Vallisneria, red light induces rotational cytoplasmic streaming, which is regulated by the cytoplasmic concentration of Ca2+. Our previous investigations revealed that red light induces Ca2+ efflux across the plasma membrane (PM), and that both the red light-induced cytoplasmic streaming and the Ca2+ efflux are sensitive to vanadate, an inhibitor of P-type ATPases. In this study, pharmacological experiments suggested the involvement of PM H+-ATPase, one of the P-type ATPases, in the photoinduction of cytoplasmic streaming. We hypothesized that red light would activate PM H+-ATPase to generate a large H+ motive force (PMF) in a photosynthesis-dependent manner. We demonstrated that indeed, photosynthesis increased the PMF and induced phosphorylation of the penultimate residue, threonine, of PM H+-ATPase, which is a major activation mechanism of H+-ATPase. The results suggested that a large PMF generated by PM H+-ATPase energizes the Ca2+ efflux across the PM. As expected, we detected a putative Ca2+/H+ exchange activity in PM vesicles isolated from Vallisneria leaves.In mesophyll cells of the aquatic monocot  Intracellular movements are closely associated with a wide spectrum of plant cell activities, including cell division ,2,3, cel2+-sensitive regulatory mechanisms for the actomyosin-dependent generation of motive force have been extensively investigated, and a number of actin-linked and myosin-linked components responsible for the Ca2+ sensitivity have been identified [2+ fluxes across the plasma membrane (PM) and the sarcoplasmic reticulum membrane of those motile cells function to control the cytoplasmic concentration of Ca2+ and excitation-contraction coupling [2+ [2+. The sources of Ca2+ are different in each stimulus-dependent response: an influx of Ca2+ across the PM is induced by electrical or mechanical stimuli [2+ source in the case of cytoplasm hydration [In muscle cells, Caentified ,12. Morecoupling . The cytling [2+ . In respling [2+  or to hyling [2+ ,16, a pr stimuli ,18, whilydration . Althougydration ,21, dissVallisneria [Vallisneria mesophyll cells is also actomyosin-dependent [2+ [Vallisneria mesophyll cells were continuously illuminated with red light, the extracellular concentration of Ca2+ increased [2+ and induction of cytoplasmic streaming were sensitive to vanadate, a general inhibitor for P-type ion-translocating ATPases, whereas blockers of PM Ca2+ channels substantially suppressed the far-red light-induced influx of Ca2+ and inhibition of cytoplasmic streaming [2+ transport systems across the PM are crucial for light-dependent changes in the cytoplasmic concentration of Ca2+, which in turn bring about the induction and cessation of cytoplasmic streaming.We have been investigating the regulatory mechanism for light-sensitive rotational cytoplasmic streaming in mesophyll cells of the aquatic angiosperm lisneria ,22. Sinclisneria ,24, phytependent ,26,27, adent [2+ . By elecdent [2+ . When prncreased . The efftreaming ,29. Cons2+-ATPase and Ca2+/H+ exchange activities play a central role in Ca2+ homeostasis in plant cells [+-ATPase is integrated in multiple signaling pathways derived from environmental cues [2+-ATPase [+-ATPase [2+ efflux across the PM. Based on the results obtained from pharmacological, electrophysiological, and biochemical approaches, we propose that photosynthesis-dependent activation of PM H+-ATPase generates a large H+ motive force, which provides a driving force for the Ca2+ efflux across the PM.In general, Cant cells ,31. On ttal cues . Since w+-ATPase  and PM H+-ATPase ,34, in t−2 s−1), as described previously [2+-ATPase when used at submicromolar concentrations [2+ but not that of H+ in the PM vesicles isolated from Vallisneria leaves  and diethylstilbestrol (DES) [+-ATPase rather than PM Ca2+-ATPase is responsible for induction of Ca2+-regulated cytoplasmic streaming. The H+ uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 2 µM also inhibited the photoinduction of cytoplasmic streaming (+ gradient (ΔpH) across the PM in the response.In contrast, inhibitors for Hol (DES) ,39,40, atreaming D, furthe+-ATPase generates the H+ motive force (PMF) across the PM, which is comprised of chemical and electrical components, namely, the H+ activity gradient (ΔpH) and the membrane potential , even when far-red light  was superimposed in order to minimize the amount of Pfr, an active form of phytochrome  as a probe revealed that the phosphorylated 94-kDa polypeptides bound to the 14-3-3 protein  and the-ATPase; ). Both aase PMA1 . Immunobpeptides A. Furthe protein A. The am protein A.−2 s−1 ethyleneglycol-N,N,N′,N′-tetraacetic acid (BAPTA) at 2 mM, inhibited ATP-dependent H+ transport to the same level of about 9%   C. The incin (∆N) B, C werey n = 2, D. When vhrosin B A, left, exchange A, right.+-ATPase plays crucial roles in the photoinduction of cytoplasmic streaming in mesophyll cells of Vallisneria. Since the cytoplasmic streaming is regulated by the cytoplasmic concentration of Ca2+ [2+ efflux were sensitive to vanadate [2+-ATPase in both responses. The photoinduction of cytoplasmic streaming, however, is insensitive to erythrosin B  and the uncoupler (CCCP), which drastically decreased PMF 2, 0.1 mM Mg(NO3)2, and 2 mM PIPES-NaOH at pH 7.0, for one cycle of the dark and light regimen, each half piece of the leaf was mounted on a glass slide with the mesophyll cell side downward. The glass slide was immersed in fresh APW and kept in complete darkness for another 12 to 18 h. After dark treatment, we confirmed that all the mesophyll cells did not show any sign of streaming (−2 s−1) on the stage of a light microscope from below through a condenser lens, as previously described [N,N,N′,N′-tetraacetic acid (EGTA) at 10 mM in cells that had been illuminated with red light in the presence of DCCD at 25 µM or DES at 40 µM. Specimens for light microscopy were prepared as described by Izutani et al. . Brieflytreaming . Then thescribed . The phoescribed , becauseµH+) was estimated by following equation:R is gas constant, T is absolute temperature, AH+in is the cytosolic concentration of H+, AH+out is the extracellular concentration of H+, z is valency of H+, F is Faraday’s constant, pHcyt is cytosolic pH, pHout is extracellular pH and ΔΨ is the membrane potential. Values of membrane potential [The PMF , 5 mM K2S2O5, 1 mM dithiothreitol (DTT), 10 mg ml−1 butylated hydroxytoluene, 1% (w/v) casein, 1.2 mg ml−1 aprotinin, 2.5 mg ml−1 pepstatin, 20 mg ml−1 polyvinylpolypyrrolidone, and 50 mM MOPS-KOH at pH 7.6. After differential centrifugation, the resultant pellet was washed with buffer A  and designated as a crude microsome fraction. A PM fraction was isolated from the crude microsome fraction by an aqueous two-phase partitioning at pH 7.8 using a polymer mixture composed of dextran T500  and polyethylene glycol P-3640 . All the procedures were carried out at 0 to 4 °C.A PM-rich fraction was prepared from the leaves according to Harada et al.  under wh+-loaded PM vesicles with inside-out orientation were prepared through a nigericin-mediated exchange of preloaded K+ with H+ as described by Kasai and Muto [+-loaded inside-out vesicles were further treated with nigericin to induce an exchange of loaded K+ with H+. 45Ca2+ transport into the H+-loaded inside-out vesicles was measured by a membrane filtration method according to Bush et al. [Hand Muto . Brieflyh et al.  in the a+ transport into inside-out PM vesicles was assayed at 30 °C according to Harada et al. [−1 of protein with a 1% volume of each of 200 mM ATP-BTP at pH 7.0 and 1175 mg ml−1 Brij 58 [−1 Brij 58. After incubation for 5 min, the reaction was started by the addition of MgSO4 to a final concentration of 5 mM. The rate of quenching of fluorescence from quinacrine was monitored with a fluorescence spectrophotometer . The excitation and emission wavelengths were 420 and 495 nm, respectively. Nigericin dissolved in ethanol was used at 1 µg ml−1 to dissipate the generated H+ gradient across the membrane of vesicles. Stock solutions of BAPTA, vanadate, and CaCl2 were buffered to pH 7.0 with 50 mM, 2 mM and 2 mM MOPS-BTP (pH 7.0), respectively.The activity of ATP-dependent Ha et al. . Briefly Brij 58 . The pre Brij 58  that con−2 s−1) or blue light  were cut into small pieces with a razor blade and then homogenized in an ice-cold homogenization buffer  using a mortar and pestle. The homogenate was solubilized by the addition of half the volume of sodium dodecyl sulfate (SDS) sample buffer . The solubilized samples were incubated at 52 °C for 10–15 min. Then the samples were centrifuged at 12,000g for 1 min and the resulting supernatant was subjected to SDS-PAGE. Red and blue light were obtained by a light-emitting diode (LED) red light illuminator  and a LED blue light illuminator , respectively.Immunoblot and protein blot analyses were performed according to previous methods ,49,66 wiArabidopsis AHA2 (anti-PM H+-ATPase), phosphorylated penultimate Thr-947 of AHA2 (anti-pThr), glutathione S-transferase (GST), and Arabidopsis GF14phi (anti-14-3-3 protein) were described previously [+-ATPase and anti-pThr recognize not only AHA2 but also other H+-ATPase isoforms in Arabidopsis [The polyclonal antibodies raised against the catalytic domain of eviously ,44. Anti


