Cardiomyopathies remain among the leading causes of death worldwide, despite all efforts and important advances in the development of cardiovascular therapeutics, demonstrating the need for new solutions. Herein, we describe the effects of the redox-active therapeutic Mn(III) in vitro and in vivo experiments, to analyze intracellular Ca2+ dynamics, L-type Ca2+ currents, Ca2+ spark frequency, intracellular reactive oxygen species (ROS) levels, and cardiomyocyte and cardiac contractility, in control and MnTE-2-PyP5+-treated cells, hearts, or animals. Cells and hearts were treated with 20 μM MnTE-2-PyP5+ and animals with 1 mg/kg, i.p. daily. Additionally, we performed electrocardiographic and echocardiographic analysis.  Wistar rats weighing 250-300 g were used in both 5+ reduced intracellular Ca2+ transient amplitude, without altering cell contractility. Whereas MnTE-2-PyP5+ did not alter basal ROS levels, it was efficient in modulating cardiomyocyte redox state under stress conditions; MnTE-2-PyP5+ reduced Ca2+ spark frequency and increased sarcoplasmic reticulum (SR) Ca2+ load. Accordingly, analysis of isolated perfused rat hearts showed that MnTE-2-PyP5+ preserves cardiac function, increases SR Ca2+ load, and reduces arrhythmia index, indicating an antiarrhythmic effect. In vivo experiments showed that MnTE-2-PyP5+ treatment increased Ca2+ transient, preserved cardiac ejection fraction, and reduced arrhythmia index and duration. MnTE-2-PyP5+ was effective both to prevent and to treat cardiac arrhythmias.  Using isolated rat cardiomyocytes, we observed that MnTE-2-PyP5+ prevents and treats cardiac arrhythmias in rats. In contrast to most antiarrhythmic drugs, MnTE-2-PyP5+ preserves cardiac contractile function, arising, thus, as a prospective therapeutic for improvement of cardiac arrhythmia treatment. MnTE-2-PyP Therapeutic improvements, lifestyle modifications, and wider adoption of evidence-based medicine have resulted in a remarkable 30–35% decline in cardiovascular mortality . However2+ was required for cardiac contraction and pacemaker activity, the role of Ca2+ as a signaling ion in the heart has been progressively dissected and better understood at molecular level, and it is clear that abnormalities in Ca2+ homeostasis play a pivotal role in the pathogenesis of many cardiovascular diseases, including cardiac arrhythmias [2+-handling proteins can contribute to the pathogenesis of arrhythmias in different categories of heart disease [Since the first observation that Cahythmias . Inherit disease . However disease .2+ handling within cardiomyocytes is widely recognized as a potential target for the treatment of cardiac disease. Whereas the role of Ca2+ channels in cardiac muscle contraction has long been elucidated [2+ channels were disclosed [Caucidated , the bioisclosed , 8, contisclosed .2+ channel blockers are able to decrease the automaticity of ectopic foci in the heart and can be used in many arrhythmias [ICa,L) result in less Ca2+ overload on the myocyte, reducing tendency to ectopy, which can trigger arrhythmias [+/K+ ATPase. An increase in intracellular Ca2+ content mediates their positive inotropic effect but has also been suggested as a trigger of life-threatening arrhythmias [Accordingly, Cahythmias . Overallhythmias . Additiohythmias .+, Na+, L-type Ca2+ channels (in plasmatic membrane), and ryanodine receptor (RyR2) in sarcoplasmic reticulum membrane [meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP5+), also known as AEOL10113 or BMX-010, is currently under phase I/II clinical trials in Canada and the USA [5+ at a dose of 1 mg/kg/day led to no statistically significant changes in heart rate or arterial blood pressure [In many tissues, including the heart, reactive oxygen/nitrogen species (ROS/RNS) are often derived from mitochondria, NADPH oxidase, or uncoupled-nitric oxide synthase (NOS) and are kept under tight homeostatic control , 11. In membrane –14. Mn-pmembrane –18. Mn. Animals were maintained at the Federal University of Paraiba (UFPB), Brazil, in accordance with NIH guidelines for the care and use of animals. Experiments were performed according to approved animal protocols from the Institutional Animal Care and Use Committee at UFPB (CEUA Protocol 016/2017). All animals were euthanized by decapitation.All experiments were performed using rats of both sexes  [5+ is extremely stable toward demetallation, even in strong concentrated acids  [in vitro and in vivo experiments, MnTE-2-PyP5+ was diluted in 0.9% NaCl sterile solution.MnTE-2-PyPlsewhere –25. Conc = 5.14) –24. Moreic acid) –28, or iic acid) –29. For 2+ analysis was performed with Fluo-4 AM -loaded cardiomyocytes. The cells were stained for 30 min and then washed to remove the excess dye. Cells were electrically stimulated at 1 Hz to produce steady-state conditions. The images were recorded in a Zeiss LSM 510META confocal microscope. As an indicator of the SR Ca2+ load, 10 mM caffeine stimulation (in a Ca2+- and Na+-free solution) and the amplitude of the Ca2+ transient evoked were recorded [2+ spark frequencies were recorded in resting ventricular myocytes. The Ca2+ level was reported as F/F0 (or as ΔF/F0), where F0 is the resting Ca2+ fluorescence.Ventricular rat cardiomyocytes were isolated and stored until they were used as previously described . Intracerecorded . PrecondμM dihydroethidium  for 30 min at 37°C and were subsequently washed with an extracellular solution to remove the excess dye. Images were acquired with a Zeiss LSM 510 META confocal microscope. Images were analyzed in ImageJ software.Isolated cardiomyocytes were incubated with 10 2+ current  measurement was done using internal solution as follows (in mM): 5 NaCl, 120 CsCl, 20 TEACl, 5 EGTA, and 10 HEPES and pH 7.2 (adjusted using CsOH 1.0 M). External solution composition was as follows (in mM): 5.4 KCl, 140 NaCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose and pH 7.2 (adjusted with NaOH 1.0 M). To measure the effect of MnTE-2-PyP5+ on ICa,L density, we used a holding potential of −80 mV. Next, to inactivate both voltage-gated Na+ channels and T-type Ca2+ channels, a prestep of 50 ms to −40 mV was applied. Then, the membrane potential was swapped to 0 mV for 300 ms. This protocol was used before, during, and after washing off each concentration of MnTE-2-PyP5+.Whole-cell voltage-clamp recordings were done at 22–25°C using an EPC-9.2 patch-clamp amplifier  as described previously , 33. L-t2, 1.8 CaCl2, 10 HEPES, and 10 glucose and pH set at 7.4. All experiments were performed at room temperature. Myocytes were stimulated to contract at 1 Hz with 4 ms square pulse. Shortening was measured using a video-edge detection acquisition system . Sarcomeric shortening was expressed as a percentage of diastolic LV myocyte length. Five consecutive myocyte contractions were averaged before analysis. Cell shortening, maximal rates of contraction and relaxation, and times to 10% contraction and relaxation were determined for all groups.Cellular contractility was evaluated as previously described . Briefly2, 1.25 CaCl2, 11 glucose, 27 NaHCO3, and 2 NaH2PO4 (pH 7.4), oxygenated with carbogen mixture (95% O2 and 5% CO2) and maintained at 29 ± 0.1°C. The atrium was electrically stimulated . Tissue was placed under 5 mN tension, and an isometric force transducer (HP FTA 10-1 Sunborn) was used to record the contraction force. After 30 min of stabilization, MnTE-2-PyP5+ was added cumulatively to the bath .The left atrium was mounted in an organ chamber and maintained in modified Krebs–Henseleit solution (KHS) containing (in mM) 120 NaCl, 5.4 KCl, 1.2 MgCl2 and 5% CO2, and the left atrium was dissected. The left atrium was tied with an isometric force transducer (Grass FT03) which was mounted vertically on a micromanipulator. Stimulation (STIMULATOR SD9 GRASS) was with pulses of 0.5 ms duration at a suprathreshold voltage, and frequency of stimulation was 1 Hz for a 30 min equilibration period. Steady level and postrest contractions following 20 s of