfusion . Howevererfusion . These fATP channels , ox-CaMKII(Met281/2) selectively phosphorylates the RyR2 and not PLB (2+ load (2+ leak; an effect that may be offset if the Ca2+ levels at the RyR2 cytoplasmic domain are sufficiently reduced (As outlined above, CaMKII can provide important inotropic support to maintain contractile function in stunned hearts subjected to a brief ischemic challenge. An important body of experimental work supports the view that CaMKII actions can transition from conferring benefit to liability in a manner which reflects the myocyte capacity to balance relative SR Calearance . The meclearance , an incr not PLB . Augment(2+ load . With a  reduced .2+ mismanagement and mitochondrial dysfunction in response to ischemic changes in cellular Ca2+ and redox status. Thus, there is potential for CaMKII inhibitor utilization as a prophylactic therapeutic intervention for “at risk” patients with ischemic heart disease. Conversely, conflicting reports suggest that in other circumstances maintained CaMKII activation can improve reperfusion recovery. To optimize and tailor therapeutic strategies involving manipulation of CaMKII activation, it is clear that further mechanistic studies are required to fully understand the nuances of CaMKII response in a range of pathophysiologic settings.In summary, CaMKII is a crucial regulatory intermediate in ischemia and reperfusion injury. CaMKII is relatively quiescent under basal conditions, but displays considerable capacity to exacerbate CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Junonia orithya, which has distinct eyespots, to examine the possible involvement of Ca2+ waves in wing development.Butterfly wing color patterns emerge as the result of a regular arrangement of scales produced by epithelial scale cells at the pupal stage. These color patterns and scale arrangements are coordinated throughout the wing. However, the mechanism by which the development of scale cells is controlled across the entire wing remains elusive. In the present study, we used pupal wings of the blue pansy butterfly, 2+ waves in vivo that propagated slowly over long distances. Some waves appeared to be released from the immediate peripheries of the prospective eyespot and discal spot, though it was often difficult to identify the specific origins of these waves. Physical damage, which is known to induce ectopic eyespots, led to the radiation of Ca2+ waves from the immediate periphery of the damaged site. Thapsigargin, which is a specific inhibitor of Ca2+-ATPases in the endoplasmic reticulum, induced an acute increase in cytoplasmic Ca2+ levels and halted the spontaneous Ca2+ waves. Additionally, thapsigargin-treated wings showed incomplete scale development as well as other scale and color pattern abnormalities.Here, we demonstrate that the developing pupal wing tissue of the blue pansy butterfly displayed spontaneous low-frequency Ca2+ waves, spontaneous low-frequency slow waves, which travel over exceptionally long distances. Our results suggest that spontaneous Ca2+ waves play a critical role in the coordinated development of scale arrangements and possibly in color pattern formation in butterflies.We identified a novel form of CaThe online version of this article (doi:10.1186/s12861-015-0067-8) contains supplementary material, which is available to authorized users. The role of ions as signaling molecules is possible partially because their distribution and trafficking in cells, tissues and organs are tightly regulated by membranous structures. Among these signaling ions, calcium ions (Ca2+) are known to contribute to numerous cellular processes in both excitable and non-excitable cells during development and differentiation [2+ signals play important roles in various physiological processes, including cellular proliferation and the coordination of neuronal metabolism [2+ waves have also been well documented in the retina [2+ waves coordinate morphogenesis in zebrafish [2+ waves has also been reported in the slime mold [2+ waves have been well studied in the blowfly salivary gland [2+ waves in insect tissues have not been elucidated [In biological systems, cellular communication is mediated by various types of molecules, including signaling proteins. However, some inorganic ions, such as Nantiation -3. For etabolism -6. Devele retina ,8, the se retina , and thee retina ,11. Sponebrafish ,13, and ime mold . In insery gland ; howeverucidated . We therButterfly wings harbor unique color patterns that are constructed by regularly arranged scales on the surface of the wing, akin to mosaic roof tiles. At the microscopic level, each scale has a single color that is produced by a single epithelial scale cell during the pupal stage . At the Bicyclus anynana [Junonia almana, which have been examined at relatively high resolution, are not explainable by a simple concentration gradient model [Although the nature of the morphogenic signals is not known, the color pattern determination process has been explained using a concentration gradient model for positional information -24. This anynana , but the anynana . Furthernt model . Such dant model . Consident model ,31.2+ waves are the most likely to be involved in the mechanism underlying this function.Over the course of butterfly evolution, repeated wing-wide systematic changes in the position, size, and shape of color pattern elements in the nymphalid groundplan may have occurred. Similar global changes in these elements can be induced by temperature shock or chemical injection -36, demo2+ waves in butterfly wing development using the blue pansy butterfly Junonia orithya, which has large eyespots on its wings  contained the prospective eyespot area. However, the prospective eyespot area was resistant to the indicators  that traveled slowly over long distances   at approximately 5–10 min intervals   was obtained. The waves that traveled long distances were faster than those that traveled short distances. The propagation of these Ca2+ waves could be described as negative acceleration with a varied initial velocity.We examined the relationship between the travel distance of a wave and its velocity Figure f. The ven = 7)  in this species   Figure a-c.Figur2+ waves in the visual field in addition to the ROIs, thapsigargin was injected into the abdomen, which caused a transient increase in the intracellular Ca2+ concentration and the disappearance of spontaneous waves at 3–10 min after injection (n = 4) , heparin (n = 5), and dimethyl sulfoxide (DMSO) (n = 3) did not cause any changes in the spontaneous Ca2+ waves  Figure d. After arin n = , and dimn = 13)  (data not shown), and heparin (n = 7) (data not shown) induced characteristic color pattern changes in the adult wings when injected into the abdomen. In contrast with normal wings, the thapsigargin-treated wings showed incomplete cover scale development  in 13 individuals , which suggests that they play developmental roles only in pupal wings.Using a unique technique for imaging developing butterfly wings, we discovered spontaneous intercellular Capatterns ,42,43, eFor many waves, we were unable to find a clear point of origin or trajectory. We believe that this was largely because our visual field was not large enough to find originating points for the waves. Additionally, wave interactions complicated this issue. Variability in waves may be due to technical reasons, such as inherent individual variation, slightly different locations, the timing of recordings, uneven loading of the indicator from individual to individual and from location to location, and the level of bleaching of the indicator . It is w2+ waves in wing development. Physical damage . It is possible that a long-range wave may reach every single cell of a given wing surface, which may occasionally result in the wing-wide synchronization of waves.Third, these waves are long-range. The maximum travel distance that we observed was approximately 1 mm, which is an underestimate because a greater distance is expected if the visual field under the microscope is wider or if the detection sensitivity of the system is higher. Nonetheless, this 1-mm distance was exceptionally long, spanning at least 50–100 cells, given that a single epithelial cell is approximately 10–20 μm in diameter in pupal wing tissue  membrane [The biological functions of Cand color , transiemembrane ,51. Thesmembrane ,51, we b2+ waves. However, we speculate that long-range, slow Ca2+ waves may play important roles in multiple aspects of normal cover scale development. The disorder of the scales suggests that Ca2+ waves may function as synchronization signals for cell growth and division, resulting in wing-wide phenotypic coordination [2+ waves in wing development, genetic manipulations may be required in the future. A relatively easy-to-use method is baculovirus-mediate gene transfer, which can be successfully used to deliver a gene into the wings of this butterfly species [The long-term effects of thapsigargin on scales in this species are known , and simdination ,41. Altedination ,42. To d species .2+ waves is currently unknown. Inside a cell, mitochondria appear to play a similar role as in cochlear supporting cells [2+ waves when octanol, which is a general gap-junctional inhibitor, was injected (data not shown), signaling may be mediated by a chemical (such as ATP) and a receptor via the extracellular space. The Ca2+ waves detected in this study are somewhat similar to progressive waves from an organizing center that have been predicted by the induction model for positional information [2+ waves and the predicted ultra-slow waves remains to be clarified. For example, Wnt ligand, which may be involved in color-pattern formation [2+ waves.The mechanism that underlies the intercellular propagation of Cang cells . As we cormation ,31. Howeormation ,55 or otormation ,57, may 2+ waves that travel slowly over long distances during butterfly wing development. These findings were made possible by our technological advancements in butterfly wing live imaging. Moreover, Ca2+ waves were generated by physical damage, which is known to produce ectopic eyespots. Thapsigargin injection abolished the Ca2+ waves and resulted in abnormal scales and patterns. These results suggest that spontaneous Ca2+ waves play critical roles in scale development and color pattern formation, which are involved in two-dimensional morphogenesis in butterfly wings. The discovery of long-range, slow Ca2+ waves that may contribute to pattern formation is a significant advancement in the developmental biology of pattern formation [This study detected, for the first time, spontaneously produced Caormation ,59 and iormation .J. orithya  . Therefore, the in vivo images that we obtained in the present study can be considered to be free from artifacts. The hindwing epithelial cells were loaded with modified low-pH Ringer’s solution with the following composition: NaCl (183 mM), KCl (20 mM), CaCl2 (5.6 mM), MgCl2 (0.83 mM), and citric acid (0.19 mM) containing Fluo-4 AM  or Fluo-8 AM  at a final concentration of 20 μM with brief sonication. We found that the addition of a small amount of citric acid to the loading solution increased the efficiency of Fluo-4 loading into the wing tissue. This addition was not necessary when using Fluo-8. Approximately 50 μL of loading solution was sandwiched between the fore- and hindwings for 60 min at approximately 27°C under high-humidity conditions to avoid the evaporation of the liquid  the operation itself is simple, but an unsuccessful operation may have occurred, mainly due to a leakage of hemolymph, which may have weakened the pupae; and (5) laser-based observations over a long period of time may have damaged the pupae.Naked hindwings were placed on cover glasses  and MitoTracker Orange (Life Technologies) to stain the nucleus and mitochondria, respectively. For SYBR Green I, the original solution from the manufacturer was diluted 2,000 fold in the final loading solution, which was used for the staining process. For MitoTracker Orange, the concentration of the final loading solution was 100 μM. The solutions were loaded similarly as the Careported .2+ waves were examined with AQUACOSMOS 2.6 software. We focused on the waves that were relatively easily identifiable during propagation. The locations and times of the wave fronts were monitored manually using serial images. We focused on 5 waves that were not mutually overlapping from a single tissue. We examined 7 individuals, resulting in the assessment of a total of 35 waves.The acquired serial images of Ca2+ waves. Physical holes were created on the surfaces of the early pupal hindwings without hemolymph leakage. This minor damage did not induce ectopic spots on the adult wings. To induce ectopic color patterns, a large insect pin  was required. Indeed, small areas of damage were able to heal quickly without leading to a color pattern change.After the loading solution was washed out, the pupal hindwings were physically damaged using an insect pin  in a background area of the basal region to induce Ca2+ waves respond to chemicals, pupae were injected with 2.0 μL of a solution containing the following chemicals into the abdomen on the opposite side of the operated hindwing after indicator loading: sodium tungstate (1.0 M in ddH2O), thapsigargin , and heparin (5.0 U/mL in dimethyl sulfoxide) using an Ito microsyringe . The exact mechanisms of action of tungstate and heparin in butterflies are unknown, but thapsigargin is a well-known inhibitor of Ca2+-ATPase in the ER membrane that increases the cytosolic Ca2+ concentration [n = 4), ddH2O (n = 3), and dimethyl sulfoxide (n = 3) were injected as controls, resulting in no changes in pupal Ca2+ waves or adult color patterns. It has been shown that no color pattern modifications in adult wings are induced by these chemicals [To examine whether Cantration ,51. In ahemicals ,35. DeteScale images were taken using a high-resolution high-depth Keyence VHX-1000 digital microscope  and a Hitachi High-Tech TM3030 tabletop microscope, which is a benchtop-type scanning electron microscope .