Mitochondrial Ca2+ accumulation stimulates aerobic metabolism by inducing the activity of three TCA cycle dehydrogenases. In detail, matrix Ca2+ indirectly modulates pyruvate dehydrogenase via pyruvate dehydrogenase phosphatase 1, and directly activates isocitrate and α-ketoglutarate dehydrogenases. Here, we will discuss the contribution of mitochondrial Ca2+ uptake to the metabolic homeostasis of organs involved in systemic metabolism, including liver, skeletal muscle, and adipose tissue. We will also tackle the role of mitochondrial Ca2+ uptake in the heart, a high-energy consuming organ whose function strictly depends on appropriate Ca2+ signaling.Recently, the role of mitochondrial activity in high-energy demand organs and in the orchestration of whole-body metabolism has received renewed attention. In mitochondria, pyruvate oxidation, ensured by efficient mitochondrial pyruvate entry and matrix dehydrogenases activity, generates acetyl CoA that enters the TCA cycle. TCA cycle activity, in turn, provides reducing equivalents and electrons that feed the electron transport chain eventually producing ATP. Mitochondrial Ca The decoy of different triggers and the well-defined spatio-temporal patterns of the Ca2+ response to different stimuli warrant the specificity of the cellular response , are generated upon Ca2+ release from the ER/SR (endoplasmic/sarcoplasmic reticulum) stores, ensuring rapid mitochondrial Ca2+ entry , determined by the activity of the electron transport chain (ETC), generates a mitochondrial membrane potential (Δψ) that is negative inside. Cagradient  and it igradient . Microdo2+ entry , 1998.2+ regulates the activity of TCA cycle dehydrogenases, thus triggering the generation of reducing equivalents that feed the ETC and eventually ATP production. On the contrary, in pathological settings, mitochondrial Ca2+ overload causes the opening of the Permeability Transition Pore (PTP) that results in the rapid collapse of the Δψ and in mitochondrial swelling, together with the release of cytochrome c and pro-apoptotic factors .To date, three different mitochondrial membrane proteins have been characterized as components of the CaCCDC109a), which encodes a 40 kDa protein composed of two coiled-coil domains and two transmembrane domains, the latter separated by a short but highly conserved loop facing the intermembrane space (IMS). This loop is enriched in acidic residues, essential to confer Ca2+ selectivity. MCU was predicted to oligomerize in order to form a functional channel, and this hypothesis was supported by gel separation of native mitochondrial proteins, that highlighted an MCU-containing complex with an apparent molecular weight of 450 kDa  is conserved in most vertebrates although it is absent in some organisms in which MCU is present . MCUb protein, with a molecular weight of 40 kDa, shares 50% sequence homology with MCU and it is similarly organized: it has two coiled-coil domains and two transmembrane domains separated by a loop. A crucial amino acid substitution (E256V) in the loop region neutralizing a negative charge is responsible for the drastic reduction in channel conductivity compared to the MCU  injury. Thus, similarly to the MCU–/– mouse model generated in a mixed background  and MICU3 (formerly named EFHA2), that similarly to MICU1 are located in the IMS . While taffected .2+ binding to these domains triggers conformational modifications resulting in channel opening. However, the entity of the Ca2+ affinity to the EF-hand domains is still an open issue. Measurements performed by isothermal titration calorimetry indicate that the Kd of the MICU1/Ca2+ interaction ranges from 4 to 40 μM  has a molecular weight of about 8 MDa and contains multiple copies of three components that catalyze the conversion of pyruvate to acetyl-CoA. The pyruvate decarboxylase (E1) and the dihydrolipoate dehydrogenase (E3) are attached to a central core formed by the dihydrolipoate acetyltransferase (E2) . The pyr subunit . The Ca2tyl- CoA . Notablynd motif , the PDP binding . Further to 1 μM .+ ratios inhibits the IDH enzyme, a feature that is shared with the other two Ca2+-dependent dehydrogenases, the pyruvate and the α-ketoglutarate dehydrogenases. The sensitivity of Ca2+ binding to IDH is controlled by the ATP/ADP ratio. A decrease in the ATP/ADP ratio increases Ca2+ binding to IDH, which in turn leads to a decrease of the Km for isocitrate , to which 2-oxoglutarate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) subunits are attached. Increases in the succinyl-CoA/CoA and NADH/NAD the IDH .2+ dynamics of rat hepatocytes revealed the oscillatory pattern of cytosolic Ca2+ upon hormonal stimulation , besides corroborating the role of MCU in maintaining muscle mass, helped to elucidate the contribution of mitochondrial Ca2+ uptake in muscle metabolism. Furthermore, this animal model was informative on how the MCU-dependent skeletal muscle oxidative metabolism systemically impinges on substrate availability  of myofibers kept in a high-glucose medium. Skeletal muscle glucose uptake was increased as well as glycogen content, indicative of unaffected insulin sensitivity but inefficient glucose utilization.A muscle-specific MCU knockout mouse  oxidation, which significantly contributed to basal OCR . A seconeriments .–/– mouse. In skeletal muscle, MPC deletion decreased glucose oxidation despite increased glucose uptake and lactate production. This triggered enhanced FA oxidation and Cori cycling, with an overall increase in energy expenditure. Thus, skeletal-muscle restricted MPC deletion accelerated fat mass loss when mice were fed again with a chow diet after high-fat diet-induced obesity  knockout mouse strikingly resembled the phenotype of skMCU obesity . Altoget2+ uptake to the regulation of cardiac energetics has been deeply investigated. Pozzan and coworkers demonstrated that mitochondrial Ca2+ transients occur beat-to-beat, and participate in shaping the systolic Ca2+ peaks in neonatal cardiomyocytes. The presence of high [Ca2+] microdomains generated at the SR/mitochondria contacts allows mitochondrial Ca2+ uptake during systole resulting in a significant buffering of cytosolic Ca2+ peaks. Ca2+ is eventually released back to the cytoplasm during diastole because of the presence of the Na2+/Ca2+ exchanger and of the Ca2+/H+ antiporter. In addition, the genetic modulation of MCU, thus the alteration of mitochondrial Ca2+ accumulation, directly affects the amplitude of the cytoplasmic Ca2+ oscillations in neonatal cardiomyocytes , which is encoded by the Slc8b1 gene  . PDC act2+ uptake and impaired pyruvate oxidation increase β-oxidation in the heart. Recent work by 2+] regulates the entry of pyruvate and glutamate into the TCA cycle, lipid oxidation is not controlled by mitochondrial [Ca2+]. Thus, in a context of mitochondrial Ca2+ impairment, fatty acids are readily oxidized to sustain energy demand.These data suggest that, like in skeletal muscle, decreased mitochondrial Ca2+ homeostasis in adipose tissue biology represents a relatively new area of research. Different pieces of evidence suggest that Ca2+ dysregulation may affect adipocyte function. Accordingly, it has been demonstrated that intracellular Ca2+ concentration regulates adipocyte lipid metabolism, thus controlling adipogenesis. 2+] plays a biphasic regulatory role in adipocyte differentiation: on the one hand, an increase in cytosolic [Ca2+] suppresses the early stages of murine adipogenesis; on the other hand, it promotes the late stages of adipose differentiation. Most importantly, elevated intracellular [Ca2+] in adipocyte appears to contribute to the insulin-resistance of elderly hypertensive obese patients. Strikingly, 1 month of therapy with nitrendipine, a Ca2+ channel blocker, reduced blood pressure, decreased plasma insulin upon glucose stimulation to control values, and restored normal adipocyte glucose uptake , early work on isolated adipocytes demonstrated that norepinephrine stimulation mobilizes intracellular Ca+ stores , and thi+ stores . Howeverogenesis . In deta2+ signaling is still largely unexplored. Recently, the link between MCU activity and the development of obesity and diabetes was investigated  of obese patients, both pre-diabetic and diabetic, compared to lean individuals. Interestingly, upon bariatric surgery, the expression of MCU complex components in patients’ VAT returned to physiological levels, suggesting that mitochondrial Ca2+ accumulation plays an active role in this pathology. Mcub overexpression, although insufficient to improve the defect in insulin-stimulated glucose uptake of insulin-resistant cells, restored ROS production to normal levels. In addition, Mcub overexpression reduced TNF-alpha and IL-6 expression, suggesting a possible role of mitochondrial Ca2+ uptake in obesity-associated inflammation , was identified by RNAi screen among genes affecting adiposity in the fly . Along with hyperphagia, the Stim-sNPF axis controls lipid metabolism, resulting in adipose tissue hypertrophy. Further evidence on the role of Ca2+ homeostasis in D. melanogaster lipid metabolism comes from studies on InsP3 receptor mutants, which are obese and display altered FA utilization (2+] (2+ levels, leading to an impairment in the fat storage. Moreover, in dSeipin mutants, mitochondrial Ca2+ uptake is reduced, triggering a decrease in TCA cycle activity and accumulation of citrate, a key component of lipogenesis. The lipid storage defect in dSeipin mutant fat cells is rescued by the recovery of mitochondrial [Ca2+] or by the restoration of normal citrate levels (Further insights into the role of Caesponses . Concern the fly . Stim dolization . In addiion (2+] . Mutatioe levels .2+ entry causes defective oxidative metabolism in liver, heart, skeletal muscle and adipose tissue, and rewiring of substrate utilization that determines increased fatty acids oxidation in the heart and skeletal muscle, and lipid accumulation in the liver. Future work will further shed light on the importance of mitochondrial Ca2+ uptake and pyruvate oxidation on critical issues of metabolism, and on the cross-talk between different organs.About 10 years after the identification of the genes encoding a fundamental MCU regulator (i.e., MICU1, GG and CM wrote the manuscript. HM prepared the figures. RR contributed to the critical insights. All authors contributed to the article and approved the submitted version.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.