pause in stimulation were observed in caffeine-treated (10 mM) or caffeine-treated containing 20 μM MnTE-2-PyP5+. Measurements were made on the last contraction before the pause and on the first contraction after the rest interval.The heart was quickly removed, placed in a Krebs–Henseleit solution continuously bubbled with 95% 02, 1.2; CaCl2, 1.25; glucose, 11; NaHCO3, 27; and NaH2PO4, 2 (pH 7.4). The perfusion fluid was maintained at 34 ± 1°C with a pressure of 10 ml/min in constant oxygenation (5% CO2 and 95% O2). Electrical activity was recorded utilizing three platinum electrodes  placed inside the chamber close to the heart for sensing electrical signals. Hearts were perfused for an initial 20 min period with KHS. After equilibration, the hearts were perfused for 12 min with KHS, 12 min with KHS+20 μM MnTE-2-PyP5+, and last 30 min period with KHS. The high-calcium model was used to determine cardiac arrhythmias. Therefore, after a 20 min stabilization, hearts were perfused for 25 min with normal perfusion KHS at 34°C, started 25 min with high calcium (HC) (3.3 mM) and 25 min with HC+20 μM MnTE-2-PyP5+.Animals were euthanized 10–15 min after intraperitoneal injection of 1000 IU heparin/kg. The heart was dissected and perfused through a 1.0 ± 0.3 cm aortic stump with Krebs–Henseleit solution (KHS) containing (in mM) NaCl, 120; KCl, 5.4; MgClIn vitro arrhythmia was determined in an isolated heart as described previously [μM MnTE-2-PyP5+ during 25 min. Arrhythmia scores were determinate as previously described [eviously . Hearts escribed . TherefoLeft intraventricular pressure was measured using a water-filled balloon introduced into the cavity of the left ventricle with a constant diastolic pressure of 15 mmHg by adjusting the volume of the balloon, connected to a pressure transducer  coupled to an amplifier . Ventricular pressures were processed using a dedicated software .in vivo safety of MnTE-2-PyP5+, animals were randomized into two groups: (1) control 0.9% saline  and (2) MnTE-2-PyP5+ , which were treated for 15 days. The dose regimen for MnTE-2-PyP5+ administration was chosen based on rat model experiments carried out previously by our group [To test the ur group , 37, 38.The animals were anesthetized with ketamine ; surface ECG measurements were conducted using subdermal electrodes placed in the DII lead arrangement connected to a cardioscope, amplified and digitalized . ECG signals were recorded for 15 min, then animals received an injection of caffeine  and epinephrine . Data were analyzed in LabChart 8  and arrhythmic score measured as described previously .5+  during the 7 days of dexamethasone administration. In the treatment protocol, the animals received only dexamethasone until day 5 and MnTE-2-PyP5+  and dexamethasone  in days 6 and 7. Rats were randomized into four experimental groups: 1: control; 2: dexamethasone; 3: dexamethasone+MnP (treatment); and 4: dexamethasone+MnP (prevention).The animals were injected with dexamethasone  for 7 days to predispose to arrhythmia. In the prevention protocol, animals received MnTE-2-PyPCardiac function under noninvasive conditions was assessed by two-dimensional guided M-mode echocardiography of halothane-anesthetized mice as previously described . Brieflyt-test for two-group analysis or one-way ANOVA followed by post hoc analysis for multiple comparisons. In all statistical tests, a p < 0.05 was used as a measure of statistical significance.Data are presented as mean ± SEM. Sample comparisons were performed using Student's 5+ modulates Ca2+ signaling in the heart, we performed Ca2+ transient analysis in isolated cardiomyocytes loaded with Fluo-4/AM (5 μM), incubated for 90 min with crescent concentrations of MnTE-2-PyP5+ (2-200 μM), and observed a significant reduction in peak Ca2+ transient in a concentration-dependent manner  mimetic (currently recognized as a broad redox modulator) [5+ effect on cardiomyocyte ROS levels. MnTE-2-PyP5+ did not change the basal levels of ROS. However, as its well-known that MnTE-2-PyP5+, physiologically, has antioxidant activity under oxidative stress conditions, we used isoproterenol (ISO) as a cell stressor. As expected, ISO induced an increase in DHE fluorescence that was prevented by MnTE-2-PyP5+  , 18, we  Figures , indicat2+ channels (LTCC) and ryanodine receptors (RyR) are critical for normal Ca2+ signaling, we investigated the MnTE-2-PyP5+ effects on L-type Ca2+ current  using whole-cell voltage-clamp recordings. Figures 5+ induced a significant reduction in ICa,L current density, compatible with the reduction in the peak Ca2+ transient. Additionally, we measured Ca2+ spark frequency, and interestingly, MnTE-2-PyP5+ reduced basal spark frequency  and considering the importance of Ca Figures . This re5+ actions on heart contractility. First, using isolated left atria preparation, it was observed that MnTE-2-PyP5+ did not evoke alterations in contraction force, dT/dt(+), and dT/dt(-)   . Additio2+ spark frequency in isolated cardiomyocytes, we also tested if MnTE-2-PyP5+ altered SR Ca2+ content in isolated atria. Consistent with isolated cell data, MnTE-2-PyP5+ increased the SR Ca2+ load in approximately 66%, which helps to explain the maintenance of the cardiac contractility  recordings were used to analyze ECG intervals and segments. The results showed that heart rate and QRS complex length were not modified by MnTE-2-PyP5+  to induce cardiac arrhythmias. 5+ decreased HC-induced cardiac arrhythmias. Furthermore, most of the arrhythmias evidenced in control situation were of lower severity, such as ventricular premature beats  for 15 days. First, we tested MnTE-2-PyP5+ effects on Ca2+ transient. Cardiomyocytes from MnTE-2-PyP5+-treated rats presented increased peak Ca2+ transient and T90 time for Ca2+ decay, different from what we observed in acutely treated isolated myocytes  when compared to control  and heart weight/tibia length (HW/TL) ratios were evaluated as markers of cardiac hypertrophy in MnTE-2-PyP control , indicat5+ in preventing cardiac arrhythmias in isolated hearts, we assessed its antiarrhythmic property in vivo. By using a dexamethasone-induced arrhythmia model, we tested both preventive and therapeutic actions of MnTE-2-PyP5+. 5+ was completely effective to reduce both arrhythmia score and duration   . Howeverce of VT .5+, 5+ both in vitro and in vivo.To better view the multiple actions of MnTE-2-PyP5+, 5+ both in vitro and in vivo.To better view the multiple actions of MnTE-2-PyPCardiac arrhythmias are important causes of sudden death; thus, proper treatment of these conditions is of utmost importance. Indeed, over the last two decades, there has been a great deal of progress in arrhythmia management. However, most antiarrhythmic drugs proved ineffective or dangerous in patients with ventricular arrhythmias , demonstβ-blockade, there has also been early evidence that blocking ICa,L with the LTCC blocker verapamil prevents ventricular arrhythmias [ICa,L results in less Ca2+ overload of the myocyte, reducing predisposition to ectopy that can trigger arrhythmias [5+ to isolated cardiomyocytes reduced the peak Ca2+ transient in these cells, in association with reduced ICa,L. Additionally, acute administration of MnTE-2-PyP5+ in isolated hearts resulted in reduction in arrhythmia index, severity, and duration of arrhythmias, demonstrating, for the first time, that MnTE-2-PyP5+ represents a new lead molecule for the treatment of cardiac arrhythmias.Although the mainstay of treatment for catecholaminergic polymorphic ventricular tachycardia (CPVT) has been hythmias . Overallhythmias . In agrein vitro acute use of MnTE-2-PyP5+ reduced Ca2+ transient in cardiomyocytes, in vivo 15-day use of MnTE-2-PyP5+ in healthy rats did not change Ca2+ transient in cardiomyocytes. Although these results may at first seem inconsistent, when we consider that MnTE-2-PyP5+ reduced Ca2+ spark rate and increased SR load, chronically, these two effects combined may account for the final increased Ca2+ transient