SMG1 was suggested as a novel potential tumor suppressor gene, particularly in hypoxic tumors. To investigate the function of SMG1 in acute myeloid leukemia (AML), we performed methylation-specific polymerase chain reaction and found that SMG1 was hypermethylated in the promoter region. SMG1 hypermethylation was found in 66% (33/50) of AML samples compared with none (0/14) of the normal controls. SMG1 mRNA was down-regulated in AML patients with hypermethylation status whereas it was readily expressed in patients without methylation. Moreover, treatment of AML cells with demethylating agent 5-aza-2'-deoxycytidine (decitabine) inhibited AML cell growth and induced apoptosis by reversing SMG1 methylation status and restoring SMG1 expression. On the other hand, knockdown of SMG1 by RNA interference inhibited apoptosis. We also found that mTOR expression level was negatively correlated to SMG1 expression in AML patients which indicated that SMG1 and mTOR maybe act antagonistically to regulate AML cell growth. In conclusion, our results indicate that SMG1 acts as a potential tumor suppressor with epigenetic regulation in AML.Suppressor with morphogenetic effect on genitalia family member (SMG1) belongs to a family of phosphoinositide 3-kinase-related kinases and is the main kinase involved in nonsense-mediated mRNA decay. Recently,  Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis characterized by the uncontrolled proliferation and accumulation of immature and dysfunctional hematopoietic progenitors. Cytotoxic chemotherapy has been widely used as the main approach for AML treatment.Recent studies have proved that, not only the successive accumulation of genetic alterations in oncogenes and tumor suppressor genes, but also the epigenetic alterations contribute to carcinogenesis . The mos5-Aza-2'-deoxycytidine , as a kind of DNA methyltransferase inhibitor, is the most widely used epigenetic modulator to date. Recently, many studies have come to a conclusion that DAC offers a promising alternative therapeutic option for AML patients who are not candidates for standard remission induction chemotherapy . CurrentSMG1  was hypermethylated in the promoter region in AML. SMG1 is a well known member of phosphoinositide 3-kinase-related kinases (PIKK) family. It is mainly involved in nonsense-mediated mRNA decay (NMD), which is the process of eliminating mRNAs that contain premature termination codons to prevent the accumulation of truncated proteins [SMG1 functional mutations, deletions, or reduced expression in human cancer is rarely studied. Recently, SMG1 was suggested to be a potential tumor suppressor [SMG1 promoter methylation status and its expression level in AML.In our study, we found that proteins . Howeverppressor  and coulppressor . Howeveret al. reported that SMG1 and mammalian target of rapamycin complex 1 (mTORC1) act antagonistically to regulate response to injury and growth in planarians and their study indicated that SMG1 is likely to be a potential human tumor suppressor gene product [Cristina  product . Mammali product . As a reSMG1 acts as a potential tumor suppressor with epigenetic regulation and highlights a new approach for the demethylating treatment of DAC in AML.In our study, we found that SMG1 expression level was negatively correlated with its methylation status and mTOR expression level respectively which indicated that SMG1 and mTOR may act antagonistically to regulate AML cell growth. In conclusion, our results indicate that SMG1 in AML.We performed quantitative Real-Time Polymerase Chain Reaction (RT-PCR) to detect SMG1 mRNA expression in bone marrow samples from 50 AML patients and 32 normal controls. The results showed that SMG1 was down-regulated in AML, but was readily detected in the controls as shown in SMG1 gene results in the reduced SMG1 mRNA expression, we performed Methylation-Specific Polymerase Chain Reaction (MSP) to analyze the methylation status of SMG1 gene in AML. The SMG1 methylation status was detected in 50 samples of AML patients and 14 samples of normal controls. We found SMG1 hypermethylation in 33 out of 50 (66%) AML samples and no SMG1 hypermethylation (0/14) in the controls. SMG1 was also methylated by varying degrees in four AML cell lines  in our study. The representative results are shown in SMG1 may be responsible for transcriptional down-regulation.In order to investigate whether the promoter hypermethylation of SMG1 caused the reduced expression of SMG1, firstly we performed MSP to test the demethylating function of DAC. As shown in SMG1 gene.To further test the hypothesis that the DNA hypermethylation in the promoter region of SMG1 gene was responsible for the decreased expression of SMG1 in AML.Then, we performed quantitative RT-PCR to detect the SMG1 expression in DAC treatment group and control group in HEL cells. As shown in SMG1 may play a role in the above functional effect of DAC in AML cells.To determine whether the effect of DAC was related to apoptosis, we performed flow cytometry and found that DAC treatment caused a significant increase in early apoptotic cells and late apoptotic cells compared with that of the control group C. These SMG1 in AML, we knocked down SMG1 in HEL cells by siRNA. Quantitative RT-PCR results showed that SMG1expression was significantly decreased by SMG1-siRNA . DNMT3A, a major player in the s in AML . Despites in AML , the bios in AML . The mecet al. found that SMG1 was required for the tight control of stem cell proliferation and differentiation caused by injury or nutrient status in planarian flatworms [SMG1 in planarian flatworms leads to lethal outgrowths which display several hallmarks of human cancers, which is similar with the knockdown of known human tumor suppressors such as PTEN or p53. These studies suggested that SMG1 might have a potential tumor suppressor role in human cancer. It will be very interesting to investigate this new role in AML.Despite being involved in NMD, SMG1 has many other biological functions, such as maintaining telomere integrity, protecting against TNF-α-induced apoptosis, regulating lifespan and oxidative stress resistance, having an essential role in embryogenesis and activating p53 and an important role in the DNA damage response network ,15,16,17SMG1 was investigated in AML. We found that mRNA expression level of SMG1 was negatively associated with the hypermethylation status, which could be reversed by DAC treatment. DAC also restored mRNA expression of SMG1 and induced AML cells apoptosis. It is plausible to assume that DAC has an effect on the cell growth and proliferation via regulating SMG1. On the other hand, knockdown of SMG1 by siRNA inhibited AML cell apoptosis. These results indicate that SMG1 functions as a tumor suppressor in AML.Thus, the tumor suppressor role of et al. also described almost polar opposite roles for mTOR and SMG1 in planarian flatworms. The study of Robert et al. suggested that the mTOR pathway may be targeted and inhibited tumor suppressor elements with regulatory effects on mRNA translation [It has been widely accepted that aberrant activation of the PIKK/AKT/mTOR pathway promoted AML cell proliferation and survival . mTOR isnslation . Future A total of 82 subjects were recruited for this study, including 50 AML patients and 32 healthy volunteers at the Qilu Hospital of Shandong University . Our research was approved by the Medical Ethical Committee of Qilu Hospital of Shandong University . A written informed consent document has been obtained from each participant including the guardians on the behalf of the minors/children participants involved in our study. Mononuclear cells from bone marrow aspirates were isolated by density-gradient centrifugation with the use of Ficoll-Paque Plus . Detailed clinical information for AML patients is summarized in l-glutamine), at 37 °C in humidified air containing 5% carbon dioxide air atmosphere and were routinely subcultured every 2–3 days.The human AML cell lines Kasumi-1, HL60, THP-1 and HEL were purchased from Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College . Cells were cultured in complete medium . The SMG1 promoter region is located approximately 1.3 kb up stream and up to 200 bp downstream of the transcription initiation site, which has a GC content up to 64.6% [SMG1 M-forward primer sequence: 5'-GCGTACGTGAATTTAAGGGTAC-3';SMG1 M-reverse primer sequence: 5'-AACAAAAAATCTCCACTACTACGAC-3';SMG1 U-forward primer sequence: 5'-GGTGTATGTGAATTTAAGGGTATGT-3';SMG1 U-reverse primer sequence: 5'-AACAAAAAATCTCCACTACTACAAC-3'.Genomic DNA from patient samples was isolated by DNA Extraction Kit  and chemically modified by using an EZ DNA Methylation-Gold Kit  according to the manufacturer’s instructions. The methylation status of 6 cells per mL in 10-cm dishes. After 24 h of culture, cells were treated with DAC for 2 days. Control cells were treated in parallel with DMSO. The medium was replaced every day. Cells were harvested and mRNA expression of SMG1 and mTOR were analyzed by quantitative RT-PCR.Demethylation was performed by using DAC. AML cells were seeded at a density of 1 × 10The synthetic SMG1–siRNA and controls were purchased from Ribobio . Transfection of siRNA was performed with Lipofectamine 2000 reagent  in accordance with the manufacturer’s protocol. AML cell lines with SMG1 expression were transfected with SMG1–siRNA or control–siRNAs. Knockdown efficiency was evaluated at 48 h after transfection by quantitative RT-PCR. The siRNA with the highest knockdown efficiency was used for the following functional studies.Total RNA was extracted from all samples using TRIzol reagent (Invitrogen). To detect SMG1, mTOR expression, cDNA was synthesized from about 1 µg total mRNA using the M-MLV RTase cDNA Synthesis Kit  according to the manufacturer’s instructions. Quantitative RT-PCR was conducted on an Applied Biosystems 7900HT System  with SYBR Green PCR Master Mix . β-Actin was used to normalize expression levels of SMG1 and mTOR. Each sample was measured three times at least and fold changes in mRNA expression levels were calculated using the comparative threshold cycle method.5 cells were harvested, resuspended in 100 µL flow cytometry binding buffer, and stained with 5 µL Annexin V/FITC followed by 1 µL PI. Cells were then incubated in the dark for 15 min at room temperature, and added 400 µL binding buffer. Cells were immediately measured by Galios flow cytometer .Apoptosis was detected using an Annexin V/FITC and PI Apoptosis Detection Kit . Briefly, after treatment with DAC or transfection with SMG1–siRNA for 48 h, 2 × 10p-mTOR  overnight at 4 °C. After washing with TBST for 3 times, membranes were incubated with secondary antibodies  for 1.5 h at room temperature. Protein bands visualized by FluorChem E Chemiluminescent Western Blot Imaging System .AML cells were lysed by Total Protein Extraction Kit  and protein samples were stored at −80 °C. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, which were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 2 h at room temperature. The membranes were then incubated with the following specific antibodies: rabbit anti-mTOR or rabbit anti-p-values of less than 0.05 were considered statistically significant. The statistical comparisons between the controls and AML patients were made with the Mann-Whitney U nonparametric tests.Data was collected from at least three experiments in duplicate. Statistical analysis was carried out using SPSS software . Differences with Taken together, our findings suggest that SMG1 acts as a novel functional tumor suppressor gene which was down-regulated by CpG island hypermethylation in AML. SMG1 methylation may serve as a potential epigenetic biomarker for early diagnosis of AML patients. SMG1 and mTOR may act antagonistically to regulate AML cell growth and proliferation. Our study highlights a new approach for the demethylating treatment of DAC in AML.