In addition, mitochondria-derived reactive oxygen species (mROS), produced as a consequence of ATP synthesis at the electron transport chain (ETC), modulate cellular signaling pathways that contribute to many cellular processes. Cancer cells modulate mitochondrial Ca2+ ([Ca2+]m) homeostasis by altering the expression and function of mitochondrial Ca2+ channels and transporters required for the uptake and extrusion of mitochondrial Ca2+. Regulated elevations in [Ca2+]m are required for the activity of several mitochondrial enzymes, and this in turn regulates metabolic flux, mitochondrial ETC function and mROS generation. Alterations in both [Ca2+]m and mROS are hallmarks of many tumors, and elevated mROS is a known driver of pro-tumorigenic redox signaling, resulting in the activation of pathways implicated in cellular proliferation, metabolic alterations and stress-adaptations. In this review, we highlight recent studies that demonstrate the interplay between [Ca2+]m and mROS signaling in cancer.Calcium (Ca Ca2+ signaling regulates many physiological functions, including muscle contraction, neuronal excitability, cell migration, and growth. In order to maintain a delicate regulation of Ca2+ homeostasis, various plasma membrane and organellar Ca2+ channels, exchangers and transporters are needed m can in turn participate in mitochondrial ROS (mROS) generation, which takes place at the electron transport chain (ETC) [2+]m flux and mitochondrial Ca2+ transport as important regulators of mROS generation and redox signaling. In addition to physiologically necessary fluctuations in [Ca2+]m, excess [Ca2+]m can also determine cell fate, since mitochondrial Ca2+ overload induces the opening of the mitochondrial permeability transition pore (mPTP), which leads to mitochondrial membrane depolarization, elevated mROS, and the release of cytochrome c, to activate pro-apoptotic factors [2+]m uptake and extrusion is necessary to ensure proper cellular functions. Calcium  . This im factors . Consequ2+]m transport; we discuss some of the recent discoveries regarding the role of [Ca2+]m in cancer cells; and focus on how [Ca2+]m transport is an important regulatory mechanism by which cancer cells elicit mROS-dependent pro-tumorigenic signaling.In this review, we briefly describe the composition and regulation of m, suggesting that it might be a negative regulator of MCU m entry m entry . It is n]m entry .2+]m homeostasis, Ca2+ cations must be extruded from the mitochondrial matrix. The Mitochondrial Na+/ Ca2+/Li+ exchanger (NCLX), also known as solute carrier family 8-member B1 (SLC8B1) is one of the main proteins responsible for Ca2+ efflux from the matrix m efflux. This effect was also confirmed by pharmacological inhibition of NCLX, using the CGP-37157 compound. Unlike other members of the NCX family, NCLX does not contain an allosteric Ca2+-binding site m efflux m extrusion, but the activity of Letm1 is crucial for maintaining the tubular shape and cristae organization m) in cancer cells.Cellular ROS include free radicals such as superoxide . Moreovchanisms ,51. In S2+ uptake and extrusion in cellular signaling is likely two-fold. First, the buffering capacity of mitochondria has been implicated in regulating cytosolic Ca2+ levels to influence Ca2+-dependent signaling outside of the mitochondria, such as the regulation of NFAT  [2+]m, as eluded to above, influence the function of mitochondrial enzymes. This can alter cellular signaling due to effects on metabolism and metabolite pools, and due to the generation of mROS. Here we focus on the role of mitochondrial Ca2+ in regulating mROS in the context of cancer.The role of regulated mitochondrial Cagnaling) ,55. Seco2+]m dynamics is store-operated Ca2+ entry (SOCE). SOCE is physiologically initiated upon agonist binding to Gq-protein coupled receptors at the plasma membrane. This leads to the activation of phospholipase C and breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to its receptor (IP3R) in the ER, leading to the release of Ca2+ from ER stores m stimulates the activity of PDP, favoring PDH de-phosphorylation and thus its activity m observed in tumor cells. Given that many cancer cells exhibit increased PDK activity, which inhibits PDH and drives metabolism towards aerobic glycolysis [2+]m decreases PDH phosphorylation in the context of underlying genetic changes that increase PDK activity in cancer cells.Pyruvate dehydrogenase (PDH) is located in the mitochondrial matrix and catalyzes a key reaction upstream of the TCA cycle. The last product of glycolysis, pyruvate, is transported in the mitochondrial matrix, where it is converted into Acetyl-CoA, which supplies the TCA cycle. This reaction is catalyzed by the E1 subunit of the PDH complex dehydrogenase complex, activity of which is regulated by phosphorylation. E1 phosphorylation by pyruvate dehydrogenase kinase (PDK) inactivates the PDH enzyme, while its de-phosphorylation activates it. Caactivity . Accordiycolysis , it rema+ ratios inhibit the activity of IDH3, while high [Ca2+]m stimulate the enzyme. IDH3’s Kd for Ca2+ varies from 5 to 50 μM depending on the ATP/ADP ratio [2+ when the NADH/NAD+ ratio decreases [2+]m and enhanced IDH3 activity has not been made in cancer cells, it is possible that increased IDH3 expression and enhanced IDH3 activity via [Ca2+]m might cooperate to drive mROS production in tumors. Interestingly, tumor cells are able to run the TCA cycle in reverse via IDH2, and to utilize reductive carboxylation to generate citrate and subsequently NADPH and acetyl CoA, important substrates for glutathione reduction and fatty acid synthesis, respectively [2+]m-driven IDH3 activity opposes or is inhibited by enhanced reductive carboxylation in cancer cells requires further investigation. Isocitrate dehydrogenase (IDH3) catalyzes the oxidative decarboxylation of isocitrate to produce α-ketoglutarate in the TCA cycle. The product of this reaction, NADH, is an essential reducing equivalent for the ETC. Rises in ATP/ADP and NADH/NADDP ratio ,63 and tecreases . A lack ecreases ,65. Althectively . Whether2+]m m. Since OGDH is an important regulator of α-ketoglutarate levels, which is a cofactor for demethylases, OGDH activity can therefore affect epigenetic transcriptional regulation. Whether OGDH activation by [Ca2+]m alters the epigenome in tumor cells has not been directly investigated. Oxoglutarate dehydrogenase (OGDH), catalyzes the next reaction of the TCA cycle, converting α-ketoglutarate to succinyl-CoA, and generating NADH. Similar to IDH3, its activity can regulate ETC function by providing the electron donor NADH. OGDH activity is enhanced by elevated [Ca2+]m ,63,67,68+ ratios . When thentation , demonst2+]m activates the above metabolic enzymes, it is generally concluded that [Ca2+]m directly mediates the synthesis of the reduced substrate NADH of the respiratory electron transport chain (ETC), and increases OXPHOS . A cons the ETC ,74 , and can activate the mitochondrial permeability transition pore (mPTP) to induce Ca2+ and cytochrome c efflux, mitochondrial swelling, and break-down of the ΔΨm, which are important aspects of apoptosis initiation (2•− can dismute to hydrogen peroxide (H2O2) spontaneously or through enzymatic dismutation by the matrix manganese superoxide dismutase (MnSOD or Sod2) or copper/zinc superoxide dismutase (CuZnSOD or Sod1) in the IMS or the cytosol m homeostasis, as well as well-adjusted ROS production and scavenging, are necessary to maintain a redox homeostasis that is required for normal function of the cell. In pathophysiological settings, including tumor cells, an imbalance of [Ca2+]m and mROS due to altered [Ca2+]m can therefore have different consequences on cell function. In addition to the above controlled increases in mROS as a consequence of m homeostasis with tumor growth and progression are starting to emerge, in particular after the molecular identification of the proteins responsible for mediating mitochondrial Ca2+ uptake and extrusion, and the findings that their expression is often altered in tumor specimens. In this section, we highlight recent findings demonstrating that mitochondrial Ca2+ transport has important roles in tumorigenesis and cancer progression by altering mROS. We should note that few studies directly compare [Ca2+]m and mROS levels between cancer and matching normal tissues and cells. Although, it is generally assumed that cancer cells have altered [Ca2+]m levels compared to normal cells based on changes in expression of [Ca2+]m transport mechanisms. As seen in 2+]m transport mechanisms and concomitant mROS changes, or demonstrate that these changes are exacerbated in cells with a more malignant phenotype. Multiple investigations have highlighted a key role for alterations in cellular Catastasis . Mechani2+. In a physiological context, VDAC is considered a regulator of apoptosis. Upon apoptotic stimuli, VDAC1 undergoes oligomerization, interacts with pro-apoptotic proteins and promotes the release of cytochrome c to the cytosol, leading to apoptosis m-dependent changes in sub-lethal mROS involved in redox signaling. Because VDAC mediates a switch toward glycolysis and affects ATP flux from the mitochondria, ETC function and ETC-dependent mROS production are likely also affected by changes in VDAC expression and regulation by HK binding. Moreover, evidence suggests that VDAC1 acts as a preferential release channel for the hydrophilic O2•− anion, produced during respiration into the IMS [2+]m in the mitochondrial matrix, which is the primary site of mROS production, but refer the reader to several review articles discussing the role of VDACs in cancer m. In most cancers levels of MCU are elevated, and while regulation of MCU expression has not been widely studied, the Cancer Genome Atlas (TCGA) demonstrates that the MCU gene is amplified in some tumor types m uptake, a decline in ROS production and HIF1-α expression [2+]m was reduced, in the TNBC cell line MDA-MB-392. However, the investigators noted that loss of MCU and MICU1 expression significantly affected cell survival of normal human mammary epithelial cells (HMECs) m and subsequent reduction in mROS decreased the migration and invasion of HCC cells and metastasis formation in vivo. Further, mROS activates the Jun N-terminal kinase (P-JUNK) pathway to drive HCC migration [2+]m levels compared to normal hepatocytes [The mitochondrial Caor types . A potenor types . The MCUltration . Upon MCpression . It was pression . In cont (HMECs) . Effects (HMECs) . MCU exp (HMECs) . The invigration . In thisatocytes .2+]m –mROS positive feed-back loop is independent from the other regulatory proteins of the MCU complex and leads to higher mitochondrial Ca2+ uptake and mROS production m uptake was MCU dependent and resulted in elevated mROS production. This stimulated degradation of the tumor suppressor p53 via activation of the AKT/Mdm2 (murine double minute 2) pathway [in vivo metastasis by inducing the expression of the EMT-transcriptional regulator Snail. MCUR1-dependent increase in [Ca2+]m and subsequent mROS generation activated the ROS/Nrf2/Notch1 pathway and elevated Snail expression. These phenotypes were reversed when [Ca2+]m was buffered with parvalbumin and when mROS was scavenged with mito-Tempo m uptake by MCU and subsequently affects ROS production and apoptotic sensitivity [2+]m accumulation, leading to ROS production and tumor progression [2+ transport to further manipulate mitochondrial redox signals. The absence of the gatekeeper protein MICU1 increases [Casitivity . A recengression . This hi2+]m by regulated activation of MCU regulators can lead to mROS production necessary for pro-tumorigenic signaling. However, if [Ca2+]m and subsequent mROS production exceeds a threshold that elicits deleterious effects in mitochondria, consequences of hyperactivation of mitochondrial Ca2+ uptake can lead to mitochondrial Ca2+ overload and cell death. There are several examples in the literature that demonstrate that tumor cells also adapt to prevent these deleterious surges in [Ca2+]m by regulating the MCU gatekeepers, MICU1, and MICU2. In ovarian cancer, the MICU1 protein is highly expressed and this is associated with a poor prognosis and chemoresistance [et al. demonstrated that