observed in vivo. Additionally, as we and others [5+ prevents oxidative stress, this effect can also contribute, chronically, to cellular restoration of basal transient kinetics. Accordingly, Almeida et al. [ICa,L and Ca2+ transient and that Angiotensin-(1–7) restored basal ICa,L albeit with a great increase in Ca2+ transient. Further investigation demonstrated that this alteration in Ca2+ transient was caused by reduction in Ca2+ spark frequency and consequent increased SR load. MnTE-2-PyP5+ apparently works via a similar mechanism by improving Ca2+ transient in the long term. In addition, the systemic antioxidant effect of MnTE-2-PyP5+ must be considered in the cardiovascular health in vivo.Additionally, while d others , 18 demoa et al.  demonstrBy reducing peripheral vasoconstriction and LV afterload, calcium channel blockers were thought to have a potential role in the management of chronic heart failure (HF). However, first-generation dihydropyridine and nondihydropyridine calcium channel blockers also have myocardial depressant activity . Several5+ prevents and treats cardiac arrhythmias while preserving contractility at both cardiomyocyte and heart levels. These combined effects respond to a large gap in arrhythmia treatments, especially in patients with HF.Although the use of calcium channel blockers for arrhythmia treatment is often plagued by myocardial depressant activity, here, we demonstrated that MnTE-2-PyP5+ preserves cardiomyocyte and heart contractility and exerts antiarrhythmic effects both in vitro and in vivo, representing, thus, a potentially new strategy to treat cardiac arrhythmias in patients with contractile dysfunctions. In addition, although reduction in peak calcium transients is usually related to reduction in cardiac contractility, modulation of proteins involved in calcium handling or contractile machinery can alter this relationship. In this way, Vanzelli el al [5+ effect may be somewhat related to the mechanism described by Vanzelli el al [5+ mechanism(s) of action.It is worth noting that our study demonstrates that MnTE-2-PyPli el al . demonstli el al .; furthe5+ (BMX-001) [5+ represents a simple, promising redox-active therapeutic for preventing and treating cardiac arrhythmias, preserving heart contractile function. Taken together, the data strengthen the therapeutic potential of Mn porphyrins in a quite unexplored field of cardiac applications.Finally, it is important to highlight that the use of Mn porphyrin-based SOD mimics for cardiovascular treatments is in its infancy. A first report  was veryBMX-001) , to supp


In humans, numerous cell stimulation pathways lead to the mobilization of sarco/endoplasmic reticulum (S/ER) stored Ca2+, resulting in the propagation of Ca2+ signals through the activation of processes, such as store-operated Ca2+ entry (SOCE). SOCE provides a sustained Ca2+ entry into the cytosol; moreover, the uptake of SOCE-mediated Ca2+ by mitochondria can shape cytosolic Ca2+ signals, function as a feedback signal for the SOCE molecular machinery, and drive numerous mitochondrial processes, including adenosine triphosphate (ATP) production and distinct cell death pathways. In recent years, tremendous progress has been made in identifying the proteins mediating these signaling pathways and elucidating molecular structures, invaluable for understanding the underlying mechanisms of function. Nevertheless, there remains a disconnect between using this accumulating protein structural knowledge and the design of new research tools and therapies. In this review, we provide an overview of the Ca2+ signaling pathways that are involved in mediating S/ER stored Ca2+ release, SOCE, and mitochondrial Ca2+ uptake, as well as pinpoint multiple levels of crosstalk between these pathways. Further, we highlight the significant protein structures elucidated in recent years controlling these Ca2+ signaling pathways. Finally, we describe a simple strategy that aimed at applying the protein structural data to initiating drug design.Calcium (Ca Under resting conditions, intracellular Ca2+ concentrations are maintained at ~100 nM by effectively segregating Ca2+ into the extracellular fluid and within intracellular stores, such as the mitochondria and sarco/endoplasmic reticulum (S/ER). Under basal conditions, Ca2+ levels within the cytosol and mitochondria are similar; conversely, in times of cellular stress or injury, mitochondria can uptake Ca2+ [2+ overload [2+ would lead to a cycle of higher respiratory chain activity and the generation of reactive oxygen species (ROS), resulting in oxidative stress, apoptosis, and/or necrosis [2+ is a cofactor that is required for mitochondrial dehydrogenase function, which are enzymes integral to the electron transport chain [2+ uptake is via the low affinity, high capacity mitochondrial Ca2+ uniporter (MCU) complex [2+/proton (H+) antiporter is involved in fine-tuning these levels [2+ uptake, dictating both mitochondrial and cellular function.Regulating cytosolic calcium ion Ca+ levels ake Ca2+ ,4 in ordoverload ,6,7. Pernecrosis ,9. Ca2+ rt chain . The pri complex ,13. Howee levels ,15. Thus2+ store, maintaining free Ca2+ in the ~400–700 µM range (depending on cell-type) [3Rs), respectively, serve as the predominant channels that release Ca2+ into the cytosol from the S/ER stores [2+ is critical for the activation of Ca2+-dependent signaling cascades that require more chronic elevations in cytosolic Ca2+ because S/ER Ca2+ stores are limited in capacity. Store-operated Ca2+ entry (SOCE) is the process whereby S/ER Ca2+ store depletion leads to the activation of plasma membrane (PM) Ca2+ channels for a sustained Ca2+ entry into the cytosol from the extracellular space [2+-sensing S/ER-membrane inserted stromal interaction molecules (STIMs) [2+ levels that are driven by SOCE not only signal downstream physiological and pathophysiological cellular responses, but also replenish the depleted intracellular Ca2+ stores.The S/ER is the largest intracellular Call-type) ,19,20,21ll-type) . In exciR stores ,24,25. Aar space ,27,28,29 (STIMs) ,31 and t (STIMs) ,34,35,363Rs and/or RyRs, leading to the depletion of the S/ER Ca2+ stores [2+ store release  [3 is a small diffusible second messenger that binds and activates IP3Rs, causing S/ER Ca2+ store release into the cytosol [3R-driven Ca2+ signaling mechanism is critical in platelets where thrombin binding to protease activated receptor (PAR)-1 or PAR4 mediates thrombosis [1 receptor (H1R) leads to bronchoconstriction [GPCRs constitute a major family of PM receptors that, when activated by the appropriate extracellular ligand, can serve to mediate the release of Caia IP3Rs ,42. Upon cytosol . This IPrombosis , in vascrombosis , and in triction , to name2+ release through IP3R activation. RTKs, such as insulin growth factor-1 receptor in skeletal myoblasts activate phospholipase C and IP3 production upon binding insulin-like growth factor-1, leading to ER Ca2+ release necessary for differentiation in myogenesis [3R activation, enabling humoral immunity signaling and apoptosis/elimination of self-reactive T-cells, respectively [3 generation and S/ER Ca2+ release [Nevertheless, it is not only GPCRs that can induce S/ER Caogenesis ,69. In sectively ,70. Likeectively ,17. Immu release ,17,55,563Rs and similarly function to release S/ER Ca2+ [2+ channels) open in response to membrane depolarization, resulting in the entry of Ca2+ into the cytosol from the extracellular space [2+ binds to S/ER RyRs, opening these channels, in contrast to IP3Rs, which require IP3. Remarkably, the RyR-mediated release of S/ER Ca2+ can propagate to adjacent channels and promote additional RyR activation in a process that is known as Ca2+-induced Ca2+ release [RyRs are much larger structural homologues of IP/ER Ca2+ . For exaar space ,61. This release ,61.2+ signaling pathway that has evolved to use S/ER Ca2+ depletion as an activation signal, facilitating further and more sustained extracellular Ca2+ influx in response