The suffix “-ase,” indicating an enzyme, is derived from “diastase” , the first enzyme discovered in 1833 by Payen and Persoz.Enzymes that catalyze the cleavage of β-lactam rings in penicillins, cephalosporins, monobactams, and carbapenems were first described by Abraham and Chain in 1940. These enzymes confer resistance to β-lactam antibiotics on bacteria that produce them. β-lactamases  are anci


To achieve ultrasensitive Ca2+ imaging for low resting Ca2+ or small Ca2+ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca2+ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca2+ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca2+ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca2+ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca2+ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from Rmax and Rmin values under in vivo condition. The mice will be useful for ultrasensitive Ca2+ imaging in vivo.Yellow Cameleons are genetically encoded Ca The up-regulation of intracellular free Ca2+ is mediated by release of Ca2+ from intracellular stores, a process known as inositol triphosphate-induced Ca2+ release (IICR) or Ca2+-induced Ca2+ release (CICR), as well as by voltage-dependent Ca2+ influx from the extracellular fluid. Exocytosis, in which the contents of secretory vesicles are released, is very important in cytophysiology  or small [Ca2+] transients less than 100 nM  transients in embryos of zebrafish and in mouse neural cells following in utero electroporation  originated in the apical region and propagated to the basal region  was estimated as 2.01 ± 0.08 .Next, we carried out a time-course analysis in the apical and basal regions of cells C–F. In t regions C,D. In Fondition . The onsl region E,F. Thisar cells  and of gar cells . Subsequsaturate C,D. The Rmin) of YC-Nano15 (2+-free buffer that did not affect the FRET ratio of the cell. When this fluid was replaced with Ca2+-free EGTA buffer containing CPA and FCCP (indicated as “EGTA”), the FRET ratio rapidly increased, and then gradually decrease to a final value of 1.63 ± 0.04 , probably reflecting release of Ca2+ from intracellular storage to the cytoplasm, followed by its leakage to the outside. Next, the fluid was replaced with Ca2+-free BAPTA-AM buffer (indicated as “BAPTA-AM”). In this case, the FRET ratio decreased below the initial value, indicating that the membrane-permeable chelator further depleted intracellular Ca2+. After that, the fluid was replaced with a Ca2+-containing buffer (indicated as “1 mM Ca2+”), but the FRET ratio continued decreasing, probably because the effect of the accumulated intracellular chelator surpassed the influx of Ca2+ from outside. Finally, the fluid was replaced with Ca2+ ionophore in Ca2+-free EGTA buffer (indicated as “A23187”). In this case, since 10 μM BAPTA-AM solution was superfused constantly, Ca2+-free BAPTA-AM could be sufficiently maintained in the cytosol, i.e., the intracellular concentration of BAPTA-AM must be enormously higher than that of YC-Nano 15, stoichemiometrically most of YC-Nano 15 could be shifted to the unbounded form. The FRET ratio then further decreased to a plateau of 0.76 ± 0.02 , reflecting complete depletion of intracellular Ca2+, i.e., Rmin under in vivo conditions. This result indicates that YC-Nano15 can sense extremely low levels of intracellular Ca2+ in mouse tissue. Nevertheless, because the transgenic mice are healthy and the homeostatic mechanism is maintained, the expression level of YC-Nano 15 is estimated to be moderate in the acinar cells. The expression level of YC-Nano 15 is critical to discuss the capability to sense the low [Ca2+] in vivo, in addition to that, the issue of the affinity in vivo is also related to the discussion. We then experimentally estimated the dK value of YC-Nano invivo, for instance, considering the ratio change by the following calibration Equation (1)  such as Ca2+ oscillation due to the limitation of the sensitivity. In this study, for the first time, we were able to successfully visualize Ca2+ oscillation under stimulation with agonist (ACh) at nanomolar levels in pancreatic acini of YC-Nano15 mice.To examine both spatiotemporal Caf 850 nm A. An incst (ACh) . In thisst (ACh) C,D. Exocst (ACh) . In acin plateau . Howevers report , both Ca 200 nM) , but conet al., the ratiometric FRET imaging of YC-Nano 15 was performed in Dictyostlium  at physiological levels in acinar cells. The transgenic mouse strains created in this study will be useful for exploration of other previously unseen physiological phenomena at low [Ca2+] in tissues and in whole animals.In summary, we demonstrated that ubiquitously expressed ultrasensitive GECIs are not toxic in transgenic mice, and that these reporters can be used to analyze intracellular [CaMain Animal experimental procedures were approved by the animal research committee of the National Institute for Basic Biology, Japan (10A091). Animals were sacrificed with cervical dislocation.BamHI and EcoRI. The 1.9-kb YC-Nano15 gene was cloned downstream of the CAG promoter, and upstream of the SV40 polyadenylation sequence, in vector pCAGGS.The YC-Nano15 gene was produced by digesting the pRSETb- YC-Nano15 plasmid with SalI-HindIII digestion of the pCAGGS-YC-Nano15 construct. This fragment was purified and microinjected into pronuclei of C57BL/6J fertilized mouse eggs, which were then transferred to pseudo-pregnant females. The transgene cloning and microinjection were performed by Unitech Co., Ltd. . Founder mice were genotyped, using tail genomic DNA, by polymerase chain reaction (PCR) using the forward primer 5'-ACTTCAAGATCCGCCACAAC-3' and the reverse primer 5'-TTGTCAAAAACACGGAATGC-3'. Genopositive founders were backcrossed into the C57BL/6J background for production of transgenic offspring.A 4.3-kb YC-Nano15 transgene was produced by GAPDH (mGAPDH) was included to check the integrity of the RNA.YC-Nano15 mice and C57BL/6J (both strains: 8–14 weeks old) were sacrificed, and organs  were harvested and cooled on ice. Each of the organs was washed with ice-cold PBS, cut into small pieces (3 mm × 3 mm × 3 mm), suspended in RNAlater , and stored at −20 °C prior to total RNA extraction. The frozen tissues were homogenized in TRIzol Reagent  using a Polytron  and a Dounce tissue grinder, and total RNA was isolated with chloroform and isopropanol. Precipitated total RNA was washed with 75% ethanol and redissolved in RNase-free water. Complementary DNA (cDNA) was synthesized from total RNA using the SuperScript VILO cDNA Synthesis Kit . PCR was performed using cDNA template, specific primer pairs, and Taq polymerase  according to the recommended cycling protocol. PCR products were detected by electrophoresis assays on 2% agarose gels. The gene encoding mouse w/v) polyacrylamide gel, and then electro-transferred onto a PVDF membrane. After blocking in TBS-T  with 5% skim milk (w/v) for 1 hour at room temperature, the membrane was incubated with anti-GFP  for 1 h at room temperature. After a TBS-T rinse, the membrane was incubated with HRP-conjugated anti-goat secondary antibody  for 1 h at room temperature. The membrane was then washed in TBS-T, and signals were detected by chemiluminescence . For reblotting, membranes were stripped using Restore Western Blot stripping Buffer , followed by incubation with anti-β-actin serum  for 1 h at room temperature. After a TBS-T rinse, the signal was detected by ECL.Various tissues were excised from YC-Nano15 mice and C57BL/6J mice (both strains: 8–14 weeks old) and immediately frozen in liquid nitrogen. To generate lysates, the tissues were homogenized in lysis buffer containing protease inhibitor, DTT, and SDS on ice. The samples were centrifuged , and the supernatants were collected and boiled for 10 min, after which 10% SDS and beta-mercaptoethanol were added. After another centrifugation , the supernatants were again collected. Proteins were separated by electrophoresis in 8%  followed by gentle trituration. The acini were dispersed in a small chamber and superfused (1 mL·min−1) with a solution termed Solution A . All chemicals, except where otherwise stated, were purchased from Nacalai Tesque .Clusters of acini were prepared as described in our previous reports ,14. In b2+ permeability of the plasma membrane, a non-fluorescent Ca2+ ionophore, 4Br-A23187 (molecular probes) was first dissolved in dimethylsulfoxide  and then diluted to 10 μM in Solution A by sonication. To saturate the intercellular free Ca2+ concentration, 1 mM Ca2+ solution  containing 10 μM 4Br-A23187 (Ca2+/A23187 solution) was applied. In order to chelate intracellular or extracellular free Ca2+ ion, 10 mM ethylene glycol tetraacetic acid (EGTA) solution or EGTA solution containing 10 μM 4Br-A23187 (EGTA/A23187 solution) was applied. In some experiments, to chelate intracellular free Ca2+ we also applied 10 μM O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester  in EGTA-solution (BAPTA-AM solution).Live-cell FRET imaging was performed using a two-photon laser scanning upright microscope  with a femtosecond pulse Ti: Sapphire laser . Acinar cells were seeded in a hand-made perfusion chamber with in and outflow channels inside a cell-culture dish , which was placed on the microscope stage. Microscopic observation was performed at room temperature. During the observation, extracellular fluid was superfused constantly, typically at a flow velocity of 0.5 mL/s. Stimulation of cells was carried out by exchanging the extracellular fluid. Acetylcholine  at a concentration of 5–500 nM in Solution A was employed as a natural agonist. To increase CaAn excitation wavelength of 850 nm was used for YFP–CFP FRET imaging and cross-section exocytotic imaging with SRB. The two-photon excited fluorescence images of CFP and YFP (from YC-Nano15) and SRB were captured in separate channels  through dichroic mirrors and emission filters, BP460-500, DM505/BP520-560 and DM570/BP575-630, respectively. Excitation laser power and high-voltage (HV) values for each channel were adjusted to obtain images with good signal-to-noise ratios and nearly equal intensities for CFP, YFP, and SRB under unstimulated conditions. The image acquisition speed was approximately 1 frame/s.i.e., before stimulation). FRET signal images were represented as pseudo color images.The public domain software ImageJ  was emplin vivo and in vitro. GECIs have been developed and widely used for live-cell calcium imaging, but they are not suitable for observation in some cells due to lack of sensitivity. In this study, we generated healthy transgenic mice ubiquitously expressing ultrasensitive GECIs and demonstrated the usefulness of these new mouse strains. Specifically, we performed FRET ratio imaging by two-photon microscopy of acinar cells in order to visualize calcium dynamics during minimal agonist stimulation. The results provide a new practical animal model for ultrasensitive calcium imaging in vivo or in vitro that could be widely utilized in biological and medical imaging analyses in the near future.Here we report on establishment and evaluation of transgenic mice which have an ultrasensitive calcium indicator in their tissues and organs to visualize spatiotemporal dynamics of intracellular free calcium