enhanced expression of the histidine triad nucleotide-binding (HINT2) protein elevates [Ca2+]m levels and subsequent ROS production leading to apoptosis m and subsequent mROS pro-tumorigenic signaling, while limiting pro-apoptotic mROS surges. It is clear that the role and regulation of MCU and its regulators is likely cancer-type specific. For example, Hall et al. failed to observe a change in ROS production or the survival of breast cancer cell lines under stress condition following MICU1 knockdown [The above studies demonstrate that MCU gatekeepers can be regulated transcriptionally and at the post-translational level to fine tune [Canockdown . More wo2+]m and mROS production have been developed in other pathologies [2+]m from the mitochondrial matrix, NCLX is able to affect OXPHOS and mROS formation [2+]m efflux and increased cytosolic Na+ levels, which results in TCA cycle activation, changes in NADH/NAD+ ratios and mROS production [2+]m levels, leading to elevated ROS production and increased NLRP3 inflammasome activation m-mROS axis in cancer and whether this also represents a viable pharmacological target.The NCLX-ROS interplay has not been examined in cancer. However, links between changes in NCLX expression, altered m by mitochondrial Ca2+ transport. While we only touch on a few examples from an ever-expanding literature, we highlighted several examples where Ca2+ within the mitochondria has an important regulatory function on mROS production and redox signaling events that are beneficial to tumor cell survival and progression. As mentioned in 2+]m influences the activity of several TCA enzymes in the mitochondria, which likely drives mROS production. As described above, several studies have demonstrated that manipulation of [Ca2+]m results in changes in the NAD+/NADH ratios. However, direct evidence that [Ca2+]m alters TCA cycle enzyme or ATP synthase activity in cancer cells requires further attention. This is of particular importance in the context of genetic aberrations that may influence the expression and activity of PDH and TCA cycle enzymes, and in the context of other metabolic changes, such as substrate flux, that could influence the action of [Ca2+]m on mitochondrial metabolism. The discovery of MCU and its regulatory proteins opens a new era in the study of MCU-mediated mitochondrial Ca2+ homeostasis in cancer. Currently, the available candidate agents targeting MCU or its regulators in cancer chemotherapy are still burgeoning [2+]m have not been explored, opening exciting avenues of investigation.Mitochondria are intracellular organelles that play key roles in energy production and in the regulation of many cellular processes from cell signaling to death. The ETC is the major source of ATP in the cell and its activity is coupled with the generation of mROS that are finely maintained at physiological levels by highly efficient mitochondrial antioxidant systems. In the past few years, several studies have demonstrated that mROS are essential for physiological signaling, but when the balance between mROS production and elimination is altered, by either overproduction of mROS or impairment of the antioxidant defensive mechanisms, mitochondrial dysfunction occurs. One important mechanism regulating mitochondrial mROS homeostasis in cancer cells is through alterations in [Cargeoning ,142, and


However, the apparent affinity for Ca2+ of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism in vivo. In this work, we compared the individual and synergistic roles of the myristic moiety, the GRK1 target and the disc membrane in modulating the calcium sensitivity of Rec. We show that the sole presence of the target or the disc membrane alone are not sufficient to achieve a physiological response to changes in intracellular [Ca2+]. Instead, the simultaneous presence of GRK1 and membrane allows the T to R transition to occur in a physiological range of [Ca2+] with high cooperativity via a conformational selection mechanism that drives the structural transitions of Rec in the presence of multiple ligands. Our conclusions may apply to other sensory transduction systems involving protein complexes and biological membranes.The prototypical Ca Acyl domains  is essen T state a, while e occurs b  varies in a narrow sub-micromolar range . Interestingly, the simultaneous presence of GRK1 and membrane allows the T to R transition of mRec to occur in a physiological range of [Ca2+] with high cooperativity.Here we used absorption, fluorescence and circular dichroism spectroscopy to show that neither the sole presence of the GRK1 target nor the ROS membrane alone are sufficient to achieve a physiological response of mRec to changes in intracellular  in the decalcified buffer and showed complete saturation at approximately 200 µM [Ca2+] (a). Under the tested experimental conditions, the detected affinity of OG BAPTA-5N for Ca2+  was suitable for competition assays with Rec, therefore we performed Ca2+ titrations of mRec both in the absence and in the presence of GRK1 target. Data fitting to a one-site model for mRec (figure 3c) estimated a Kd of 18.0 µM (figure 3d), displaying a similar behaviour to that of nmRec, although to a lesser extent. No fitting to a two-site model was possible in this case.As no competition could be detected between mRec and BrBAPTA-5N a. The hiM [Ca2+] a. Under  18.0 µM , identic 18.0 µM  in the p2.2.2+ binding called myristoyl-switch, which results in a substantial increase in exposure of hydrophobic surface, mainly due to the externalization of aromatic residues, in particular of W31, W104 and W156  in the absence (a) and in the presence of GRK1 peptide (b). Specifically, we monitored the ratio between the emission peak of the intrinsic fluorescence and the emission peak of ANS. Results summarized in figure 4c highlight a minor but significant decrease in EC50 exhibited by mRec in the presence of the target , in line with previous results  to 120 nM and caused a decrease in ellipticity in the Phe band and a sign inversion of the signal in the Tyr and Trp bands, indicative of a substantial structural variation towards a conformation different from the apo form in the absence of the target . Interestingly, the inversion in the order of ligand additions (figure 5b) showed differences between apo nmRec in the absence and in the presence of the GRK1 peptide in all spectral bands, suggesting an interaction even in the absence of Ca2+ . Further addition of Ca2+ induced the same Ca2+-loaded and GRK1-bound conformation as in figure 5a . Chelation of Ca2+ upon addition of EGTA again resulted in a negative shift of the spectrum to a conformation similar to that of the apo form in the presence of the GRK1 peptide, with small differences in the Trp band, probably due to the slightly higher free Ca2+ concentration under this condition.Near UV CD spectra of nmRec showed a relatively small conformational change upon Ca results ,34 and w2+ binding (c). Addition of the GRK1 peptide to Ca2+-bound mRec resulted in a positive shift involving all three spectral regions (blue lines), very similar to the relative cases shown in figure 5a and c. Moreover, Ca2+ chelation by EGTA triggered the transition to a different conformation, suggesting again a potential interaction between apo mRec and the GRK1 peptide. Such interaction was confirmed by inverting the addition of the interactors, as the presence of GRK1 caused a positive spectral shift especially in the Phe and Tyr bands . Addition of Ca2+ resulted in a further positive shift with a fine structure comparable to that exhibited in figure 5c and in the case of nmRec . Finally, addition of EGTA caused a negative shift, reverting the fine structure of spectrum to that of the apo GRK1-bound form, with small differences in intensity attributable to the residual free Ca2+.Similarly to nmRec, also mRec displayed its typical change in the fingerprint of the protein tertiary structure upon Ca binding c. Additi2+ binding  accompanied by an increase of the θ222/θ208 ratio from 0.84 to 0.88 , suggesting again a mild interaction of the GRK1 peptide with nmRec even in the absence of Ca2+. In line with the near UV CD spectra, also far UV CD spectra displayed a small but significant spectral variation upon addition of the GRK1 peptide in the absence of Ca2+ , both in terms of intensity and shape . A similar behaviour was displayed by mRec under the same conditions, albeit with numerical differences. Binding of Ca2+ induced an ellipticity rise at 208 nm  yielding a shift of the θ222/θ208 ratio from 0.81 to 0.91 , pointing towards an interaction between mRec and its target even in the absence of Ca2+. Analogously to nmRec, the order of addition of the two interactors Ca2+ and GRK1 peptide did not matter, as the addition of Ca2+ enhanced both the intensity of the CD signal and the θ222/θ208 ratio to 0.95 (θ222/θ208 = 0.85).As far as the compactness and the secondary structure of the Rec variants are concerned, far UV CD spectra were substantially in line with the behaviour exhibited in the near UV. Indeed, nmRec displayed an increase in α-helix content and compactness upon Ca to 0.88 , which iing 0.94 , while a to 0.91 , while t to 0.91 . Also in to 0.95 , which w2+-dependent structural changes occurring in Rec variants and the effects exerted by the target peptide, we evaluated the evolution patterns of the near UV spectra at increasing Ca2+ concentration. Interestingly, results for nmRec highlighted a completely different evolution of the spectra upon Ca2+ titration between GRK1-free (a) and GRK1-bound (b) forms, which started and ended with substantially different conformations, suggesting an interaction between nmRec and GRK1 also in the absence of Ca2+. By monitoring the dichroism signal at 292 nm as a function of the Ca2+ concentration (c), we could estimate the concentration at which the conformational transition was half-maximal by means of fitting to a three-parameter Hill sigmoid. Results revealed not only a 3.5-fold decrease of the EC50 from 156.8 ± 37.3 nM to 44.9 ± 2.3 nM  regardless of the presence of the target. On the other hand, mRec exhibited larger variation in the Phe and Tyr spectral bands upon Ca2+ binding in the presence of the target compared to the isolated protein (d and e); nevertheless, the overall spectral shape features were not substantially perturbed. The evaluation of the ellipticity signal at 279 nm (c) after fitting to a three-parameter Hill sigmoid pointed to a decrease of the EC50 for mRec from 25.12 ± 7.28 µM to 9.44 ± 1.90 µM  even in the presence of GRK1 (see the grey-shaded regions in figure 6c and f).To further investigate the CaRK1-free a and GRKK1-bound b forms, ntration c, we cou± 2.3 nM  in the p± 2.3 nM  due to t protein d and e; t 279 nm c after f 1.90 µM  in the p 1.90 µM . Notably2.4.2+ affinity and decreased EC50 of mRec, such values fell out of the physiological concentration of Ca2+ operating in photoreceptor ROS, therefore, we tested whether the presence of membranes would be able to further decrease the EC50.While the presence of the GRK1 peptide increased the apparent Cain vitro the presence of membranes, we employed liposomes (LP) with the same composition as the ROS membranes. To ensure the stoichiometric excess of theoretical binding sites for mRec on LP, we measured the hydrodynamic diameter and the concentration of the vesicles by means of dynamic light scattering and nanoparticle tracking analysis , resulting in 75.3 ± 1.3 nm diameter LP and estimated the maximum number of mRec proteins potentially bound to LP (approx. 