to the release of intracellular S/ER Ca2+ [2+ sensor and SOCE activator [2+ changes  deactivates Orai1 channels to terminate SOCE [S-Glutathionylation, for example, is the covalent attachment of a glutathione (GSH) moiety to free cysteine (Cys) thiols. GSH modification of STIM1 at Cys56 has been shown to evoke constitutive Orai1 channel activation and Ca2+ entry independent of luminal Ca2+ concentrations [A process known as Caate SOCE ,86. Multate SOCE ,88,89,90ate SOCE ,102,103.trations . It is wSeveral heritable mutations in STIM1 and Orai1 cause disease. The loss of function mutations in STIM1 occur as frameshift i.e., E128RfsX9 ) and intRfsX9  anGain of function mutations occur in STIM1  potential or depletion of sodium (Na+) could inhibit SOCE [2+ uptake diminishers [2+ uptake near store-operated Ca2+ channels is believed to prevent Ca2+-dependent inactivation by limiting the formation of high Ca2+ level domains. Interestingly, one study showed that the distance between mitochondria and PM SOCE channels decreases upon SOCE activation, allowing for a more sustained Ca2+ entry due to reduced CDI [2+ uptake, but not mitochondrial translocation/motility, was required for STIM1 and Orai1-mediated SOCE [2+ levels [The first link between mitochondrial Cabit SOCE . Indeed,inishers . Mitochouced CDI . In contted SOCE . Further+ levels .2+ uptake following SOCE [+/Ca2+/lithium (Li+) exchanger (NCLX) on the IMM has been shown to be vital for clearance of mitochondrial Ca2+ . SOCE iand SOCE . The actits SOCE . It is bits SOCE .2+ uptake plays a central role in regulating this energy production and it is also instrumental in regulating apoptosis and shaping cytosolic Ca2+ transients [2+ can readily move through the voltage dependent anion channel (VDAC) of the outer mitochondrial membrane, Ca2+ uptake into the mitochondrial matrix is more precisely controlled [2+ uptake into the matrix, functioning as a multimeric protein complex that forms a Ca2+ selective pore through the IMM [2+ into the matrix, despite the highly negative IMM potential (~−180 mV); following a rise in cytosolic Ca2+ levels, the MCU channel open probability increases, allowing for Ca2+ to move into the matrix, being driven by the IMM potential [2+ uptake 1, 2, and 3  [Mitochondria are primarily recognized for their role in adenosine triphosphate (ATP) production, a process that requires an electrochemical gradient across the IMM . Mitochoansients . While Cntrolled . MCU pla the IMM ,13. Undeotential ,149,150.otential ,154,155,d MICU3) ,157; a sd MICU3) ; an MCU d MICU3) ; and a cd MICU3) .2+ selectivity and controls permeability through the channel due to binding of Ca2+ to the acidic residues [2+ uptake, which is likely due to the removal of these negative charges [2+-permeable pore [Caenorhabditis elegans MCU was suggested to exist as a pentamer, with the DIME motifs forming an unstructured loop at the opening of the channel [C. elegans NMR model have established metazoan MCU as a tetramer with the DIME motifs lining the pore as part of the helical transmembrane regions [The MCU pore forming subunit is an ~40 kDa protein  composed of two transmembrane domains spanning the IMM, with both the N- and carboxyl (C)-termini localized in the mitochondrial matrix ,161 FigA. An Aspresidues . Amino a charges . MCU subble pore . Initial channel . Several regions ,152,154 2+ uptake [Interestingly, the C-terminal domain of MCU alone can form a channel, because it contains the TM1 and TM2 helices that create the IMM channel pore ,162,163;+ uptake ,153,155 + uptake .2+-sensing properties to MCU channel activity. The presence of an Asp-rich C-terminus is thought to be important in MCU complex regulation [2+ uptake in mammals; subsequent overexpression of MCU is unable to recover the channel function [EMRE is a metazoan-specific ~11 kDa  single-pass membrane protein that spans the IMM . EMRE ingulation ,166; howgulation . The mosgulation   that shares ~50% sequence identity with MCU  Figure A. Much lble pore . Interesble pore . Mechanible pore . Once ineability ,180. Nev2+ uptake, reduces the IMM potential, and disrupts oxidative phosphorylation [2+ uptake, but also plays a key role in controlling cellular bioenergetics.MCUR1 has been identified as a positive regulator of MCU, functioning as a scaffold factor that binds to both MCU and EMRE ,181. MCUrylation . Collect2+ signaling has been implicated in a variety of pathophysiological disorders. Aberrant mitochondrial Ca2+ uptake not only affects processes reliant on maintaining tight cytosolic Ca2+ concentrations, but also impacts oxidative metabolism and can trigger cell death pathways [2+ overload and/or oxidative stress results in an opening of the mitochondrial permeability transition pore (mPTP) [2+ uptake has dire implications for cell survival and has been associated with various diseases, including cancer, neurodegenerative pathologies, diabetes, and learning and muscular disorders, among others [Mitochondrial Capathways ,147,183.e (mPTP) . High coe (mPTP) ,186. Ulte (mPTP) . Thus, dg others ,190,191.MICU1 mutations , which result in intronic insertions causing frameshifts, nonsense mediated mRNA decay, and loss of MICU1 protein [MICU1 exon 1 deletions  [MICU1 nonsense mutations  has been discovered, which eliminates full-length MICU2 protein [2+ uptake. Further, MICU2 protein abrogation also suppressed mitochondrial Ca2+ uptake rates [2+ levels due to either MICU1 or MICU2 protein downregulation [Patients have been identified with heritable  protein ,192,193.eotides)  and MICU:p.Q185* ) have be protein . All of  protein ,195,196. protein  rates ofgulation ,196, whi2+ uptake can suppress cytosolic Ca2+ signals in fibroblasts from MICU1-deficient patients [2+ signals [2+ uptake may be related to work showing deletion of MICU1 in mouse hepatocytes causes sensitization to Ca2+-overload-induced mPTP opening [Enhanced mitochondrial Capatients , which i signals ,199,200. opening . The ide2+ and K+ ion homeostasis, regulating key facets of mitochondrial physiology, such as osmotic balance and ATP production [LETM1 as one of ~2000 essential genes in human cell cultures [LETM1 is an essential IMM protein linked to mitochondrial ion transport, regulation of cell cycle, mitochondrial oxidative stress and bioenergetic function . Interesoduction ,207,208.oduction . Homozygoduction . Clinicaoduction ,210 , LETM1 has been shown to transport Ca2+ at ~nM cytosolic Ca2+ levels, suggesting that LETM1 provides cells with a high affinity/low capacity pathway necessary for precise mitochondrial Ca2+ homeostasis [2+/H+ exchange, independent of MCU [2+ uptake, decreases ATP production, and increases ROS production [In the context of mitochondrial Caeostasis ,214,215.t of MCU ,206,208.oduction ,216.2+-binding EF-hand motif [Human LETM1 is an ~83 kDa transmembrane protein  that localizes in the IMM ,207 FigA. LETM1 nd motif . This tond motif ,206,207.nd motif   [2+ ATPase (SERCA) pumps [2+ toolkit structural data, yet reinforces a major question at the nexus of the structural biology, cell biology, and clinical research fields. How can we use this wealth of protein structural information to design new diagnostic tools and drugs for research and treatment of disease?Over the past ~15 years, a number of protein structures involved in regulating cytosolic and stored Ca as SOCE ,31 and m+ uptake ,13, but tion NMR ,113, thetion NMR , and X-rtion NMR ,108  , S/ER CaA) pumps ,240, andA) pumps ,242. Thi2+ signaling proteins, there is a disconnect between applying this protein structural knowledge and rationally developing research tools or drugs. Perhaps this disconnect exists due to the relatively static insights provided by many of the resolved structures. Most  protein function involves dynamic changes in conformation and stability. Rationalizing effective drugs without a detailed understanding of the conformational ensembles present in the inactive, transition, and active states of proteins is practically challenging.Indeed, while