Stomatal opening or closure is accompanied by the modulation of guard cell turgor. Among the events leading to stomatal closure by plant hormones or microbial elicitors, three signaling components stand out as the major converging points. These are reactive oxygen species (ROS), cytosolic free Ca2+, and ion channels. Once formed, the ROS and free Ca2+ of guard cells regulate both downstream and upstream events. A major influence of ROS is to increase the levels of NO and cytosolic free Ca2+ in guard cells. Although the rise in NO is an important event during stomatal closure, the available evidences do not support the description of NO as the point of convergence. The rise in ROS and NO would cause an increase of free Ca2+ and modulate ion channels, through a network of events, in such a way that the guard cells lose K+/Cl−/anions. The efflux of these ions decreases the turgor of guard cells and leads to stomatal closure. Thus, ROS, NO, and cytosolic free Ca2+ act as points of divergence. The other guard cell components, which are modulated during stomatal closure are G-proteins, cytosolic pH, phospholipids, and sphingolipids. However, the current information on the role of these components is not convincing so as to assign them as the points of convergence or divergence. The interrelationships and interactions of ROS, NO, cytosolic pH, and free Ca2+ are quite complex and need further detailed examination. Our review is an attempt to critically assess the current status of information on guard cells, while emphasizing the convergence and divergence of signaling components during stomatal closure. The existing gaps in our knowledge are identified to stimulate further research.Dynamic regulation of stomatal aperture is essential for plants to optimize water use and CO Plant hormones  and polyamines (PAs) induce stomatal closure. Elicitors such as salicylic acid (SA), chitosan, and Flg22 also cause stomatal closure  on stomatal movements have been studied in detail. ABA induced stomatal closure is mediated by many signaling components like cytoplasmic pH, reactive oxygen species (ROS), reactive nitrogen species , cytosolic free Ca2+ may act at either downstream or upstream of ROS/NO. The changes in cytosolic pH of guard cells may be important at either downstream or upstream of ROS or NO. The production of NO precedes that of ROS, but NO can act as antioxidant as well. Despite these ambiguities, it is clear that a rise in ROS or NO triggers a rise in free Ca2+ of guard cells, modulate the ion channels and cause an efflux of K+/Cl−/malate, leading to loss in turgor of guard cells. We emphasize that the signaling events during stomatal closure converge at ROS, cytosolic Ca2+, and ion channels. Similarly, ROS, NO, and Ca2+ form the points of divergence.There are yet questions about the sequence of the signaling events during stomatal closure. For e.g., cytosolic free Ca2+, and anion channels. For e.g., plant hormones (such as ABA or MJ) and microbial elicitors invariably cause an increase in the levels of ROS or NO in guard cells, leading to rise in free Ca2+ within the guard cells  sensitive peroxidase or amine oxidases   is an important secondary messenger during stomatal closure  mutants. In gpa1 mutants, ABA-induced ROS production was disrupted, but Ca2+ channels were activated by exogenous H2O2 application.Calcium , quick anion channel 1 (QUAC), slow anion channel homolog 3 (SLAH), and even aluminum activated malate transporters (ALMT)  is a small, gaseous molecule involved in growth, development and even disease resistance of plants  and calcium sensing receptors (CAS)  induced stomatal opening, by causing cytoplasmic acidification, and lowering of NO levels, even in presence of ABA , the product of phospholipase C/D (PLC/PLD) induced stomatal closure by inhibiting 2+, and pH are pointed out (Zhang et al., 2+ and modulate ion channels. However, the feedback relationship between NO and ROS is obscure. Similarly, cytoplasmic pH may act directly on ion channels, particularly 2+ also can interact with NO and pH (Wang et al., 2+-dependent and Ca2+-independent signaling pathways are considered to function during stomatal closure (Kim et al., 2+-dependent and independent pathways during guard cell signal transduction are yet to be elucidated.Signaling components, particularly ROS and NO, play an important role in not only stomatal closure but also in integrating stimuli from abiotic or biotic stress (Song et al., 2 induced stomatal closure can also be mediated by ABA (Chater et al., 2 and ABA. An increase in ROS due to elevated CO2 in guard cells (Kolla et al., Interactions of guard cell signaling components with environmental factors are not only interesting but are essential for adaptation. Drought raises the levels of ROS and ABA levels in plant tissues, with both these phenomena leading to stomatal closure (Saxena et al., 2+ of guard cells, modulate ion channels, and cause an efflux of K+/Cl−/malate from guard cells, leading to stomatal closure. We emphasize that ROS, cytosolic Ca2+, and ion channels are the points of convergence (Figure The patterns and action sequence of signaling components during stomatal closure have been worked out using different triggers, such as ABA, MJ, and chitosan (Gonugunta et al., e Figure . The cytAR proposed the topic. AR and AS collected the literature, critically assessed the information, and wrote the manuscript together.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Any changes in Ca2+ storage and release from the sarcoplasmic reticulum (SR), or sensitivity of the contractile apparatus to Ca2+ may underlie the mechanism linking IUGR to an increased risk of CVD. This study aimed to explore the effects of maternal nutrient restriction on cardiac function, including Ca2+ handling by the SR and force development by the contractile apparatus. Juvenile Long Evans hooded rats born to Control (C) and nutrient restricted (NR) dams were anaesthetized for collection of the heart at 10–12 weeks of age. Left ventricular bundles from male NR offspring displayed increased maximum Ca2+-activated force, and decreased protein content of troponin I (cTnI) compared to C males. Furthermore, male NR offspring showed a reduction in rate of rise of the caffeine-induced Ca2+ force response and a decrease in the protein content of ryanodine receptor (RYR2). These physiological and biochemical findings observed in males were not evident in female offspring. These findings illustrate a sex-specific effect of maternal NR on cardiac development, and also highlight a possible mechanism for the development of hypertension and hypertrophy in male NR offspring.Intrauterine growth restriction (IUGR), defined as a birth weight below the 10th centile, may be caused by maternal undernutrition, with evidence that IUGR offspring have an increased risk of cardiovascular disease (CVD) in adulthood. Calcium ions (Ca At the end of the depolarization phase the SR rapidly re-accumulates Ca2+ via the SR Ca2+-ATPases  resulting in relaxation of the cardiomyocyte.In an attempt to understand the basis of the increased risk of disease described above, several previous studies have examined the effects of IUGR on the contractility of the heart. These studies have predominately examined whole heart function from IUGR animals  which have typically found both increased ventricular pressure –14 and d2+ handling and myofilament contractility in both male and female offspring. Furthermore, we have measured relative protein abundance and phosphorylation of key proteins related to the physiological processes associated with these steps in the ECC cascade.Using a global maternal nutrient restriction (NR) model  in WistaAll experimental protocols within this study were approved by the Animal Ethics committee La Trobe University and comply with the National Health and Medical Research Council: Guidelines to promote the wellbeing of animals used for scientific purposes.ad libitum access to standard rat chow. Dams were mated overnight with LE males and mating confirmed by the presence of a vaginal plug on the subsequent morning. Following confirmation of mating, dams were housed individually, with food intake and body weight measured daily throughout pregnancy. At day 14 of pregnancy, 5 dams were randomly selected to undergo a 60% NR diet  for the remaining trimester of gestation. This model has previously shown to produce both morphological and physiological changes to the heart , made by mixing appropriate volumes of Solution B and solution C together, until the maximum Ca2+-activated force was achieved (2+) and the procedure repeated. The average of each comparable force response was taken from the two treatment runs. Submaximal force responses were normalized to the maximum Ca2+-activated force and plotted against the-log free [Ca2+] (pCa). Non-linear regression, specifically one-phase exponential association, was used to fit the normalized data of each bundle, allowing for the Ca2+ sensitivity to be ascertained. The pCa generating 50% force (pCa50) and the Hill coefficient was measured from fitted curves in each bundle and averaged. The maximum Ca2+-activated force was measured by calculating the force (mN) per CSA (Area = πr2) of the bundle.Following Triton X-100 treatment, bundles were exposed to a pre-contraction solution  for 2 mins and then exposed to a series of heavily buffered Caachieved . Bundles-1. 