1400) as elucidated in the Methods section.To mimic 2+-free mRec in the presence of LP showed small but significant differences in the Phe band  between GRK1-free and GRK1 bound, although the overall spectra were similar. It is noteworthy that even a low concentration (5 nM) of LP caused prominent signal scattering in the near UV; therefore, spectra were noisy due to the considerably lower concentration of mRec (7 µM) compared to experiments without LP (43 µM). Nevertheless, the experimental conditions ensured a reasonable signal-to-noise ratio.Near UV CD spectra of Ca50 of mRec in the presence of LP, resulting in 0.37 ± 0.68 µM (a). Noticeably, the same experiment performed in the presence of GRK1 (b) resulted in a threefold decrease in EC50 to 0.13 ± 0.02 µM  suggested a highly cooperative conformational change in mRec induced by the proximity of membranes and by the presence of the peptide target. Overall, near UV CD titrations showed that ROS membranes increase the affinity of mRec for Ca2+ to a larger extent with respect to the sole presence of the target GRK1 ; figure 7b, grey shaded area).Again, by monitoring the ellipticity at 279 nm, we estimated the EC 0.68 µM  with a H 0.68 µM a. Notice of GRK1 b resulte 0.02 µM , 72-fold3.in vitro studies reported that the half-maximal inhibition of GRK1 by Rec occurs between 1.5 and 6 µM free Ca2+  changes upon rod illumination  window (0.13 µM) and allowed the conformational transition to occur with high cooperativity (Hill coefficient = 2.15).In the present work, we investigated the mutual influence of these parameters on the structural and functional aspects in Camination  and thatfor Ca2+  and decr figures  and 6f, ficantly , as the  to mRec ,17,19. O2+-saturating conditions, the binding between Rec and GRK1 is driven by conformational selection rather than induced fit [2+ ions, GRK1 target, myristoyl group and disc membrane, where the modified fatty acid can spontaneously insert. A formal description of the conformational selection mechanism is particularly challenging in the case of multiple equilibria as it would require both specific structural information on each state and complete knowledge on the kinetics of reaction pathways [2+ binding, as the interaction between the GRK1 peptide and both nmRec and mRec was observed prior to Ca2+ binding. While this is not surprising for nmRec, which is already in the R state, this implies that the GRK1 peptide, per se unstructured in solution [2+, but rather by the presence of GRK1, which may facilitate the completion of the T to R transition that can occur with slightly decreased EC50  structures  cells previously co-transformed with the plasmids encoding for Rec (pET11a-Rec) and N-myristoyl transferase (yNMT1) from S. cerevisiae (pBB131- yNMT1). Protein expression and purification were performed according to the protocols elucidated in [−1 ampicillin and 30 µg ml−1 kanamycin until OD600 reached 0.4, when the medium was added with 50 µg ml−1 myristic acid (in 50% EtOH) to allow myristoylation. Protein expression was induced at OD600 = 0.6 with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After 4 h at 37°C cells were harvested by centrifugation at 5000 × g for 15 min. Bacterial pellets were resuspended in 50 mM Tris–HCl pH 8 containing 1 mM MgCl2, 1X EDTA-free Protease Inhibitor Cocktail (Sigma), 0.1 mg ml−1 lysozyme and 5 U ml−1 DNAse I and incubated at 30°C for 30 min. After 30 min centrifugation at 20000 × g at 4°C, the supernatant was added with 0.5 mM CaCl2 and loaded onto an HiPrep Phenyl HP 16/10  column previously equilibrated with 50 mM Tris–HCl pH 7.5, 0.5 mM CaCl2, 1 mM DTT, then eluted with 50 mM Tris–HCl pH 7.5, 2 mM EGTA, 1 mM DTT. Fractions purity was checked by SDS-PAGE. Rec was dialyzed against decalcified NH4HCO3, aliquoted after measuring the concentration by Bradford assay [Myristoylated Rec was heterologously expressed in dated in ,41 with rd assay , lyophilThe peptide encompassing the first 25 amino acids of GRK1  in the presence of 28 µM Rec upon addition of 3 µM Ca2+ for each titration point. Decalcified lyophilized proteins were solubilized in decalcified 20 mM Tris–HCl pH 7.5, 150 mM KCl buffer , protein concentration was calculated by Bradford assay [A263 is the absorbance of each titration point, Amin and Amax are the absorbance values at the lowest and highest Ca2+ concentration, respectively.The Cachelator  as previchelator . The firrd assay , tempera4.2.2.2+  and a maximum fluoresce emission at λ = 524 nm following excitation at 494 nm. Fifteen μM Rec was used in competition assays upon addition of 3 µM Ca2+ for the first 15 titration points followed by addition of 30 µM Ca2+ to verify Ca2+ saturation. Decalcified lyophilized proteins were dissolved in 10 mM MOPS pH 7.5, 100 mM KCl , temperature was set at 25°C, protein concentration was estimated by Bradford assay. Emission fluorescence spectra were recorded at 25°C between 500 and 560 nm after excitation at 494 nm in 1 cm quartz cuvettes, using a Jasco FP-750 spectrofluorometer, at a scan rate of 1 nm s−1, data pitch 2 nm. Data refer to an average of three accumulations subtracted with the spectrum of the buffer, and were normalized as followsF524 is the fluorescence emission of the dye at each titration point, Fmin and Fmax are the fluorescence values at the lowest and highest Ca2+ concentration, respectively. In all experiments with the GRK1 peptide, the stoichiometric ratio Rec : GRK1 peptide was set to 1 : 1.5.The second method was based on monitoring the fluorescence emission increase of 0.5 µM Oregon Green-conjugated BAPTA-5N (hereafter ‘OG BAPTA-5N’). The dye has a relatively low affinity for Ca2+ titration was fitted using CaLigator [Kdi, The normalized signal decrease upon CaaLigator  to estim4.3.A mixture of lipids of the same composition of bovine ROS membranes  consisti4.4.Liposome (LP) size was assessed by dynamic light scattering (DLS) measurements using a Zetasizer Nano-S instrument  with the same experimental settings as in , briefly4.5.−1 at 25°C. Additional parameters: flow rate 20 µl min−1, camera level 16, detect threshold 5. Liposome size is reported as the mean of the mode in each of the three independent measurements ± standard error, liposome concentration is reported as mean ± standard error, data shown in electronic supplementary material, figure S2B refers to the average of three independent measurements.Liposome size and concentration were estimated by Nanoparticle Tracking Analysis using a NanoSight instrument , by acquiring three 1-min videos, each consisting of 25 frames s4.6.NRec) bound to LP was calculated according to the same geometrical model as explained in [rLP is the radius of LP  and rRec is the radius of Rec (2.2 nm). Under such assumptions, the maximum number of Rec molecules bound to a 75.3 nm LP is approximately 1400.The maximum number of Rec  consists of monitoring the conformational changes upon Ca2+ binding, which can be achieved by exploiting the fluorescence resonance energy transfer (FRET) phenomenon between fluorescence donors and acceptors that become very close to each-other upon interaction. The intrinsic fluorescence emission of Rec following excitation at 280 nm is essentially due to the three Trp residues  to a final concentration of 2 µM Rec estimated by Bradford assay. In all experiments with the GRK1 peptide, the stoichiometric ratio Rec : GRK1 peptide was set to 1 : 1.5. Free Ca2+ concentration for each titration point, spanning from 3 nM to 800 µM, was obtained using Ca2+-EGTA buffer solution as previously explained [I is the fluorescence ratio of each titration point, Imin and Imax are the fluorescence ratios at the lowest and highest Ca2+ concentration, respectively.The emission fluorescence spectra were recorded between 300 and 550 nm at 25°C in 1 cm quartz cuvettes using a Jasco FP-750 spectrofluorometer, after excitation at 280 nm, at a scan rate of 1 nm sxplained . Data re4.8.50, consisted in monitoring changes in secondary and tertiary structure of Rec upon Ca2+ titrations.The second indirect method to evaluate the EC2+ concentration for each titration point (between 420 pM and 1 mM) was calculated by MaxChelator (maxchelator.stanford.edu/CaEGTA-NIST.htm) using Ca-EGTA NIST database, additional parameters were temperature: 25°C, pH 7.5, ionic strength 0.15 M, EGTA 300 µM.CD spectra were recorded essentially with the same methodology described in  using a −1, bandwidth: 1 nm, response time: 4 s. Rec concentration in far UV spectra was adjusted to 10 µM, while for near UV spectra was set to 43 µM in the absence of LP and 7 µM in the presence of LP, since the LP suspension generated a prominent scattering of the incident light resulting in low signal-to-noise ratio, which prevented the collection of spectra at higher protein concentrations. In all experiments with the GRK1 peptide, the stoichiometric ratio Rec : GRK1 peptide was set to 1 : 1.5. Reported data refer to the average ± standard deviation after the following normalization:λθ is the ellipticity of each titration point at fixed λ , θmin and θmax are the ellipticity values at the lowest and highest Ca2+ concentration, respectively.The spectrum of the solvent  was considered as blank and subtracted; for the experiments with LP, the spectrum of 5 nM LP was considered as blank. Each spectrum represents five accumulations collected by setting scan rate: 50 nm min


Ostericum citriodorum is a plant with a native range in China used in herbal medicine for treating angina pectoris. In this study, we investigated the vasodilatory effects of isodillapiolglycol (IDG), which is one of the main ingredients isolated from O. citriodorum ethyl acetate extract, in Sprague–Dawley rat aortic rings, and measured intracellular Ca2+ ([Ca2+]in) using a molecular fluo-3/AM probe. The results show that IDG dose-dependently relaxed endothelium-intact or -denuded aortic rings pre-contracted with noradrenaline (NE) or potassium chloride (KCl), and inhibited CaCl2-induced contraction in high K+ depolarized aortic rings. Tetraethyl ammonium chloride (a Ca2+-activated K+ channel blocker) or verapamil (an L-type Ca2+ channel blocker) significantly reduced the relaxation of IDG in aortic rings pre-contracted with NE. In vascular smooth muscle cells, IDG inhibited the increase in [Ca2+]in stimulated by KCl in Krebs solution; likewise, IDG also attenuated the increase in [Ca2+]in induced by NE or subsequent supplementation of CaCl2. These findings demonstrate that IDG relaxes aortic rings in an endothelium-independent manner by reducing [Ca2+]in, likely through inhibition of the receptor-gated Ca2+ channel and the voltage-dependent Ca2+ channel, and through opening of the Ca2+-activated K+ channel. There are two main types of channel in VSMCs: the receptor-operated Ca2+ channel (ROCC) and the voltage-dependent Ca2+ channel (VDCC). Activation of ROCC causes extracellular Ca2+ influx and intracellular Ca2+ ([Ca2+]in) release from the sarcoplasmic reticulum (SR), and activation of VDCC causes extracellular Ca2+ influx in in VSMCs.In our previous study, we found that isoapiole, which was separated from the petroleum ether extract of t manner . A divert manner , and we y effect ,16. In tIn this primary experiment, we examined the vasodilatory effects of eight major fractions (Fr. A–H) obtained from OCE. The aorta rings were pre-contracted with noradrenaline (NE) (3 μM), before adding Fr. A–H (1000 μg/mL). Among the eight fractions, Fr. F had the strongest vasodilatory effect .12H12O6, electron ionization mass spectrometry (EI–MS) (positive) m/z: 279.085 [M + Na] +. 