obtaining structural information has been invaluable for informing the broad scientific community on the molecular and structural basis for the function of hundreds or thousands of Ca2+ uptake protein machinery are appealing drug targets for several reasons. First, mutations causing disease have been identified in the proteins mediating these processes (see above). Second, complexes of multiple different proteins regulate SOCE and mitochondrial Ca2+ uptake; hence, the modulation of function, in some cases, could be achieved by targeting native protein interaction partner components. Third, drugs targeting ion channels are demonstrated to be effective at treating a wide range of human diseases and acute conditions [2+ uptake molecular machinery provide a unique opportunity for the development of new diagnostics and/or therapeutics that can biasedly target different aspects of these pathways.The SOCE and mitochondrial Canditions ,244,245.nditions ,247). Finditions ,250,251.Saccharomyces cerevisiae as an MCU-null organism to screen for small molecule modulators of MCU function. Human MCU, human EMRE, and a mitochondrial-targeted Ca2+ indicator  were expressed in these yeasts to establish a reporter system for the screening of ~600 clinically approved drugs [2+.Small molecule screens targeting MCU function have already been performed. An initial study employed ed drugs . In a seed drugs . In the ed drugs . All of 2+ or NFAT translocation to the nucleus [Since the identification of STIM1 and Orai1, published drug screens targeting SOCE have largely involved the over-expression of STIM1 and Orai1 in mammalian cells or the use of cells with endogenous levels of STIM1 and Orai1, in combination with indicator dyes or proteins monitoring cytosolic Ca nucleus ,257,258. nucleus . In sili nucleus ,261.2+ uptake have been performed, these published approaches have not used the available protein structure information as initiation points to drug discovery.Thus, while drug screens targeting the protein machinery that mediates both store-operated and mitochondrial CaA starting point for drug development could be initiated by a simple high-throughput small molecule in silico screen of millions of compounds. This computational screen would use available protein structures and knowledge of the vital interfaces modulating activity. The structures of the small molecules/drugs would also need to be known. Such small molecule databases and computational docking programs exist and are freely available .2+ signaling, these types of FRET-based sensors have been engineered and used in screens for the identification of SERCA and RyR modulators. A green fluorescent protein (GFP) and red fluorescent protein (RFP) FRET pair was fused into the cytosolic headpiece of SERCA, which undergoes large scale structural changes during pump activity. This intramolecular FRET sensor was successfully used to identify the small molecule modulators of SERCA function [High resolution structural information can also be used to develop tools for high-throughput drug discovery. For example, fluorescence-resonance energy transfer (FRET)-based biosensors can be rationalized based on proximity information that is revealed by isolated and co-complexed structures. With respect to Cafunction ,266. Sinfunction ,268. In 2+ signaling axis, providing several possible initiating points to protein structure-based drug design. The elucidation of Ca2+ signaling protein structures is vital for understanding the fundamental mechanisms underlying cellular processes that are integral to life and death. The structural information can also provide an entry point for the development of tools and therapeutics targeting these pathways.Not discussed herein are the countless additional experimental , logistical, and administrative hurdles that are required to move any small molecule lead into clinical use. Nevertheless, we provide a simple strategy for applying high-resolution structural knowledge of proteins as a starting point to drug development. Excitingly, several key protein structures are available that inform on the mechanisms regulating the SOCE-mitochondrial Ca


Although a similar role for Ca2+ in prokaryotes has been difficult to demonstrate, there is increasing evidence for Ca2+ as a cell regulator in bacteria. The purpose of this study was to investigate Ca2+ signaling and the effect of Ca2+ on the Staphylococcus aureus multidrug resistant efflux pump LmrS. We hypothesized that antibiotics act by increasing Ca2+ concentrations, which in turn enhance the efflux activity of LmrS. These Ca2+ transients were measured by luminometry in response to various antibiotics by using the photoprotein aequorin reconstituted within live bacterial cells. Efflux associated with LmrS was measured by the increase in fluorescence due to the loss of ethidium bromide (EtBr) from both S. aureus cells and from E. coli cells in which the lmrs gene of S. aureus was expressed. We found that addition of antibiotics to cells generated unique cytosolic Ca2+ transients and that addition of CaCl2 to cells enhanced EtBr efflux whereas addition of Ca2+ chelators or efflux pump inhibitors significantly decreased EtBr efflux from cells. We conclude that antibiotics induce a Ca2+ mediated response through transients in cytosolic Ca2+, which then stimulates LmrS efflux pump.Calcium ions (Ca Since cpression  and virupression , we hypoStaphylococcus aureus ATCC 25923 and clinical isolate EBSA54 were grown in Brain Heart Infusion (BHI) broth . E. coli strain JM109 containing the expression vector pMMB66EH with the apoaequorin coding sequence was used to conjugate S. aureus cells. E. coli cells were a gift from Dr. Anthony K. Campbell . E. coli cells were grown in Luria-Bertani (LB) broth  with carbenicillin (100 μg/ml) . Confirmation of S. aureus after conjugation was done by culture in Mannitol Salt Agar (MSA)  and testing for coagulase production  and latex agglutination . Mueller-Hinton broth  was used to determine the Minimum inhibitory concentration (MIC) assays. The vector pRMC2 was purchased from Addgene to clone the amplified lmrS gene. DH5α E. coli cells were obtained from Promega, Madison, WI, United States).2, Carbonyl cyanide m-chloro phenylhydrazone (CCCP), Calmidazolium (CDZ), Verapamil, coelenterazine, Trifluoperazine (TPZ), Chlorpromazine (CPZ), Phosphate Buffer Solution (PBS), Triton X-100, Tris-Acetate EDTA buffer (TAE), and aequorin oligonucleotides were all purchased from Sigma-Aldrich . Taq polymerase master mix for PCR , agarose molecular grade and 100 bp molecular ruler were obtained from Bio-Rad, Hercules, CA, United States). Kanamycin, Gentamicin, Streptomycin, Vancomycin, Ciprofloxacin, and Erythromycin . BamHI and HindIII , T4 ligase .Ethidium bromide (EtBr), ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), ethylenediaminetetra acetic acid (EDTA), isopropyl-β-D-thiogalactoside (IPTG), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) coelenterazine, carbenicillin, CaClStaphylococcus aureus cells were transformed by conjugation using E. coli cells containing the apoaequorin coding sequence . Conjugation was done according to the protocols for tri-parental mating from The Samuel Miller Lab (2019) Conjugating plasmids into bacteria http://miller-lab.net/Miller Lab/protocols/bacterial-genetics/conjugating-plasmids-into-bac teria/with modifications. E. coli and S. aureus were grown in 250 mL triple-baffled flasks containing 50 ml LB (Becton Dickinson DifcoTM) broth with 100 μg/mL carbenicillin (Sigma-Aldrich) and BHI broth (Remel Thermo Fisher) respectively, at 37°C, in a rotatory shaker at 220 rpm overnight. After 18–20 h of incubation, E. coli and S. aureus cultures were adjusted to an OD600 of 0.5. A 1:5 dilution (100 μL of E. coli and 400 μL S. aureus cells) of the bacterial cultures were incubated in 10 mL of BHI with 100 μg/mL carbenicillin and incubated for 3 h at 37°C (220 rpm). The culture was dotted (25 μl) onto BHI agar plates containing 100 μg/mL