50 μl aliquots of homogenates were diluted with 25 μl 3x SDS solution  and then further diluted to 2.5 μg wet weight.μl-1. Ca2+ handling proteins associated with both the uptake and storage of Ca2+ within the SR, namely calsequestrin (CSQ2) RyR2, SERCA2a, and contractile proteins responsible for force production, namely troponin I (cTnI), tropomyosin (Tm), troponin T (cTnT) and troponin C (TnC) were analyzed by Western blotting. Briefly, 25 μg wet weight of each sample was loaded onto 4–15% Criterion Stain Free™ SDS-PAGE gels , proteins were separated (200 V for 45 min), and Stain Free images captured using Stain Free™ Imaging System (Bio-Rad) for total protein quantification. Proteins were then transferred to nitrocellulose membranes (100V for 30 min). Following a series of rinses, membranes were immersed in antibody extender solution  for 10 min, and rinsed. Membranes were washed in a blocking buffer containing 5% skim milk in phosphate-buffered saline with 0.025% Tween (PBST) for 2 hr at RT, and then exposed overnight at 7°C to rabbit monoclonal anti-SERCA2a, the first of six primary antibodies to be investigated , as previously described ,30. This2+ content and rate of release data. As no grouping effects were evident for any physiological parameters investigated, biochemical data were analyzed using Student’s independent samples t-tests in samples from each sex. All findings were deemed statistically significant at P < 0.05.Data were analyzed with a combination of Prism 5 (GraphPad), LabChart 7 (ADInstruments) and SPSS 19 (IBM). G*Power 3.1.2 was utilized for post hoc power analysis. Mean physiological data were analyzed with ANCOVA . Linear regression was employed to analyze SR Card trimester gain of ~2.8% BW, compared with 18.5% in Controls, with NR dams significantly smaller than Controls at parturition  of both Control and NR dams increased by ~15% during the first 2 trimesters. Following the induction of NR protocol, subsequent weight gain in NR dams was reduced, with a 3F  = 15.724, P = 0.004; th percentile of control pups . At the time of physiological experimentation, 10–12 week old NR male and female offspring had no difference in heart weight  = 13.74, P = 0.01; 2+ release was not significantly different between female Control and NR offspring  = 8.925, P = 0.017; 2 ± 0.18 vs. NR, 8.38 ± 0.27; F  = 2.966, P>0.05; 0.03; Male Control and NR samples showed no significant difference in the relative amount of the regulatory protein TnC, however the inhibitory cTnI protein showed a significant decrease compared with Controls  and the species, including rats, sheep and guinea pigs –33. Manyture see . Our res2+, and subsequently release it, enabling relaxation and contraction respectively, is essential to maintaining contractile function in cardiomyocytes. Factors affecting the quantity of Ca2+ loaded and stored in the SR may include the buffering of the intracellular environment, the spatial size of the SR, and quantitative and functional properties of SERCA2a, CSQ2 and RyR2. To date no studies have investigated the properties of the SR and its associated structural and functional proteins in relation to IUGR directly. We show for the first time that the effects of global nutrient restriction leading to IUGR resulted in changes in SR properties visible only in male offspring. We show that NR did not affect the net SR Ca2+ content in either male or female offspring (as assessed by the area of the caffeine-induced force responses associated with different Ca2+ loading periods) and similarly, the expression of two key proteins that are responsible for SR Ca2+ uptake (SERCA2a) and SR Ca2+ content (CSQ2) were also unaffected. These data initially suggest that NR has little impact on both SR Ca2+ uptake and SR Ca2+ storage. However, when we examined the rate of caffeine-induced Ca2+ release, we note a significant decrease in the rate of rise in male offspring only. This was associated with a concomitant decrease in the protein expression of RyR2. These data suggest that NR decreases the ability of the SR to release Ca2+ likely through a reduction in the amount of RyR2. When we reconsider that the SR Ca2+ content data described above, one would expect that in NR males, the net accumulation of Ca2+ (which is a function of the difference between SR Ca2+ leak via RyR2 and SR Ca2+ uptake via SERCA2a) should be reduced given the reduced amount of RyR2 observed and the reduced rate of caffeine-induced Ca2+ release. Given that the SR Ca2+ contents were the same between NR and Controls it is plausible that there has been some proportional decrease in the rate of SR Ca2+ uptake not evident from the similar amount of SERCA2a measured between treatments. Although we did not measure the degree of phosphorylation of phospholamban , it is possible that there may be differences in the amount of phosphorylation of phospholamban that would alter SERCA2a activity between treatments that could explain these data.The ability of the SR to load and store Ca2+ handling, the effects of maternal NR leading to IUGR in the current study bare similarities to several disease states long associated with IUGR. Previous studies in diabetic rats [Although there are no prior studies that have directly examined the effects of IUGR on SR Catic rats , a conditic rats , have shtic rats ,39, howetic rats . Converstic rats .2+-activated force (>25%) than Controls. In skinned cardiomyocyte bundles from females, there was no difference in the maximum Ca2+-activated force. In addition, the Ca2+ sensitivity of the contractile apparatus was not different between NR and Control offspring of either sex. These data are consistent with several functional studies in whole perfused heart. When comparing both chronic hypoxia and NR during late gestation, Xu and colleagues reported an increase in LV end-diastolic pressure in 7 month old NR rats. However, in younger animals (4 months), which were around the same age as our own (10–12 weeks), NR did not produce a significant change in cardiovascular parameters measured [In our current study, we show that skinned cardiomyocyte bundles from male NR offspring produced significantly greater maximum Cameasured . These dmeasured . Similarmeasured  however,measured . Similar our own –12 weeks2+-activated force observed in the current study and these appear to be linked with the functional state of the troponin complex. For the first time, we show a significant reduction in cTnI levels in the NR male, but not female rats. It is becoming clearer that cTnI plays an important role in the normal functional dynamics of the heart muscle with changes in the pattern of expression during development [2+-sensitivity of the contractile apparatus [2+-activated force development simply through its direct interaction with TnC—with a diminished cTnI content resulting in less inhibition of TnC leading to a more primed myofilament [2+-sensitivity [2+-sensitivity [2+-sensitivities in both Control and NR animals. Furthermore, previous studies in skinned rat cardiomyocytes [2+ activations and this was also coupled with a reduction in Ca2+ sensitivity [2+ sensitivity in NR animals, which may counter an expected increase in the Ca2+ sensitivity if there is also a greater expression of the fetal ssTnI isoform. However, as the current study found no evidence of increased passive force (a likely scenario given the reduced inhibitory effect of cTnI), and TnC appear reduced , it is possible that if one views the troponin complex as a whole, a reduction in the overall amount of the troponin sub-units may change the spatial alignment of the sarcomeres, resulting in a state of enhanced contractility.There are several possible mechanisms that could explain the increased maximum Caelopment ,44 as weelopment ,46). Durpparatus ,44. Thispparatus ,47. Thusfilament . Alternafilament . Fetal ssitivity ,44, whicmyocytes  showed tsitivity . Thus, i2+ handling properties of the SR and the functional state of the contractile apparatus in only male IUGR offspring. These data support the notion that IUGR contributes to long-term changes in the expression and function of key cardiac proteins  that may contribute to an increased risk of CVD later in adult life.In conclusion, we have shown sex-dependent differences in ECC parameters for the first time, with changes to both the CaS1 Dataset(XLSX)Click here for additional data file.S1 ImageFemale CSQ2 Western blot.(SCN)Click here for additional data file.S2 ImageFemale cTnI Western blot.(SCN)Click here for additional data file.S3 ImageFemale p-cTnI Western blot.(SCN)Click here for additional data file.S4 ImageFemale RyR2 Western blot.(SCN)Click here for additional data file.S5 ImageFemale SERCA2a Western blot.(SCN)Click here for additional data file.S6 ImageFemale TnC Western blot.(SCN)Click here for additional data file.S7 ImageMale CSQ2 Western Blot.(SCN)Click here for additional data file.S8 ImageMale cTnI Western blot.(SCN)Click here for additional data file.S9 ImageMale p-cTnI Western blot.(SCN)Click here for additional data file.S10 ImageMale RyR2 Western blot.(SCN)Click here for additional data file.S11 ImageMale SERCA2a Western blot.(SCN)Click here for additional data file.S12 ImageMale TnC Western blot.(SCN)Click here for additional data file.