1H-NMR : δH 6.48 , 5.96 , 4.52 , 3.85 , 3.92 , 3.85 , 1.07 . In the heteronuclear multiple bond correlation (HMBC)spectrum, we found correlations between H1′ and C1/C2/C6, and between H2′ and C1, indicating that C1 was connected to C1′. The HMBC correlations between H4′ and C4/C5 suggested the connection of C4, C5, and the methylene dioxy groups of C4′ via an oxygen bond, and the position of the methoxy group could be defined by the HMBC correlations between H2–OCH3 and C2, and between H3-OCH3 and C3. We found HMBC correlations between H6 and C1/C2/C4/C5. Thus, the planar structure was established  data: δC 139.1 (C3), 138.4 (C4), 136.6 (C5), 136.0 (C2), 126.1 (C1), 106.8 (C6), 101.9 (C4′), 75.6 (C1′), 71.8 (C2′), 60.1 (3—OCH3), 57.2 (2—OCH3), 18.9 (C3′).The eviously ; thus, t18 column  was determined as the stationary phase. The mobile phase was 35% methanol–water detected at 210 nm. We detected that the purity of IDG was 97.0% and the content in O. citriodorum was 0.2955 mg·g−1 -7-hydroxy-6-mehtoxy-chromen-2-one (1) was isolated from Fr. B. Decursidin (2) (CAS: 23027-48-7) and 9-angeloyloxy-10-senecioyloxy-9,10-dihydroxanthyletin (3) were isolated from Fr. C. Additionally, 6,7-Dimethoxy-1,3-benzodiox-ole-4-methanol (4) (CAS: 245421-64-1) was isolated from Fr. E. Nodakenetin (5) (CAS: 495-32-9) was isolated from Fr. F. Lariciresinol (6) (CAS: 27003-73-2) was isolated from Fr. G. Ostercitriodin A (7) was isolated from Fr. H D (see Sup < 0.01), which indicated the stronger vasodilation of IDG compared to that of OCE at the same dose (We investigated the relaxation effect of OCE or IDG on the vasoconstriction induced by NE stimulation. The endothelium-denuded aortic rings were pre-contracted with NE (3 μM), and after this IDG  or OCE  was added. Within our designed dosage range, the vasorelaxant effect of IDG was significantly different from that of OCE (ame dose .50) was calculated.In these experiments, we investigated the vasorelaxant effects of IDG in aortic rings pre-contracted with NE (3 μM) or KCl (60 mM) in Krebs solution, and the half maximal effective concentration .At concentrations ranging from 0.7 to 2.8 mM, IDG was observed to remarkably relax the aortic rings pre-contracted with NE or KCl in a dose-dependent manner, and the ECed rings . On the 2+-free and high K+ Krebs solution, the cumulative addition of CaCl2 (0.1–10 mM) induced a progressively increased vascular tension in aortic rings in fluorescence intensity in VSMCs. In the first instance, the increase of [Ca2+]in fluorescence intensity generated by NE in VSMCs pre-treated with IDG was significantly lower (p < 0.05) in Ca2+-free Krebs solution than that observed in the control (2 (2.5 mM) in VSMCs of these groups showed a significant elevation in the [Ca2+]in fluorescence intensity; however, compared with CON group, the [Ca2+]in fluorescence intensity in VSMCs pre-treated with IDG  produced decreases of 11.93%, 24.23% and 65.95%, respectively  and SNP (0.1 μM), respectively . We investigated the vasodilating effect of these fractions and found that Fr. F had the strongest vasodilatory effect. Then, we further separated and purified these fractions. Using Sephadex LH-20 columns or ostade-cylsilane (ODS)columns, we obtained IDG and 7 other compounds, among which 8--7-hydroxy-6-mehtoxy-chromen-2-one and ostercitriodin A are the newly isolated compounds. In a preliminary experiment, IDG, which was obtained from Fr. F, showed a stronger vasodilatory effect than other compounds. We then investigated the vasorelaxant effects of IDG in rat aortic rings and the possible mechanisms of this action.2+ into the cytoplasm, and activates ROCC on the cell membrane, leading to extracellular Ca2+ influx in in VSMCs, a molecular fluo-3/AM probe was used to measure real-time changes in [Ca2+]in induced by KCl or NE. In the Ca2+-free Krebs solution, the average fluorescence intensity of VSMCs was detected after stimulation by NE and subsequent supplementation of CaCl2. The average mean fluorescence intensities of different doses of IDG groups significantly decreased compared with the CON group, indicating that IDG inhibited the extracellular Ca2+ influx and intracellular Ca2+ release through ROCC. Different concentrations of IDG attenuated the increase in [Ca2+]in fluorescence intensity induced by KCl, which suggests that IDG could act on VDCC, inhibiting Ca2+ influx. Therefore, these findings demonstrated that IDG inhibits the KCl- or NE-induced increases in [Ca2+]in in VSMCs, possibly through VDCC and ROCC.Cain VSMCs ,23. To p2+ channels, K+ channels also play an important role in vasodilation in in VSMCs. Therefore, IDG shows promise for the development of subsequent Ca2+ channel blockers. However, further studies are needed to investigate the precise mechanisms of IDG action and its pharmacokinetics in vivo.In conclusion, our major novel finding is that IDG, which is one of the main ingredients isolated from OCE, relaxed aortic rings in an endothelium-independent manner, and we provided evidence that the mechanism of its vasorelaxation was via reducing [CaO. citriodorum need further development. Previous preliminary experiments have shown that these components also have a certain vasodilation effect, meaning O. citriodorum or other medicinal ingredients have great potential for treating angina pectoris or other cardiovascular diseases, which deserves intensive scientific exploration.At the same time, we only studied the vasodilation effect of IDG, and the research on other compounds in


Human platelets aggregate at sites of blood vessel damage in response to a rise in their cytosolic calcium concentration. Controlling these cytosolic calcium rises would provide a method to inhibit platelet activation and prevent the unwanted blood clots that causes heart attack and strokes. Previously we have predicted that calcium accumulation within the lumen of an infolded portion of the platelet plasma membrane called the open canalicular system (OCS) is essential for maintaining this cytosolic calcium rise. Due to its nanometer dimensions of the OCS, it has been difficult to measure or interfere with the predicted luminal calcium accumulation. Here we utilise iron oxide magnetic nanoparticles coated with the known calcium chelator, citrate, to create calcium-binding nanoparticles. These were used to assess whether an OCS calcium store plays a role in controlling the dynamics of human platelet activation and aggregation. We demonstrate that citrate-coated nanoparticles are rapidly and selectively uptaken into the OCS of activated human platelets, where they act to buffer the accumulation of calcium there. Treatment with these calcium-binding nanoparticles reduced thrombin-evoked cytosolic calcium rises, and slowed platelet aggregation and clot retraction in human platelets. In contrast, nanoparticles that cannot bind calcium have no effect. This study demonstrates that the OCS acts as a key source of calcium for maintaining cytosolic calcium rises and accelerating platelet aggregation, and that calcium-binding nanoparticles targeted to the OCS could provide an anti-platelet therapy to treat patients at risk of suffering heart attacks or strokes. These calcium rises are triggered by thrombin and collagen  binding to receptors on the platelet surface. The activated receptors then initiate an intracellular signalling cascade that results in a rapid increase in the [Ca2+]Cyt to trigger platelet activation and aggregation  of the solution was increased. In contrast, the citrate-coated IONPs made in house were found to significantly reduce the increase in the Fura-2 fluorescence relative to its maximum value—suggesting that the citrate coating of this particle allows it to act as an effective Ca2+ chelator.Citric acid is a widely-used anti-coagulant due to its Ca2+ chelating capacity of the Cit/1-IO and Cit/2-IO nanoparticles. To assess if the different Ca2+-binding capacities were due to differences in the stability of these IONPs in solutions of different Ca2+ concentrations, measurements of ζ potential and hydrodynamic size (DH) were also performed when the IONPs were dispersed in solutions with a variety of Ca2+ concentrations. As can be seen in 2+ concentration was increased into the micromolar range. In contrast, the Cit/1-IO maintained their ζ potential until the external Ca2+ concentration reached millimolar levels. Both Cit/1-IO and Cit/2-IO reached near zero ζ potentials at high millimolar levels suggesting that both particles have their negatively charged surfaces modulated by Ca2+, but requiring orders of magnitude different Ca2+ concentrations to do so. This difference in the reduction of the ζ potentials of the IONPs correlated with differences in the Ca2+ sensitivity of the DH of the IONPs, with DH of the particles starting to increase at lower Ca2+ concentrations in Cit/2-IO IONPs than Cit/1-IO  and thus interfere with platelet activation cyt over basal to 73% ± 4% and 52% ± 3% of control, respectively . As can be seen in 2+ signals to 41.8% + 4.0% of control, respectively . These data therefore demonstrate that the Cit/1-IO Ca2+ nanoparticles can significantly inhibit human platelet cytosolic Ca2+ signalling when stimulated in physiological conditions. In contrast, the PEG-IO and Cit/2-IO nanoparticles were found to be ineffective and unable to be used as Ca2+-binding agents. Subsequent experiments focused on assessing whether the Cit/1-IO nanoparticles functioned through buffering Ca2+ rises within the OCS.Further experiments assessed the ability of Cit/1-IO nanoparticles to inhibit thrombin-evoked cytosolic Ca2.52+-chelating Cit/1-IO nanoparticles could successfully act as Ca2+ nanochelators by effectively buffering the thrombin-evoked pericellular Ca2+ accumulation in human platelets when loaded into the OCS. This was initially examined by assessing the effect of preincubation with these IONPs on thrombin-evoked rises in the extracellular Ca2+ concentration  or in the presence of near-physiological levels of extracellular Ca2+ .Experiments were performed to assess whether the Ca2+]ext both in the absence of extracellular Ca2+ . These data therefore indicate that the Cit/1-IO IONPs were able to effectively buffer thrombin-evoked rises in pericellular Ca2+ and thus are functional as effective Ca2+ nanochelators. Our previous single cell imaging of pericellular Ca2+ rises using Fluo-4 salt under the same conditions using the same methodology demonstrated that pericellular Ca2+ rises are not observed around the platelet surface membrane—but are instead found in an extracellular fluid compartment contained within the interior of the platelet, consistent with its presence in the OCS  concentration of the buffered solution was calculated using MaxChelator software .300 μM of the IONPs were dispersed in 1.2 mL ddH4.7g, to which 100 μM Aspirin and 0.1 U/mL apyrase were then added. Washed platelet suspensions were obtained by centrifugation of PRP at 350g for 20 min, and the platelet pellet was then resuspended in a HEPES-buffered saline  supplemented on the day with 1 mg/mL bovine serum albumin, 10 mM glucose, 0.1 U/mL apyrase, and 200 μM CaCl2 (Sup-HBS).This study was approved by the Keele University Research Ethics Committee. Blood was donated by healthy volunteers who had provided written informed consent. Blood was collected by venepuncture and mixed with one-sixth volume of acid citrate dextrose (ACD) anticoagulant . Platelet-rich plasma (PRP) and washed platelets were prepared according to previously-published protocols . PRP was4.82+]cyt measurements. 1 min after the activation, all platelet samples were fixed following the protocol described by White et al. peri was monitored in platelets loaded with FFP-18 salt using a method to specifically label extracellular membrane [g for 30 s and supernatant was removed and the pelleted cells were resuspended in sup-HBS. Fluorescence was recorded as for Fura-2 above. Changes in [Ca2+]peri were monitored using the 340/380 nm fluorescence ratio.Thrombin-evoked changes in [Camembrane . Sup-HBS4.112+ concentration ([Ca2+]cyt) were estimated using Fura-2-loaed human platelets. PRP was incubated with Fura-2/AM (2 μM) for 45 min at 37 °C. PRP was then centrifuged at 350g for 20 min, and the platelets were then re-suspended in sup-HBS. Fluorescence was recorded from 1.2 mL stirred aliquots of platelet suspension at 37 °C with a Cairn Research Spectrophotometer  using λex of 340 and 380 nm, and λem of 515 nm. Changes in [Ca2+]cyt were calibrated using the methodology of Grynkiewicz et al. [Thrombin-evoked changes in cytosolic Caz et al. .4.122+]cyt, [Ca2+]peri and [Ca2+]ext were quantified by integration of the change in fluorescence records above basal fluorescence with respect to time for 3 min after thrombin addition. Basal fluorescence was defined as the mean [Ca2+] recorded for 10 s prior to thrombin addition . Platelets were then stimulated by addition of 0.5 U/mL thrombin and light absorbance read every 10 s for a further 10 min. A minimum absorbance (Amin) was measured simultaneously from a platelet-free supplemented HBS sample to which 300 μM Cit/1-IO nanoparticles or an equivalent volume of ddH20 had been added. Data were normalised to % optical density using the following formula Washed human platelet suspension resuspended in supplemented HBS were preincubated with either 300 μM Cit/1-IO nanoparticles or an equivalent volume of ddHThe maximum decrease in optical density obtained during the experiment was compared between samples.4.141.2 mL of washed platelets suspensions resuspended in supplemented HBS were aliquoted into cuvettes and incubated with the different IONPs ([Fe] = 300 μM) at 37 °C for 10 min. Platelets were then stimulated by the addition of 0.5 U/mL thrombin and clot formation and retraction was then monitored taking photos every 15 min for 90 min at room temperature. Images were later analysed with ImageJ by calculation of the relative area occupied by the clot in the front face of cuvettes.4.15n signifies independently tested platelet samples. Values are reported as mean ± SEM, except for IONPs physicochemical and morphological characterisation, which are mean ± SD. Statistical significance was assessed by one-way ANOVA with a post hoc Tukey test of repeated measurements. A P < 0.05 was considered significant.Blood from two to five donors were used in each experiment. Supplementary material