carbenicillin  and incubated for 48–72 h at 37°C. Transformed S. aureus cells were isolated by streaking onto MSA (Becton Dickinson DifcoTM) for 48–72 h at 37°C. Individual bacterial colonies that fermented mannitol were selected and further tested for coagulase (Becton Dickinson BBLTM) and anti-S. aureus latex agglutination (ProlexTM Pro-Lab Diagnostics). The presence of the plasmid containing the apoaequorin coding sequence was confirmed by PCR . The oligonucleotides, AQ440LICS: Forward: AAGGAGGAAGCAGGTATGGTCAAGCTTACATCAGACTTC GAC and AQ440LICS-CAS Reverse: GACACGCACGAGG TTTAGGGGACAGCTCCACCGTAG were used for DNA amplification (2+. Aequorin is a protein that luminescence (λmax = 469 nm) when it binds to Ca2. The Ca2+ concentration of the cytosol was measured at rest  and after adding increasing concentrations of CaCl2 to the culture media. The luminescence produced is directly proportional to the concentration of free Ca2+ within the cytosol.fication  under thS. aureus were grown in BHI-carbenicillin broth overnight at 37° (220 rpm). Cells were diluted 1:100 in BHI-carbenicillin broth and incubated (same conditions) until the culture reached an OD600 of 0.3. The aequorin gene was induced by adding IPTG at 1 mM final concentration and re-incubated for additional 2 h. After 2 h, cells were washed twice with 20 mL of ice cold HEPES buffer  or at the respective experimental pHs  and pelleted by centrifugation at 4500 rpm for 5 min . Bacterial cells were re-suspended in 1 mL of HEPES and 2.5 μM of coelenterazine and incubated in the dark for 1 h at room temperature. After incubation, cells were washed twice first in 20 mL HEPES buffer and resuspended in 1 mL of HEPES. The buffer was adjusted to an OD600 0.4 at the appropriate pH (according to the experiments performed) and stored on ice for subsequent readings or 4°C overnight for stabilization. To address possible contamination from trace amounts of Ca2+ and other divalent cations, 0.05 mM of EGTA was added to the HEPES media. All glassware and labware were washed with 0.05 mM of EGTA to rinse off trace levels of calcium from the surfaces.The transformed 2+ levels. Cells were then injected with CaCl2 to a final concentration of 1.0 mM CaCl2 and chemiluminescence was monitored for an additional 300–500 s according to the appropriate experimental pHs 5, 7, and 9. At the end of each experiment the remaining amount of aequorin was determined by adding equal volumes of discharge buffer  as described by 2+. To determine the possible effect of pH on aequorin activity, cell lysates of S. aureus, expressing apoaequorin, were reconstituted with coelenterazine as described before. Aliquots of 100 μl were treated with each chemical using the same concentrations as those used in the treatments (different pHs). After each treatment, aliquots were taken to the luminometer and aequorin was completely discharged by adding an equal volume of 100 mM CaCl2 and the discharge buffer to determine the total light output. To rule out the possibility that the luminescence obtained would be due to aequorin being released into the medium by lysed cells or lysis of cells due to addition of calcium, light was measured after addition of Ca2+ to the medium in which reconstituted cells were present after removing the cells by centrifugation. We also checked for cell lysis by microscopy. Negative control used for these experiments were cells without the apoaequorin plasmid. Relative light units were converted to μM Ca2+ concentrations utilizing a matrix created by Dr. Anthony Campbell [pCa = 0.612(−logk) + 3.745 where k is the rate constant for decay of chemiluminescence (s−1)]  equipped with two dispensers allowing the reading on 96-well Microfluor microtiter plates (Thermo Fisher Scientific). Measurements were done in triplicates on 100 μL aequorin-loaded cells once every 10 s for 60 s to determine resting cytosolic free Cae (s−1)] .S. aureus cells containing the aequorin plasmid were grown overnight in BHI then subcultured into Mueller-Hinton and grown to an OD600 0.8. The culture was then adjusted to a 0.5 McFarland standard (aprox. 108 CFU/mL) in 5 mL. The MIC was determined using MicroScan autoSCAN-4 automated system using MIC panels for Gram positive bacteria PBCP20. To measure antibiotic stimulus-response, S. aureus cells were grown as stated above. S. aureus cells containing the aequorin were loaded into a microtiter plate and basal intracellular Ca2+ was monitored for 10 s followed by injection of each antibiotic to a final concentration of 0.5 μg/mL in a final volume of 100 μl. Intracellular Ca2+ was then monitored for an additional 250 s. We used a standard concentration for all antibiotics to detect unique responses of cytosolic Ca2+ transients elicited by each antibiotic. This allowed for elimination of concentration as a variable affecting cytosolic responses.The antibiotics used for the signaling experiments included: erythromycin, gentamicin, kanamycin, vancomycin, streptomycin, and ciprofloxacin. To determine the MIC for each antibiotic, S. aureus cells. In the indirect method bacterial cells were pre-incubated in (EtBr) and Ca2+ or a Ca2+ inhibitor before the beginning of the assay. Fluorescence was monitored after incubation for 20 min. Fluorescence increases as the EtBr accumulates within the cells until it reaches a steady state of efflux/accumulation. In the direct method, bacterial cells were incubated with dye in the presence of an inhibitor such as the proton decoupler CCCP, then washed to remove excess dye and inhibitor. Fluorescence was measured for 5 min after which 1 mM of Ca2+ was injected into the media. Efflux was then measured for an additional 20 min. The purpose of this is to observe the rate of efflux of EtBr that is inside the cells and to record the change in slope as they respond to the addition of external Ca2+.Efflux pump activity was measured indirectly and directly in 2+ in the efflux systems of S. aureus, EtBr was used as a substrate, and cells were treated with various concentrations of CaCl2, Ca2+-chelators, and Ca2+-dependent inhibitors. The MIC for EtBr was done using the broth dilution method and as recommended by the Clinical Laboratory Standards Institute. A final concentration of 2.5 mg/L EtBr was used for both direct and indirect efflux experiments. Bacterial strains were cultured overnight in BHI broth at 37°C in a Gyromax rotatory shaker (Amerex Instruments) at 220 rpm. Overnight cultures (1:100) were transferred into 250 ml triple-baffled flasks containing 50 mL fresh BHI broth and grown to reach an OD600 of 0.8. Bacterial cells were harvested by centrifugation at 5000 rpm for 5 min . Cells were washed three times in Phosphate buffer solution PBS  adjusting to the different experimental pHs 5.0, 7.0, and 8.0 (pH for efflux experiments was adjusted at 8.0). Bacterial cell concentration was adjusted to an OD600 of 0.6 for each pH. Experimental procedures for EtBr efflux were done following the protocols of 2+ on efflux, media calcium levels were manipulated by addition of CaCl2 (Sigma-Aldrich) to a final concentration of 1.0 and 5.0 mM. Lack of calcium was also investigated by the addition of Ca2+-chelators, EGTA, and EDTA (Thermo Fisher), which were added at final concentrations of 5 and 10 mM. To investigate Ca2+-dependent transport inhibitors, the phenothiazines, CPZ and TFP (Sigma-Aldrich), were added to final concentrations of 0.1 μM and 30 μM, respectively. The Ca2+ channel blocker, verapamil (Sigma-Aldrich) was added at 30 μM. Efflux assays were conducted at the different pHs, 5.0, 7.0, and 8.0. Glucose (0.4%) was used as control for the contribution of metabolic energy to efflux. CCCP (Sigma-Aldrich), a proton uncoupler was used to a final concentration 50 μM to illustrate proton motive force disruption.Indirect measurements. To determine the effect of Ca600 of 0.6 and incubated in EtBr at 2.5 mg/L and the indicated Ca2+ inhibitor. The cells were then pelleted by centrifugation and the supernatant containing excess EtBr and