In addition to the action of Ca2+ within mitochondria, Ca2+ released from mitochondria is also important in a variety of cellular functions. In the last 5 years, the molecules responsible for mitochondrial Ca2+ dynamics have been identified: a mitochondrial Ca2+ uniporter (MCU), a mitochondrial Na+–Ca2+ exchanger (NCLX), and a candidate for a mitochondrial H+–Ca2+ exchanger (Letm1). In this review, we focus on the mitochondrial Ca2+ release system, and discuss its physiological and pathophysiological significance. Accumulating evidence suggests that the mitochondrial Ca2+ release system is not only crucial in maintaining mitochondrial Ca2+ homeostasis but also participates in the Ca2+ crosstalk between mitochondria and the plasma membrane and between mitochondria and the endoplasmic/sarcoplasmic reticulum.Mitochondrial Ca They also serve as master switches determining cell fate on exposure to different stimuli [2+ homeostasis in mitochondria have been extensively studied over the last half century, so the importance of mitochondrial Ca2+ in regulating mitochondrial functions is well recognized. Ca2+ enters mitochondria mainly via a mitochondrial Ca2+ uniporter, a protein known as MCU or CCDC109A [2+ release by mitochondria have just begun, although functional characterization of the release system started in the 1970s [2+ release system mainly consists of an Na+–Ca2+ exchanger and an H+–Ca2+ exchanger. The molecule responsible for the former (NCLX) was identified in 2010 [ Mitochondria are crucial organelles in ATP production as well as in Ca stimuli –3. MechaCCDC109A , 5. The CCDC109A –8. On th in 2010 , althougIt is now well understood that some mitochondria are in close contact with the plasma membrane and others with the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR). Although the molecular mechanisms of tethering of mitochondria to the plasma membrane are not well understood, several tethering protein complexes involved in interactions between mitochondria and the ER/SR have been identified, and details of the molecular mechanisms have been reviewed , 13. It 2+ release system, specifically, interactions between mitochondria and the ER/SR and interactions between mitochondria and the plasma membrane, and discuss their physiological and pathophysiological significance, focusing on the destiny of Ca2+ released by mitochondria.In this paper we review recent progress in the study of mitochondrial Ca2+ is important in regulation of diverse cellular functions . In fact, an increase of mitochondrial Ca2+ has been reported to increase production of ROS in the heart and in neurons, resulting in impaired respiration and in cytotoxicity [2+ activates F1-Fo ATP synthase, balancing ATP utilization increased by Ca2+, and that it is probably the intra-mitochondrial Ca2+ which regulates F1-Fo ATP synthase, although this is still controversial [2+ results in opening of a non-specific pore called the mitochondrial permeability transition pore (PTP). PTP opening is followed by inner mitochondrial membrane depolarization, uncoupling of oxidative phosphorylation, and massive mitochondrial swelling, resulting in necrosis [2+, contributing to NO-mediated cardioprotection against PTP opening [2+ is crucial in tuning a variety of mitochondrial and, thus, cellular functions.It is now well accepted that mitochondrial Caons Fig. . For exaf a cell –16. Mitotoxicity , 18. Sevoversial , 20. A lnecrosis –23. PTP necrosis –26. Furt opening . Accordi2+? The location of the mitochondria within the cell seems important for mitochondrial Ca2+ uptake. Some mitochondria are located in proximity to the plasma membrane or the ER/SR. Ca2+ flowing through the plasma membrane or released from the ER/SR predominantly enters neighbouring mitochondria. In other words, there is Ca2+ crosstalk between mitochondria and the plasma membrane and between mitochondria and the ER/SR  and the Ca2+ release-activated Ca2+ channel (CRAC) or store-operated Ca2+ channel. The Ca2+ which enters the cell is efficiently taken up by mitochondria located near the plasma membrane [2+, re-addition of extracellular Ca2+ evoked an increase in mitochondrial Ca2+ but not in cytoplasmic Ca2+ in human umbilical vein endothelial cell line ECV304, in which 14 % of mitochondria are located within 700 nm of the inner surface of the plasma membrane. They suggested that Ca2+ levels increase in microdomains beneath the plasma membrane, causing predominant Ca2+ uptake by mitochondria facing the microdomains. However, the contribution of the Ca2+ microdomains depends on cell type, because this phenomenon was not observed for a clone of a HeLa cell line, in which <6 % of mitochondria are located in the proximity of the plasma membrane. Park et al. [2+ influx via CRAC channels through the basolateral membrane led to predominant Ca2+ uptake by sub-plasmalemmal mitochondria in pancreatic acinar cells. These reports suggest Ca2+ communication, through plasma membrane Ca2+ channels, with mitochondria located in proximity to plasma membrane. Ca2+ communication in the opposite direction has also been suggested. Mitochondrial membrane depolarization suppressed store-operated Ca2+ entry in T lymphocytes and in rat basophilic leukaemia cells [2+ crosstalk between mitochondria and the plasma membrane is important in the regulation of cellular functions. As will be described below, the mitochondrial Na+-Ca2+ exchanger NCLX is involved in this Ca2+ crosstalk.Camembrane –33. Lawrmembrane  found thk et al.  reportedia cells , 35. Acc2+ store within the cells. The ER/SR is located close to mitochondria, approximately 5–30 nm, and narrow interorganellar spaces exist between the organelles in a variety of cell types, including B lymphocytes and cardiomyocytes [2+ signalling [2+, these narrow interorganellar spaces may serve to create Ca2+ level much higher than in the bulk cytosol, up to 9–50 μM [2+ uniporter, the Ca2+ affinity of which is relatively low, to transport enough Ca2+ into mitochondria. Several studies reported that an inositol 1,4,5-trisphosphate (IP3) receptor of the ER and a voltage-dependent anion channel of the outer mitochondrial membrane are located in the mitochondria–ER region, creating a Ca2+ pathway from the ER to the mitochondria. This may aid regulation of energy metabolism and apoptosis in such cells as HeLa cells, CHO cells, fibroblasts, and yeast [2+ released from the ryanodine receptor (RyR) on the SR also accumulates in mitochondria of rat ventricular myocytes, indicating preferential coupling of Ca2+ transport from the SR to mitochondria [2+ movement, from mitochondria to the ER/SR, is important for refilling ER/SR Ca2+ after ligand stimulation of such cells as HeLa cells, endothelial cell line, and vascular smooth muscle cells, to minimize cytosolic Ca2+ elevation and to prevent depletion of ER/SR Ca2+ [2+ from mitochondria to the ER/SR was clearly demonstrated in our recent studies [2+ crosstalk between mitochondria and the ER/SR is also important for regulating cellular functions.The ER/SR is major Camyocytes . Figure myocytes , 13. Mitgnalling , 37. Wit 9–50 μM , 39, enand yeast –42. Ca2+chondria . It has /SR Ca2+ –45. The  studies –48, as d2+ crosstalk between mitochondria and the plasma membrane and between mitochondria and the ER/SR is important in controlling cellular functions. Understanding the roles of each molecule participating in the Ca2+ crosstalk is particularly important for fully understanding cellular physiological and pathophysiological functions.Taken together, Ca2+ dynamics started more than half a century ago. The existence of a respiration-dependent pathway of Ca2+ into isolated rat kidney mitochondria was reported in the early 1960s [2+ out of isolated rat heart mitochondria (Na+-dependent) and out of isolated rat liver mitochondria (Na+-independent and H+-dependent) were discovered [2+ dynamics have been extensively studied [Studies of mitochondrial Caly 1960s , 50. In scovered . Since t studied –54.2+ uptake into and efflux out of mitochondria are summarized in Fig. 2+ uptake into mitochondria is mainly mediated by a mitochondrial Ca2+ uniporter driven by a highly negative membrane potential  [2+ uptake from fast cytosolic Ca2+ transients. A mitochondrial ryanodine receptor is also reported to mediate Ca2+ uptake into rat heart mitochondria. Details are available in other reviews [2+ uniport were revealed. A regulator of the Ca2+ uniporter, MICU, was discovered in 2010 before cloning of the mitochondrial Ca2+ uniporter [2+ uniporter [Trypanosoma brucei resulted in marked dysregulation of mitochondrial bioenergetics, causing autophagy and cell death [The major pathways of Ca−180 mV) . Another reviews . It was niporter . MCU  pathway and an Na+-independent  pathway. Under pathophysiological conditions in which the PTP opens, PTP functions as a Ca2+ transporter from mitochondria. In the sections below we describe, in detail, the two physiological pathways of mitochondrial Ca2+ release.Ca+–Ca2+ exchanger was first discovered by Carafoli et al. [+–Ca2+ exchange activity is found in a wide variety of tissues and is dominant in the heart, brain, skeletal muscle, parotid gland, adrenal cortex, and brown fat [+–Ca2+ exchanger is also present in liver, kidney, and lung mitochondria, although its activity is weak [+–Ca2+ exchange activity is low, H+–Ca2+ exchange activity is of dominant importance in the release of Ca2+ from mitochondria [The mitochondrial Nai et al.  in 1974 rown fat , 59. The is weak . In tisschondria .+–Ca2+ exchanger, which is distinct from the plasmalemmal Na+–Ca2+ exchanger (NCX), is that Li+ can substitute for Na+ [+–Ca2+ exchanger, as will be described in the next section. The stoichiometry (ion-exchange ratio) and the electrogenicity of the mitochondrial Na+–Ca2+ exchanger were controversial, but it was believed to be electroneutral [+–Ca2+ exchanger is voltage-dependent and electrogenic, which suggests the stoichiometry is >3Na+ for one Ca2+ [+–Ca2+ exchanger changes, the affinity becoming lower with mitochondrial membrane depolarization [+–Ca2+ exchanger dynamically changes the exchange mode (forward or reverse) and modulates the mitochondrial Ca2+ concentration in a manner dependent on cytosolic Na+ concentration and mitochondrial membrane potential. Recently, several molecules which may modulate the mitochondrial Na+–Ca2+ exchanger have been identified. Gandhi et al. [2+/calmodulin family, causes impairment of mitochondrial Ca2+ efflux via the mitochondrial Na+–Ca2+ exchanger. Mutations in the PINK1 gene are known to cause autosomal recessive Parkinson’s disease [+–Ca2+ exchange activity in HeLa cells, regulating the capacity of mitochondria to store Ca2+. Although detailed mechanisms underlying the regulation of mitochondrial Na+–Ca2+ exchange activity by these proteins have not yet been clarified, recent identification of NCLX as a mitochondrial Na+–Ca2+ exchanger will surely accelerate understanding of the mechanisms.One interesting characteristic of the mitochondrial Na for Na+ . This unoneutral , 63. Ourone Ca2+ . We alsorization . Becausei et al.  reported disease . Da Cruz disease  showed t+–Ca2+ exchange, which was subsequently identified as a mitochondrial Na+–Ca2+ exchanger [+-dependent Na+–Ca2+ exchanger (NCKX) gene family 2 (NCKX2) [+-dependent Na+–Ca2+ exchange activity. Very soon after the publication by Cai and Lytton [+–Zn2+ exchanger gene [+-independent Na+–Ca2+ exchange activity and did not have Na+–Zn2+ exchange activity when heterologously expressed in HEK293 cells. They also found that Li+ can substitute for Na+ to release Ca2+ from the cells, so they named the clone “NCLX”. The discrepant results obtained by the