Coptis Chinensis Franch, might exert a positive inotropic effect on the heart. However, the underlying mechanisms were unclear. Here, we reported that berberine at 10–20 µM increased the left ventricular (LV) developed pressure and the maximal rate of the pressure rising, and it increased the maximal rate of the pressure descending at 20 µM in Langendorff-perfused isolated rat hearts. These effects diminished with the concentration of berberine increasing to 50 µM. In the concentration range of 50–300 µM, berberine increased the isometric tension of isolated left ventricular muscle (LVM) strips with or without electrical stimulations, and it (30–300 µM) also increased the intracellular Ca2+ level in the isolated LV myocytes. The removal of extracellular Ca2+ hindered the berberine-induced increases in the tension of LVM strips and the intracellular Ca2+ level of LV myocytes. These suggested that berberine might exert its positive inotropic effects via enhancing Ca2+ influx. The blockade of L-type Ca2+ channels (LTCCs) with nifedipine significantly attenuated 300 μM berberine-induced tension increase in LVM strips but not the increase in the intracellular Ca2+ level. Berberine (300 μM) further increased the LVM tension following the treatment with the LTCC opener FPL-64716 (10 μM), indicating an LTCC-independent effect of berberine. Lowering extracellular Na+ attenuated the berberine-induced increases in both the tension of LVM strips and the intracellular Ca2+ level of LV myocytes. In conclusion, berberine might exert a positive inotropic effect on the isolated rat heart by enhancing the Ca2+ influx in LV myocytes; these were extracellular Na+-dependent.Previous studies showed that berberine, an alkaloid from  Coptis Chinensis Franch, and has been used for the treatment of gastrointestinal infections and diarrhea in Asian countries, particularly in China. The interest in its beneficial effects on significant risk factors of cardiovascular diseases has been growing over the last two decades  is the dominant transporter mediating Ca2+ efflux , and the hearts were then quickly removed from the thoracic cavity by thoracotomy.Adult male Sprague-Dawley rats (200–300 g) were purchased from Da Ping Hospital  and were housed at Southwest University . All rats were exposed to 12/12 h light/dark cycles at room temperature (24 ± 2°C) and were free to food and water. Rats were euthanized by CO2/5% CO2 mixture, yielding a final pH of 7.4 at 37°C. For assessment of the performance of the left ventricle, a balloon connected to a pressure transducer was inserted into the left ventricle. It was used to set the left ventricular end-diastolic pressure (LVEDP) at 5–10 mmHg. After a 30-min stabilization period, berberine was added to the perfusion buffer with accumulative concentrations. Left ventricular systolic pressure (LVSP), LVEDP, LVDP (defined as LVDP = LVSP—LVEDP), the maximal rates of LVDP rising and descending during isovolumetric contraction and relaxation (+maxdp/dt and |−dp/dt|max) were analyzed with LabChart 8.1.5 .Langendorff-perfused isolated heart recordings were carried out as previously described . Briefly2/5% CO2 mixture . The strips were electrically driven with rectangular pulses  using a CS4 stimulator . Pre-load was applied until the tension reached 5 mN, and then LVM strips were washed with fresh K-H buffer every 15 min during an equilibration period of 1 h. The contraction of LVM strips was induced by epinephrine (10 μM) or EFS . Each berberine-treated strip had its vehicle control strip from the same ventricle.Isometric tension recordings in LVM strips were performed as previously described . Briefly2+-free Tyrode's buffer using a Langendorff system . The heart was then perfused with nominally Tyrode's buffer containing collagenase II (0.17 mg/ml), collagenase IV (0.10 mg/ml), bovine serum albumin (1.0 mg/ml), and 50 μM CaCl2 until it became soft and spongy. The myocytes were mechanically dispersed and stored in the Kraft-Brühe (K-B) buffer. The concentration of Ca2+ was raised from 0.2 mM to 1.5 mM using a 4-step ladder. The freshly isolated cardiomyocytes were stored at room temperature (25°C) for Ca2+ imaging.The LV myocytes of a rat were isolated as described previously . Briefly2+ levels were monitored using a fluorescent calcium probe Fluo4-AM, as previously described , and epinephrine bitartrate was from Adamas , and they were dissolved in deionized water. Nifedipine , FPL-64716 , and Fluo4-AM  were dissolved in DMSO, and the final concentration of DMSO in the buffer did not exceed 0.1%. All other chemicals were purchased from Sigma-Aldrich .3 25.0, KCl 4.6, D-(+)-glucose 11.0, CaCl2·2H2O 1.5, MgSO4·7H2O 1.6, and KH2PO4 1.2.The K-H buffer for Langendorff-perfused rat heart recording was prepared freshly and contained the following (in mM): NaCl 119.0, NaHCO3 25.0, KCl 4.6, D-(+)-glucose 11.0, CaCl2·2H2O 1.5, MgSO4·7H2O 1.6, KH2PO4 1.2, and sodium pyruvate 1.0.The K-H buffer for isometric tension recording experiment contained the following (in mM): NaCl 119.0, NaHCO2+-free K-H buffer was K-H buffer, excluding CaCl2·2H2O and containing 1 mM ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetraacetic acid.The Ca+-containing Na+-free K-H buffer was prepared freshly and contained the following (in mM): CsCl 119.0, CsHCO3 25.0, KCl 5.8, D-(+)-glucose 11.0, CaCl2·2H2O 1.5, and MgCl2 1.6.The Cs+-free K-H buffer contained (in mM): NMDG 119.0, CsHCO3 25.0, KCl 5.8, D-(+)-glucose 11.0, CaCl2·2H2O 1.5, and MgCl2 1.6.The N-methyl-D-glucamine (NMDG)-containing Na2/5% CO2 mixture except those explicitly indicated.The pH of all buffers was adjusted to 7.40 with a 95% O2+-free Tyrode's buffer for the isolation of LV myocytes contained the following (in mM): NaCl 140.0, KCl 5.4, MgCl2 1.0, D-(+)-glucose 10.0, and 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid (HEPES) 10.0; pH was adjusted to 7.35 with NaOH.The nominally Ca2PO4 25.0, taurine 20.0, HEPES 10.0, ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetraacetic acid 0.1, and MgSO4 3.0, D-(+)-glucose 10.0; pH was adjusted to 7.40 with KOH.The K-B buffer for maintaining isolated LV myocytes contained the following (in mM): KOH 80.0, KCl 40.0, L-glutamic acid 50.0, KH2+ imaging experiments contained the following (in mM): NaCl 125.0, KCl 4.0, CaCl2 1.5, MgCl2 1.0, D-(+)-glucose 10.0, HEPES 25.0, and NaH2PO4 1.2; the Ca2+-free modified K-H buffer was the modified K-H buffer excluding CaCl2·2H2O and containing 1 mM ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetraacetic acid; the low Na+ modified K-H buffer contained the following (in mM): NaCl 62.5, NMDG 62.5, KCl 4.0, CaCl2 1.5, MgCl2 1.0, D-(+)-glucose 1.0, HEPES 25.0, and NaH2PO4 1.2; pH was adjusted to 7.30 with 12 mM HCl.The modified K-H buffer for CaAll buffers were filtered through a membrane with a pore size of 0.22 µm.2+ level. The 5-min recordings before each addition of berberine were analyzed. The parameters were normalized to the recording before the first addition of berberine (taken as 100%) in Langendorff-perfused heart recordings. The isolated LVM strip tension in the 5-min recording before the addition of any compound was taken as the basal level (0%), and the effect of vehicle, berberine, or inhibitors was normalized to the basal level. The average fluorescence intensity in the last 3 min of the 15-min perfusion of vehicle, berberine, or inhibitors was analyzed and normalized to that of the last 3-min recording with the modified K-H buffer (taken as 100%). Statistical analysis was performed with GraphPad Prism 7.02 . All values are summarized as means ± SEM; n = the number of cells, LVM strips, and N = the number of rats. Statistical significance was performed with the two-way analysis of variance (ANOVA) with Sidak's multiple comparisons test or Tukey's multiple comparisons test, or the unpaired Student's t-test.LabChart 8.1.5  was used to analyze the parameters of perfused heart function and developed tension. NIS-Elements 4.3  and Clampfit 10.5  was used to analyze the intracellular CaThe Langendorff-perfused isolated-heart recordings provide information on heart functions without interference from the sympathetic nervous system . The isomaxdp/dt and |−dp/dt|max are important parameters used for the evaluation of heart contractility  (2+ (1.5 mM)  increased the fluorescence intensity of Fluo4-Ca2+ level. In the presence of nifedipine, berberine increased the fluorescence intensity of Fluo4-Ca2+ to 130.7 ± 3.2% of the basal level in LV myocytes. It rose to 130.0 ± 2.5% in the absence of nifedipine  raised the fluorescence intensity of Fluo4-Ca2+ to 132.5 ± 18.1% of the basal level in myocytes perfused with the modified K-H buffer containing 125 mM NaCl  elevated the fluorescence intensity of Fluo4-Ca2+ to 116.2 ± 22.5% of the basal level  increased LV ejection fraction in patients with chronic congestive heart failure who also took medications of angiotensin-converting enzyme inhibitors, digoxin, diuretics, and nitrates . The LV  studies .Besides, Tan investigated the tissue distribution of berberine and its metabolites in rats with a single oral dose of 200 mg/kg . The tim2+ level of LV myocytes in the concentration range from 30 or 50 to 300 μM  is the primary channel for Ca2+ entry in cardiac myocytes. Previous studies showed that berberine decreased or increased LTCC currents in guinea pig ventricular cells (2+ level of rat LV myocytes (2+ entry through pathways not dependent on LTCCs.L-type Caar cells , but hadar cells . Our res2+ concentration of LV myocytes bathed in the Ca2+-free buffer (2+ release from the intracellular stores, but further research is needed to determine the mechanism. These suggested that berberine might also exert a positive inotropic effect through an LTCC-independent mechanism.The LTCC inhibitor nifedipine significantly attenuated the berberine-induced tension increase in the rat LVM strips (+ buffer decreased (+.Sodium is also a critical cation regulating the excitation-contraction coupling in cardiomyocytes. The positive inotropic effect of berberine on LV strips bathed in the low Na+ level is elevated (+ influx during the action potential activated reverse NCX and promoted the Ca2+ entry in cardiomyocytes and neurons (+ inhibits the increase in the intracellular Ca2+ level of human epidermoid carcinoma A-431 cells by NaCN (+ concentration in the buffer suppressed the elevation of the intracellular Ca2+ level in LV myocytes by berberine (via interrelated mechanisms, including the lengthening of ventricular action potential duration, which was partially due to stimulating the reverse mode of NCX (via a Na+-dependent Ca2+ entry, which might be through activating the reverse mode of NCX.The acutely isolated cardiomyocytes are depolarized, and their intracellular Naelevated . The Na+ neurons . The rem by NaCN . The cure of NCX . The dat2+ level in LV myocytes by increasing Ca2+ influx. It consequently increased the LV contractility. These were extracellular Na+-dependent.The current study revealed that berberine elevated the intracellular CaAll datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by College of Pharmaceutical Sciences, Southwest University, Chongqing, China.WX, YW, and JZ contributed to the study design. JZ, YW, and YJ carried out literature research. JZ, YW, JG and YJ performed experiments. WX, JZ, YW and JG contributed to data analysis. WX, JZ, and YW contributed to manuscript preparation and manuscript revision. The first two authors contributed equally to the manuscript.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.