treatment was removed. The cells were then resuspended in PBS at the appropriate pH. 100 μl were loaded into a 96 microtiter plate and fluorescence was monitored for 5 min at 585 nm to establish basal levels. After 5 min, CaCl2 was added to a final concentration of 1 mM and fluorescence was monitored for an additional 20 min.Direct measurements. For direct efflux experiments, the cells were grown and cultured as described before. Briefly, the cells were grown overnight in BHI broth at 37°C. The cells were subsequently washed and pelleted twice in PBS and adjusted to a final OD A2+ had the ability to restore efflux activity inhibited by EDTA, and alkaline pH of 8, S. aureus cells were grown overnight in BHI following the methods of 600 was adjusted to 0.6 and EDTA and EtBr were added to a final concentration of 5 mM and 2.5 mg/L, respectively. Fluorescence was then measured at 585 nm for 20 min. After 20 min the experiment was stopped and cells were treated with one of the following: CPZ (0.1 μM), CPZ + CaCl2 , and CaCl2 (1 mM) in a final volume of 100 μl, loaded to a 96 well microtiter plate. Fluorometry was resumed for additional 20 min.To investigate if Ca2+ on an individual S. aureus efflux pump, the lmrS gene was amplified and cloned into the expression vector pRMC2  The MICs for the antibiotics tested were determined using the MicroScan autoSCAN-4 automated system using PBCP34 panels.Both the wild-type and 2+ treatments , Ca2+ inhibitor treatments  compared to controls in fluorescent assays. The same test was used to determine significant differences in intracellular Ca2+ levels at concentrations: 0.5 mM, 1.0 mM, and 5.0 mM. To determine significant differences of cytosolic Ca2+ at each pH  a one sample t-test and Wilcoxon test was conducted. Both the efflux and cytosolic free Ca2+ experiments were analyzed using the statistical and graphics software, Graph Pad 8.0.The Kruskal–Wallis test was utilized to determine the differences in Ca2+ acts as a messenger in prokaryotes is based on the observation that environmental signals induce changes in the level of cytosolic free Ca2+. Therefore, measurement of [Ca2+]i is essential in establishing that Ca2+ might serve as an intracellular signal  showed an increase in luminescence directly proportional to the amount of CaCl2 injected followed by a rapid decline within seconds    and luminescence was measured as before. A rapid cytosolic Ca2+ transient was observed after injection of 1 mM CaCl2 with a fast decline to basal levels. However, cultures maintained at differing pH values had differing cytosolic Ca2+ showing different amplitudes (peaks)  was added after the injection of 1 mM CaCl2 the removal of Ca2+ caused a sharp decline to basal levels  . These r studies . The effound/H+) . Externa (peaks) . At pH 9l levels . S. aure2+ . Antibiotics used in this study included: erythromycin, gentamicin, kanamycin, vancomycin, streptomycin, and ciprofloxacin. Concentrations of antibiotics were adjusted according to a standard concentration described in “Materials and Methods” section. All antibiotics tested elicited a rapid increase in cytosolic Ca2+  and trifluoperazine (TFP), which are known to inhibit efflux pump activity in various pathogens including S. aureus , which is involved in modulation of pumps in eukaryotes, were also used in this study (2 treated cells and control cells (cells without EtBr and cells with EtBr no treatment), which showed low fluorescence. Assays in panel A were conducted at slightly higher (pH 7.6) than panels B-D, showing a slight effect in efflux. A Kruskal–Wallis statistical analysis was used to determine significant differences. Each of the inhibitor treatment was significantly different as compared to 1 mM of Ca2+ and to the positive control where the cells were incubated in EtBr alone (data not shown). Each treatment represents the average of three replicates with the mean and ±SD . These results show that Ca2+ ions have the ability to enhance efflux in S. aureus cells. Since proton availability and metabolic energy affect efflux, measurements were also done at different pHs. At pH 5  and the Ca2+ chelator EDTA. We also examined the effect of adding both CPZ and CaCl2 together on efflux of EtBr. Our results showed that addition of 1 mM CaCl2 decreased the accumulation of EtBr caused by the CPZ. However, when CPZ + CaCl2 were added the decrease in fluorescence was less pronounced . The LmrS (lincomycin resistance protein of S. aureus) belongs to the MFS superfamily, which transport diverse molecules across the membrane using an electrochemical gradient. The lmrS gene was identified by lmrS gene was amplified and cloned as described in “Materials and Methods.” To investigate if recombinant E. coli cells (DH5α-lmrS) have acquired resistance through the incorporation and overexpression of the lmrS gene, we evaluated the susceptibility of the recombinant E. coli cells to various antibiotics by determining the MIC using the micro broth dilution method . The results indicate that the E. coli cells carrying the lmrS gene were resistant to all antibiotic tested in contrast to E. coli cells without the lmrS gene  were incubated in EtBr (2.5 mg/L). Cells were pelleted and washed twice with PBS and fluorescence was monitored for 5 min at 585 nm. After 5 min 1 mM CaCl2 was added and efflux was monitored for an additional 30 min. E. coli cells without LmrS were also measured as control. Significant differences in efflux  were observed between recombinant E. coli cells (DH5α-lmrS) and E. coli cells (DH5α) without the lmrS pump . Recombinant mrS pump . The exp2+ chelator EGTA and the phenothiazine chlorpromazine (CPZ) were also evaluated as described in “Materials and Methods.” The cells were pre-incubated in the Ca2+ chelator, EGTA  suggests that S. aureus cells sense pH environmental conditions through changes in cytosolic Ca2+. These signals are essential for the organism to adapt and survive under wide variety of conditions, in the human host, during infection and colonization and in different environments. pH plays an important role in skin colonization, wound healing and immune cell chemotaxis. Moreover, the phagosome-lysosome environment is highly acidic. S. aureus must have sensor systems in response to the acidic conditions to adjust gene expression for survival (2+ exchangers in prokaryotes utilize Ca2+/H+ and Ca/Na+ gradients to transport Ca2+ into the cell via an electrogenic mechanism (2+ transient (influx) developed at pH 5 (9.5 μM Ca2+) suggests proton competition for translocation of Ca2+ into the cell compared to pH 9 (23 μM Ca2+). These findings are in agreement with those presented by E. coli where a pH of 5 resulted in lowest luminescence. Our Ca2+ efflux results, however, differ slightly to those reported by 2+ decline was fast for the three pHs, at pH 9, Ca2+ levels did not reached basal levels compared to pH 5 and 7. It is interesting to note that pH triggers gene expression in various bacteria  showed different transients. Rapid increases in cytosolic Ca2+ in response to environmental pollutants and antibiotics have also been reported in Saccharomyces cerevisiae  and EDTA, various efflux pump and Ca2+ binding proteins inhibitors such as the phenothiazines CPZ and TFP, which all had a strong inhibitory effect on efflux and accumulation of EtBr. The reversal of efflux inhibition by addition of Ca2+, indicated by the levels of fluorescence, showed the significant role that Ca2+ play in S. aureus efflux systems. These results are consistent with previous studies  Institutional Biosafety Committee and all protocols were done according to the rules and regulations of the Environmental and Safety Office at UTEP. Project # 1104408-4.DD and AN: conceptualization and design. DD: administration and coordination. AN, NM, and AS: experimental work. AN and DD: data analysis and interpretation, writing, and editing. 