two groups might be a result of different experimental conditions, i.e. Cai and Lytton [+ as substituent for Na+ to examine K+-dependent Na+–Ca2+ exchange activity. This might have affected the responsiveness of the clone. In addition, FLAG epitope might have interfered with the sorting system in the Cai and Lytton [+–Ca2+ exchange activity and broad tissue distribution, for example heart, pancreas, skeletal muscle, stomach, spleen, and brain.In 2004, two independent research groups reported the cloning of a new transporter mediating Naxchanger , 67. Cai (NCKX2) . The amid Lytton , Sekler’ger gene , both lod Lytton  used Li+d Lytton  experime+–Ca2+exchanger. In 2010, Sekler’s group beautifully demonstrated that NCLX was located in mitochondria and its characteristics corresponded well to those of the mitochondrial Na+–Ca2+ exchanger [2+ by use of the mitochondria-targeted Ca2+ sensing protein Pericam-mito [2+ efflux from mitochondria was accelerated by overexpressing NCLX in an Na+ and Li+-dependent manner and was decelerated by knocking down NCLX. The fact that Li+ can substitute for Na+ in releasing Ca2+ from mitochondria corresponds well to the unique characteristic of mitochondrial Na+–Ca2+ exchange. This NCLX-mediated Ca2+ efflux from mitochondria was inhibited by the mitochondrial Na+–Ca2+ exchange inhibitor CGP-37157. It was also shown that mitochondrial Na+, measured by use of the mitochondrial Na+-sensing dye CoroNa Red, increased during the mitochondrial Ca2+ efflux phase, and the rate became much faster with NCLX overexpression. These results confirm that NCLX is the gene responsible for the mitochondrial Na+–Ca2+ exchanger. Since then, information about the involvement of NCLX in the physiological and pathophysiological functions of different types of cell has accumulated. We will describe, in detail, recent findings on the roles of NCLX in pancreatic β-cells, astrocytes, B lymphocytes, and cardiomyocytes. Experimental results from NCLX knockout/knockdown in each type of cell are summarized in Table It took another 6 years, however, for Sekler’s group to discover that NCKX/NCLX is the long-sought mitochondrial Nacam-mito  clearly + channels consequently close, resulting in depolarization of the plasma membrane. Subsequently, voltage-gated Ca2+ channels open, causing Ca2+ influx into the cell, and exocytosis of insulin granules occurs [2+, Nita et al. [2+ influx, ATP-mediated ER Ca2+ depletion, and treatment with 20 mM glucose all caused a mitochondrial Ca2+ transient. In all cases, silencing of NCLX by use of siRNA or dominant negative mutant resulted in a more rapid increase in mitochondrial Ca2+ and slower mitochondrial Ca2+ decline. The resting mitochondrial Ca2+ level was higher in NCLX knockdown cells. These results not only suggest that NCLX functions as a mitochondrial Na+–Ca2+ exchanger, but also show that Ca2+ efflux via NCLX is already activated at the early phase of mitochondrial Ca2+ influx. Interestingly, silencing NCLX caused a smaller and slower cytosolic Ca2+ increase induced by membrane depolarization or by treatment with 20 mM glucose. Furthermore, NCLX knockdown was followed by a delay in glucose-dependent insulin secretion, whereas ATP production was little affected. These results indicate that NCLX is a critical component of the Ca2+ crosstalk between mitochondria and the plasma membrane in modulation of the insulin secretion pattern. Tarasov et al. obtained results essentially similar to those of Nita et al. [2+ response and subsequent ATP/ADP increase was impaired under the glucolipotoxic conditions often observed with type 2 diabetes. Because mRNA expression levels of MCU and NCLX were unaltered, it was suggested that changes in the intracellular distribution of mitochondria [2+ dysfunction and ATP production. In 2013, Proverbio et al. [When the plasma glucose level increases, ATP production in pancreatic β-cells increases. ATP-dependent Ks occurs . In addis occurs , 71, Tars occurs , 73 demoa et al.  found thchondria  were invo et al.  used a m2+. It is also well known that astrocytes release neurotransmitters called gliotransmitters, for example glutamate, ATP and d-serine, which bind to neuronal receptors to modulate synaptic transmission and activity [2+ [2+ in astrocytes occurs via the phospholipase C (PLC)/IP3 pathway. That is, upon GPCR activation, PLC hydrolyses the membrane lipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and IP3, activating the IP3 receptor (IP3R) and releasing Ca2+ from the ER. Parnis et al. [2+ dynamics and the release of gliotransmitters. They used pericam-mito to sense mitochondrial Ca2+ and found that the resting mitochondrial Ca2+ level increased, and that the rise and decline of mitochondrial Ca2+ transients caused by extracellular ATP application became faster and slower, respectively, on knocking down NCLX. These results were very similar to those observed for pancreatic β-cells [2+ entry into the cytosol both from extracellular space and from the ER, but the effect on the former was stronger. Detailed analysis revealed that NCLX knockdown impairs store-operated Ca2+ entry (SOCE), indicative of strong Ca2+ crosstalk between mitochondria and the plasma membrane, as in pancreatic β-cells. NCLX knockdown also significantly reduced such processes as exocytotic glutamate release, in vitro wound closure, and proliferation, which may be regulated in a Ca2+-dependent manner [Brain is another tissue highly expressing NCLX. Among the different kinds of cell constituting the brain, astrocytes constitute approximately half the volume. They express a large number of G protein-coupled receptors (GPCRs), linked to a diverse array of intracellular cascades including elevation of intracellular Caactivity , 77. Thuvity [2+ . The incs et al. , by use  β-cells . Interest manner .2+, resulting in rapid proliferation and differentiation or apoptosis, depending on the differentiation stage of the cells [3 increases and facilitates Ca2+ release from the IP3R on the ER membrane. Ca2+ release from the ER causes the initial cytosolic Ca2+ increase after receptor activation. Subsequent depletion of Ca2+ in the ER causes translocation of stromal interaction molecule 1 (STIM1) to the vicinity of plasmalemma, inducing a sustained and oscillatory cytosolic Ca2+ increase by activation of SOCE through CRAC channels encoded by ORAI1 [2+ signalling from a combined study of mathematical simulations and NCLX knockout/knockdown in DT40 and A20 B lymphocytes [+-dependent mitochondrial Ca2+ efflux in saponin-permeabilized cells loaded with the mitochondrial Ca2+-sensitive dye Rhod-2, confirming that NCLX is responsible for mitochondrial Na+–Ca2+ exchange in B lymphocytes. Interestingly, mitochondrial membrane potential, evaluated by JC-1 staining, was more depolarized and DNA fragmentation (sub G1) was increased in NCLX knockout cells, suggesting that the progress of apoptosis was accelerated in NCLX knockout cells. Mathematical model revealed that NCLX inhibition reduces basal ER Ca2+ content and suppresses BCR-mediated cytosolic Ca2+ rise. These predictions were validated by experiments. i.e., ER Ca2+ content decreased and the cytosolic increase of Ca2+ was diminished in NCLX knockout/knockdown cells after the BCR activation. Comparable with the results obtained by use of astrocytes, SOCE activity in B lymphocytes was also diminished by silencing NCLX, probably further contributing to impairment of the cytosolic Ca2+ increase after BCR activation [2+ content was a result of impaired Ca2+ supply from mitochondria via NCLX, because ER Ca2+ uptake via the ER Ca2+ pump SERCA was decelerated by NCLX reduction when mitochondrial respiration was intact  and the hyperpolarization-activated cation channel (Iha), which are known to be involved in the automaticity of cardiac pacemaker sinoatrial (SA) node cells [+-dependent mitochondrial Ca2+ efflux, confirming that NCLX is responsible for mitochondrial Na+–Ca2+ exchange in HL-1 cells. Mitochondrial Ca2+ content, evaluated as the Ca2+ chelation-responsive fraction of the intensity of mitochondrial Ca2+ sensor pCase12-mito, was larger in NCLX knockdown cells. Although beat-to-beat change of mitochondrial Ca2+ was not observed in HL-1 cells, the result indicated that NCLX contributes to the steady state mitochondrial Ca2+ content in intact HL-1 cells. Cellular energetics seemed to be unaffected by NCLX knockdown, because there were no differences in cellular ATP content, mitochondrial membrane potential, or mitochondrial ROS between control cells and NCLX knockdown cells. This may be because protein expression of NCLX knockdown using siRNA was reduced by 50 % only.The heart is one of the most studied organs for investigation of the characteristics of the mitochondrial Nae system , 88. Theoduction , 90. In as 1–2 % . Howeveras 1–2 % . The expde cells –95. NCLX2+ transients [2+ transients obtained by line scanning cells loaded with cytosolic Ca2+ indicator Fluo-4 revealed that NCLX knockdown slowed the upstrokes of both action potentials and cytosolic Ca2+ transients. SR Ca2+ dynamics, which is known to contribute to the upstroke of cytosolic Ca2+ transients, was evaluated by use of ER/SR Ca2+ FRET protein cameleon D1ER [2+ content of NCLX knockdown cells was smaller and SR Ca2+ reuptake rate was slower , activation of the inward current of the plasma membrane NCX (INCX), and thus the timing of activation of voltage-dependent Na+ current (INa) and voltage-dependent T and L type Ca2+ channels (ICaT and ICaL) was thus delayed, resulting in prolongation of the cycle length. Taken together, our combined experiments and simulations indicated that NCLX regulates the rhythmicity of HL-1 cells via crosstalk between mitochondria and SR Ca2+ dynamics. Considering that NCLX reduction resulted in modification of plasma membrane NCX activity, NCLX may also be indirectly involved in mitochondria–plasma membrane Ca2+ crosstalk in HL-1 cells. Interestingly, Opuni and Reeves reported the functional coupling of mitochondrial function, possibly mitochondrial Na+–Ca2+ exchange activity, and plasma membrane NCX activity in Chinese hamster ovary cells stably transfected with bovine cardiac NCX [An interesting finding was that NCLX knockdown caused marked prolongation of the cycle length of spontaneous action potentials and Caansients . Kineticeon D1ER . As a rewer Fig. b, sugges-1 cells . The HL-diac NCX . Furtherdiac NCX , 99 is c+–Ca2+ exchange inhibitor CGP-37157 slowed the generation of automaticity of rabbit SA node cells, suggesting that mitochondrial Na+–Ca2+ exchange is involved in generation of the automaticity of SA node cells. However, because CGP-37157 also blocks ICaL, which is related to generation of the automaticity of SA node cells, a nonspecific effect of CGP-37157 on ICaL cannot be ignored. In addition, the automaticity of the SA node cells they used significantly depends on spontaneous and local subsarcolemmal Ca2+ releases from SR, the so called “Ca2+ clock” mechanism [Iha and a variety of other inward membrane currents determine the automaticity [Whether NCLX participates in the automaticity of normal pacemaker cells, sinoatrial (SA) node cells, is a big issue. Recently, Yaniv et al.  reportedechanism . Howevermaticity –104. Thi+–Ca2+ exchange resulted in prolongation of cycle length by 2.7 % only. The larger effect reported by Yaniv et al. [2+ dynamics into two representative SA node cell models: a membrane clock model by Himeno et al. [2+ clock model by Maltsev and Lakatta [2+ content, supporting the idea of Ca2+ communication between mitochondria and the SR in SA node cells. However, the effect on the automaticity was different between the models. The cycle length was prolonged in the Maltsev and Lakatta model whereas it was shortened in the Himeno model [+ and Na+-permeable inward current (sustained inward current Ist) [2+, which decreased the inward INCX thus reducing the cytosolic Na+ concentration. This increased the amplitude of the inward Ist and overcame the decrease of inward INCX, accelerating the firing rate. In contrast, the amplitude of Ist is set smaller in the Maltsev and Lakatta model so that NCLX reduction only reduces inward INCX and slows diastolic depolarization, decelerating the firing rate [2+ communication between mitochondria and the SR via NCLX functions also in SA node cells. However, it is necessary to investigate quantitatively how much the ‘‘Ca2+ clock’’ mechanism, cytoplasmic Na+, and Ist contribute to SA node automaticity.We examined the contribution of NCLX using mathematical models of SA node cells . In the v et al.  is probao et al. , 103 and Lakatta . In bothno model . Furthernt Ist)  are crucing rate . Accordi+–Ca2+ exchange has a dominant effect on release of Ca2+ from mitochondria in tissues in which mitochondrial Na+–Ca2+ exchange activity is low, for example the liver, kidney, lung, and smooth muscle [+–Ca2+ exchanger also occurs in the heart, though the activity is weak [Hh muscle . The H+– is weak , 107.