Introduction: In ventricular myocytes, spontaneous release of calcium (Ca2+) from the sarcoplasmic reticulum via ryanodine receptors (“Ca2+ sparks”) is acutely increased by stretch, due to a stretch-induced increase of reactive oxygen species (ROS). In acute regional ischemia there is stretch of ischemic tissue, along with an increase in Ca2+ spark rate and ROS production, each of which has been implicated in arrhythmogenesis. Yet, whether there is an impact of ischemia on the stretch-induced increase in Ca2+ sparks and ROS has not been investigated. We hypothesized that ischemia would enhance the increase of Ca2+ sparks and ROS that occurs with stretch.Methods: Isolated ventricular myocytes from mice  were loaded with fluorescent dye to detect Ca2+ sparks  or ROS , exposed to normal Tyrode (NT) or simulated ischemia (SI) solution , and subjected to sustained stretch by the carbon fiber technique . Ca2+ spark rate and rate of ROS production were measured by confocal microscopy.Results: Baseline Ca2+ spark rate was greater in SI  than NT . Stretch resulted in an acute increase in Ca2+ spark rate in both SI  and NT , with the increase in SI being greater than NT . Baseline rate of ROS production was also greater in SI  than NT , but there was an acute increase with stretch only in SI .Conclusion: Ischemia enhances the stretch-induced increase of Ca2+ sparks in ventricular myocytes, with an associated enhancement of stretch-induced ROS production. This effect may be important for premature excitation and/or in the development of an arrhythmogenic substrate in acute regional ischemia. The frequency of Ca2+ sparks is determined by RyR open probability, which under normal conditions is primarily influenced by the free intracellular Ca2+ concentration ([Ca2+]i) and the concentration of Ca2+ in the SR  production by NADPH oxidase 2 (NOX2) with stretch, which has been termed X-ROS  and graded by stretch amplitude and frequency, which may additionally tune the mechanical activity and redox state of cardiac myocytes to changes in physiological demand  activity  reporting standard , followed by 5-6 min of perfusion with 30 mL of Ca2+-free solution that included 6 mg of the enzyme blend Liberase TM Research Grade . The ventricles were harvested by cutting along the atrioventricular border, and then cut into 1–2 mm3 cubes and gently agitated in oxygenated Ca2+-free solution. The supernatant containing ventricular cells was collected, the remaining tissue was resuspended in fresh Ca2+-free solution, and the above procedure was repeated in triplicate. The collected supernatant was passed through a nylon mesh and centrifuged at 15 × g for 3 min, followed by resuspension of the resulting cell pellet in normal Tyrode's solution . Cells were kept at room temperature (~22°C) until ready to be used.Ventricular myocytes were isolated from mice as previously described  were subjected to a controlled, axial stretch at a single time point using the carbon fiber technique, as has been previously described for axial stretch of single ventricular myocytes  and one “stiff” (0.6 mm) carbon fiber were used, while for the ROS experiments two compliant (1.2 mm) carbon fibers were used . Tri-axial positioning of the carbon fibers was performed by custom-made three-axis hydraulic manipulators , with the compliant carbon fibers mounted on piezo-electric translators  and the stiff carbon fiber held stationary. While the unidirectional stretch using one stiff stationary and one compliant translating carbon fiber was sufficient for the Ca2+ spark experiments, this produced too much motion of the cell under the detector during stretch for ROS production to be measured. The bi-directional stretch using two compliant translating carbon fibers reduced the level of cell motion, so allowed for more accurate measurements of ROS. Whole-cell axial stretch was applied by a 20 μm increase in the separation of the piezo-electric translators and held for 15 s, controlled by custom-written LabView software and a fast analog-to-digital converter , which has been shown previously to result in a ~10% increase in sarcomere length  and a simulated ischemia (SI) group [exposed to an ischemic solution containing [in mM]: 140 NaCl, 15 KCl, 1.8 CaCl2+ sparks in ventricular myocytes was performed similar to a previously described technique  and reported as sparks·s−1·100 μm−2. The rate of image acquisition limited additional measurements of spark dynamics. Ca2+ spark rate was measured over three 5 s intervals: immediately before stretch, during stretch (immediately after its application), and immediately following complete release of stretch. Any cell in which an intracellular Ca2+ wave or synchronized SR Ca2+ release occurred during the analysis window was excluded (8% of cells in both conditions).Detection of CaThe rate of intracellular ROS production was measured using 2′,7′-dichlorofluorescein diacetate (DCF) as previously described . For the Ca2+ sparks in single mouse ventricular myocytes exposed to either NT , however values only differed between the 6-10 min and 26-30 min time points , and there was no effect of time on the change in Ca2+ spark rate with stretch  or SI  group. The average Ca2+ spark rate was higher in SI than NT  and during stretch . With stretch, Ca2+ spark rate was acutely increased in both NT  and SI , with a larger increase in Ca2+ spark rate with stretch in SI compared to NT  or SI . The rate of ROS production was greater in SI than in NT both at baseline  and during stretch . With stretch, the rate of ROS production was acutely increased in SI , but not in NT (+2.1 ± 2.7%).To determine whether the observed enhancement of the stretch-induced increase in Ca2+ spark rate and ROS production that occurs with stretch in ventricular myocytes. It was found that in simulated ischemic conditions, the basal level of both Ca2+ spark rate and ROS production were greater than in control, and that the increase in spark rate with stretch was enhanced in ischemic conditions. The difference in the response of Ca2+ spark rate to stretch represents a potential contributing mechanism to aberrant Ca2+ handling in ischemia and could be a source of premature excitation and the arrhythmogenic substrate in this pathological state.This study sought to determine the effect of ischemia on the increase in CaAcute regional ischemia is a major cause of sudden cardiac death  an increase in intracellular Ca2+ and sodium concentrations; and (iii) activation of the ATP-sensitive potassium current. At the same time, extracellular potassium concentration and catecholamine levels are also increased. All of the above changes (except for the increase in catecholamine levels), which have been shown to result in arrhythmogenic alterations of the ventricular action potential, including: (i) depolarization of the resting membrane potential; (ii) a decrease in the action potential upstroke, amplitude, and duration; and (iii) an increase in the effective refractory period, were simulated or present in the current study.The arrhythmogenic changes in ventricular myocyte electrophysiology during acute ischemia have been extensively studied , which is more similar to what occurs in the regularly beating heart .In the present study, we observed an acute increase in basal Ca2+ sparks in ischemic conditions may represent an arrhythmogenic mechanism, we observed few stretch-induced intracellular Ca2+ waves or synchronized SR Ca2+ releases, and the amount did not differ between ischemic and control cells .While it is speculated that the observed enhancement of the stretch-induced increase in Ca2+ sparks and ROS with stretch in ischemia may result from effects on various factors involved in the stretch-induced response . These possibilities warrant further investigation, as they may represent novel anti-arrhythmic targets in ischemia.The enhancement of the increase in Ca2+ spark rate with axial stretch of isolated mouse ventricular myocytes, as has been previously reported by others in both rat , yet this increase was still much lower than the previously reported control values. The reason for this discrepancy is unclear. It may relate to methodological differences or differences in data handling. Our experiments involved the stretch of ventricular myocytes using 10 μm diameter compliant carbon fibers, which stick to the cells through biophysical interactions , including hyperkalemia, acidosis, and metabolic inhibition. Previous studies that exposed isolated ventricular myocytes to simulated ischemic conditions have used a similar approach but have simulated hypoxia by the use of a 90% nitrogen-10% carbon dioxide gas phase directed over the cell chamber . Related to this concern, the increase in Ca2+ spark rate in ischemia compared to control, as well as its increase with stretch could be partly related to changes in intracellular Ca2+, however this was not measured in the current study (it would require the use of a ratiometric Ca2+- imaging approach).Temperature has been shown previously to affect Ca2+-induced arrhythmias in acute ischemia, and thus represent potential therapeutic targets for lethal arrhythmias.In the current study, we have demonstrated that ischemia enhances the acute increase in calcium spark rate that occurs with stretch of isolated ventricular myocytes, with an associated enhancement of stretch-induced ROS production. While the mechanism(s) of these enhancements remains unknown, they may contribute to the generation of CaThe datasets generated for this study are available on request to the corresponding author.This animal study was reviewed and approved by Animal Subjects Committee of Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences.BC contributed to the design of the study, performed the experimental work, analyzed the data, and wrote the manuscript. HK and KK provided technical support for the experiments. GI and TAQ contributed to the design of the study and revised the manuscript. All authors approved the final submission.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. The handling editor is currently organizing a Research Topic with one of the authors (GI) and confirms the absence of any other collaboration.