+–Ca2+ exchanger is regarded as 2H+ for 1Ca2+, being electroneutral. However, because the rate of efflux via the H+–Ca2+ exchanger decreases with increasing pH gradient in rat isolated liver mitochondria, it is suggested that the mechanism is not a passive Ca2+ for 2H+ exchanger, but an active Ca2+ for 2H+ exchanger [The stoichiometry of the Hxchanger .+–Ca2+ exchanger is still controversial. The candidate is the Letm1 (leucine–zipper–EF hand-containing transmembrane region). Jiang et al. [Drosophila genes and identified a gene affecting mitochondrial Ca2+ and H+, the human homolog of which is Letm1. By using digitonin-permeabilized S2 or 293 cells expressing mitochondrial Ca2+ sensor protein pericam, or by using purified Letm1 reconstituted in liposomes, they found that Letm1 mediates H+–Ca2+ exchange. However, because a drastic reduction of mitochondrial Ca2+ uptake was observed when Letm1 protein expression was suppressed, and because H+–Ca2+ exchange via Letm1 was sensitive to an inhibitor of mitochondrial Ca2+ influx, ruthenium red, Letm1 is regarded as mediating Ca2+ influx into mitochondria, at least at low cytosolic Ca2+ level. This idea was confirmed by subsequent work by Jiang et al. [2+ content. In the same work they produced Letm1 knockout mice and found that Letm1 homozygous knockout mice were embryonic lethal and so were half of the heterozygous knockout mice. The surviving mice had altered glucose metabolism, impaired control of brain ATP levels, and increased seizure activity, suggesting involvement of Letm1 in the pathology of Wolf–Hirschhorn syndrome, in which Letm1 is known to be one of the genes deleted [2+ flux through the mitochondrial membrane in energized mitochondria with different H+:Ca2+ stoichiometry. They proposed that Ca2+ enters mitochondria with 1H+:1Ca2+ whereas Ca2+ leaves mitochondria with 2H+:1Ca2+ or 3H+:1Ca2+ stoichiometry under physiological respiration conditions. Tsai et al. [+:1Ca2+ antiport and is insensitive to ruthenium red, by using Letm1 reconstituted proteoliposome. These results combined with the theoretical analysis by Nowikovsky et al. [+–Ca2+ exchanger. There is, currently, no reasonable explanation of the discrepancy of the sensitivity of Letm1 to ruthenium red. We independently found that a substantial part of the Ca2+ efflux from mitochondria was independent of cytosolic Na+ when mitochondria were loaded with a lower concentration of Ca2+ than when examining Na+–Ca2+ exchange activity using A20 B lymphocytes [+–Ca2+ exchange system, was sensitive to ruthenium red. Further analysis is necessary to determine whether Letm1 is, indeed, the H+–Ca2+ exchanger mediating Ca2+ extrusion from mitochondria.The molecular identity of the mitochondrial Hg et al.  conducteg et al.  in which deleted . Nowikov deleted  analysedi et al.  reportedy et al.  stronglyphocytes . This fr2+, not only inside mitochondria but also released from mitochondria, is crucially important in regulating a variety of cellular physiological functions. Identification of the molecules responsible for the pathways of Ca2+ release enables us to isolate the contributions of these molecules. For example, NCLX has been shown to participate in insulin secretion in pancreatic β-cells, glutamate release in astrocytes, Ca2+ responsiveness to BCR stimulation in B lymphocytes, and generation of the spontaneous rhythmicity of cadiomyocytes, via Ca2+ crosstalk between mitochondria and the plasma membrane and/or between mitochondria and the ER/SR (Fig. 2+ release proteins are indeed located in the tethering spots between mitochondria and the ER/SR or between mitochondria and the plasma membrane. Another is the contribution of the mitochondrial Ca2+ release system to mitochondrial energetics. Distinct phenotypes of NCLX knockdown cells related to mitochondrial energetics in cardiomyocytes and in pancreatic β-cells have not been observed, while Letm1 knockout mice had impaired mitochondrial energetics. Complete knockout of NCLX in mice or cells will clarify the matter.Ca/SR Fig. . However


The heart's regular electrical activity is initiated by specialized cardiac pacemaker cells residing in the sinoatrial node. The rate and rhythm of spontaneous action potential firing of sinoatrial node cells are regulated by stochastic mechanisms that determine the level of coupling of chemical to electrical clocks within cardiac pacemaker cells. This coupled-clock system is modulated by autonomic signaling from the brain via neurotransmitter release from the vagus and sympathetic nerves. Abnormalities in brain-heart clock connections or in any molecular clock activity within pacemaker cells lead to abnormalities in the beating rate and rhythm of the pacemaker tissue that initiates the cardiac impulse. Dysfunction of pacemaker tissue can lead to tachy-brady heart rate alternation or exit block that leads to long atrial pauses and increases susceptibility to other cardiac arrhythmia. Here we review evidence for the idea that disturbances in the intrinsic components of pacemaker cells may be implemented in arrhythmia induction in the heart. Defects in these cell-intrinsic capacities to elicit spontaneous action potentials (APs) can lead to disturbances of the rate and rhythm of heart beats, and can induce numerous clinical arrhythmia syndromes: (i) SAN dysfunction has been postulated to be a source of sinus nodal re-entry  tachyarrhythmia  components of pacemaker cells may be implicated in arrhythmia induction in the heart.To understand abnormal SAN function it is essential to first understand the normal function of intrinsic properties of pacemaker cells and their modulation by brain-heart signaling. Experimental and theoretical data over the past two decades indicate that pacemaker cells residing in the SAN entrain their AP BI variability (BIV) by regulation of intracellular electric and mechanical coupling  close to the cell surface membrane; LCRs activate membrane electrogenic clock molecules (“M clock”), mainly the Na+/Ca2+ exchanger. Na+-Ca2+ exchange current, the f-channel current, and K+ channel current, other components of the M clock, concurrently drive the diastolic membrane depolarization to ignite the next AP. The Ca2+ and M clocks entrain each other through electrical and chemical signaling: Ca2+ activation of calmodulin -adenylyl cyclase (AC)-dependent protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Both of these signaling pathways affect phosphorylation of proteins of both clocks . Additionally, cAMP positively shifts the f-channel activation curve. Based on the coupled-clock theory, a change in the activity or in the quantity of every molecule within the M or the Ca2+ clock or a change in the chemical coupling signaling of both clocks will perturb the function of the other clock, and thus alter the degree of entrainment between them. For example, in rabbit pacemaker cells, reduction in Ifactivity or in Ca2+ clock proteins leads to reduction in the coupled-clock phosphorylation activity and LCR signal . Based on the coupled-clock theory, the stochasticity of LCR periods  not only depends upon stochasticity of spontaneous RyR activation, but also upon stochastic sarcolemmal ion channel openings and closings that regulate the cell Ca2+ balance. The amplitude and timing of LCR Ca2+ signals to M clock proteins report the efficiency of clock coupling, i.e., a weaker LCR signal to M clock proteins that occurs later in time reports less-efficient clock coupling. Consequently, changes in the steady-state AP BI and the BIV embody contributions of both clocks. Reduction in the degree of synchronization between the clocks disturbs the ability to maintain the basal average AP BI, leading not only to a reduction in the average AP BI, but also to an increase in variability around the prolonged average AP BI.Even under normal conditions, spontaneous AP BIs of mammals including human pacemaker cells are not constant, but vary around the average AP BI, due to stochastic properties of intrinsic mechanisms of the coupled-clock system  of pacemaker cells residing in SAN tissue also entrain the rate and rhythm of electrical impulses that emanate from the SAN Jalife, . These iin vivo. Although mechanisms of HRV may vary from one patient to another, documentation of the relationship between HRV and different arrhythmias in human patients  are analyzed in patients with arrhythmogenic events: during the events when the entropy of the system increases  or Ca2+ clock  produces increases in AP BI and AP BIV that are related to increases in LCR period and LCR period variability  caging of intracellular Cain vivo.Similar to direct pharmacological inhibitors of coupled-clock proteins, mutation and genetically induced gene deletion of different components of the coupled-clock system are associated with arrhythmias fI) consists of three HCN members   mediate L-type Ca2+ channels that play distinct roles in mediating Ca2+ balance in the pacemaker cell. Both Cav1.2 and Cav1.3 channels are expressed in SAN, and Cav1.3 expression in the atria and SAN cells is higher than in ventricular myocytes  and is characterized by severe bradycardia  an increase in adenosine level in human SAN tissue, an endogenous metabolite of the heart, through adenosine A1 receptor upregulating, can lead to SAN dysfunction (Lou et al., Changes in heart rate and rhythm are harbingers of the appearance of arrhythmogenic events. Reduction in the degree of synchronization of any intrinsic clock functions of pacemaker cells or in the synchronization among pacemaker cells residing in the SAN can be associated with arrhythmia occurrence. The extent to which restoring normal synchronization of intrinsic clock periods within pacemaker cells and among pacemaker cells can prevent arrhythmogenic events awaits further elucidation. In our opinion, future work requires a focus on the connection between dysfunction of inherent intrinsic mechanisms associated with different cardiac diseases and cardiac arrhythmias. This connection can be explored in genetically manipulated mouse models, in animals like rabbit, dog and sheep with heart failure or atrial fibrillation, and in human-derived cardiomyocytes or human SAN. This knowledge will contribute greatly to our understanding of cardiac impulse initiation in health and in cardiac disease.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.