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 mammals, there are three TRPML proteins, TRPML1, 2, and 3, encoded by MCOLN1-3 genes. Among these channels, TRPML1 is a reactive oxygen species sensor localized on the lysosomal membrane that is able to control intracellular oxidative stress due to the activation of the autophagic process. Moreover, genetic or pharmacological inhibition of the TRPML1 channel stimulates oxidative stress signaling pathways. Experimental data suggest that elevated levels of reactive species play a role in several neurological disorders. There is a need to gain better understanding of the molecular mechanisms behind these neurodegenerative diseases, considering that the main sources of free radicals are mitochondria, that mitochondria/endoplasmic reticulum and lysosomes are coupled, and that growing evidence links neurodegenerative diseases to the gain or loss of function of proteins related to lysosome homeostasis. This review examines the significant roles played by the TRPML1 channel in the alterations of calcium signaling responsible for stress-mediated neurodegenerative disorders and its potential as a new therapeutic target for ameliorating neurodegeneration in our ever-aging population.Mucolipins (TRPML) are endosome/lysosome Ca Neurodegenerative diseases entail progressive destruction and loss of neural cells and impairment of both motor and cognitive functions. They include Parkinson’s disease (PD), Alzheimer’s disease (AD) and Amyotrophic lateral sclerosis (ALS), as well as pathologies caused by lysosomal accumulation, such as Mucolipidosis type IV (MLIV) and Niemann-Pick disease (NPD).All neurodegenerative disorders are marked by the accumulation of abnormally aggregated proteins and mitochondrial dysfunction. Some genes involved in PD or ALS are related to mitochondria, the main source of reactive oxygen species (ROS) in aging cells . In addiSeveral studies demonstrated the importance of maintaining the balance between oxidative stress and the antioxidant system . Under pThese antioxidant defenses are generally classified as enzymatic or non-enzymatic. Among the former are superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, while the latter include glutathione, selenium and vitamins A, E and C .MCOLN1 gene. TRPML1 is permeable to Ca2+, Na+, Fe2+, Mg2+, and K+ (2+ release induces nuclear translocation of transcription factor EB (TFEB)  channels play a central role in the modulation of oxidative stress and lysosome functions, in particular by regulating calcium ion influx and efflux . In the , and K+ , 2009. I, and K+ . It is a, and K+ . It is i, and K+ . Some st, and K+ . TRPML1 , and K+ . Autopha, and K+ . Zhang e, and K+ . In thisB (TFEB) , followeMCOLN2 and MCOLN3 genes. Like TRPML1, they are active in late endosomes/lysosomes; in addition, TRPML2 and TRPML3 are active in early endosomes, and TRPML2 also in recycling  or in presenilin (PS) 1/2, characteristics of familial AD, are associated with aberrant Ca2+ concentrations responsible for apoptosis and excitotoxicity in neurons  is a neurodegenerative disorder characterized by marked cognitive disabilities, ranging from memory loss to synapse disappearance. Pathologic changes occur in the brain such as pyramidal neuron damage, extracellular accumulation of β-amyloid aggregates and neurofibrillary tangles containing hyperphosphorylated Tau protein . A centr neurons . In partganelles . Autophaganelles . Related2+ concentration was reduced. The upregulation of TRPML1 expression is able to strongly attenuate these effects, and thus it is possible that the channel plays an important role in the maintenance of lysosomal homeostasis  cells, used as model of early AD, display elevated lysosomal pH due to vATP-ase deficiency. This alkaline lysosomal pH inhibits the function of the two-pore channel 2 (TPC2) and stimulates an abnormal TRPML1-mediated depletion of lysosomal Ca2+ . Their r2+ inside lysosomes and autophagic compartments. The activation of TRPML1, by its agonists, induces Ca2+ efflux from lysosomes with consequent pH acidification that promotes the clearance of intraneuronal β-amyloid/sphingomyelin deposits , responsible for the increase in LDL flux from the peripheral circulation into the brain, has been described. Moreover, high plasma levels of cholesterol are found to be able to compromise the BBB. Once inside the brain, LDL can enter into endolysosomes and deacidify them, thus blocking their function. This mechanism is responsible for the LDL-induced increases in β-amyloid peptides generation in neurons. It has been demonstrated that the TRPML1 agonist ML-SA1 is able to prevent LDL-induced increases in β-amyloid peptides, while TRPML1 silencing potentiates LDL-induced effects .PTEN-induced kinase 1, Parkin, Protein deglycase-1, and Leucine-rich repeat kinase 2 (LRRK2) genes that regulate mitochondrial and ROS homeostasis (Parkinson’s disease (PD) is characterized by the progressive degeneration of the dopaminergic neurons located in the substantia nigra pars compacta (SNc) . The maieostasis .2+ dynamics are altered when impaired formation of membrane connections between mitochondria and the endoplasmic reticulum (ER) or other components of Ca2+ signaling cause neurodegeneration in SNc neurons, which are already vulnerable due to excessive Ca2+ influx. Indeed, SNc neurons are subjected to an excessive influx of Ca2+ through voltage-gated calcium (Cav1.3) channels  , the accmal Ca2+ . If thisiated PD  probablyMoreover, in a PARK9-mutant PD model, the loss of lysosomal type 5 P-type ATPase function leads to α-synuclein accumulation. Indeed, PARK9 regulates lysosomal exocytosis, a pathway that could be potentiated to reduce α-synuclein accumulation. Tsunemi demonstrated that TRPML1 agonists are able to increase lysosomal exocytosis, thus impairing α-synuclein intracellular levels .Since neuroinflammation seems to be essential for PD pathogenesis , Gao et 2+ flux inside the cell. In particular, lysosomal Ca2+ can be released by intracellular signals, such as NAADP P2 levels are impaired in ALS. In the ALS mouse model, chronic exposure to the neurotoxin L-BMAA impairs autophagy in primary motor neurons, leading to ER stress and cell death  is a neurodegenerative disease that leads to progressive loss of motor neurons in the anterior horn of the spinal cord, with muscle weakness, wasting, and spasticity . It is cas NAADP  and PI is an autosomal recessive lysosomal storage disorder due to utations . Neurodeutations .+ and thus it dissipates high H+ concentration to maintain lysosomal homeostasis under normal condition  substrate proteins for degradation. The intraluminal loop of TRPML1 seems to interact directly with heat shock cognate protein of 70 kDa and heat shock proteins of 40 kDa, members of a molecular chaperone complex required for protein transport into the lysosome during CMA . Of noteTRPML1-knockdown HEK-293 cells  or NPD type C2 (NPC2) genes, responsible for cholesterol transport. This causes an increase in concentration of cholesterol with accumulation of unesterified cholesterol in late endosomes/lysosomes. Accumulation of sphingomyelin and cholesterol affects lysosomal Ca2+ release and blocks endocytosis and fusion between late endosomes and lysosomes, resulting in endocytosis and autophagy dysfunction (Niemann-Pick diseases (NPD) are lipid storage pathologies associated with central nervous system impairment due to lipid accumulation . Three tfunction .2+ efflux through the TRPML1 channel. Therefore, by inhibiting TRPML1 activity, the accumulation of sphingomyelin could influence both lysosomal pH and Ca2+ signaling through ER and mitochondria (2+-activated K+ channels (BK) in lysosomes. The BK channels are activated by TRPML1-mediated Ca2+ release to maintain the negative membrane potential needed for sustained lysosomal Ca2+ release (2+ accumulation, defective lysosomal membrane trafficking, and lysosome storage. Furthermore, upregulation of TRPML1 or BK reverses the impaired lysosome Ca2+ release and membrane trafficking in NPC1 fibroblasts. Moreover, in NPC1 or NPC2 KO HeLa cells, cholesterol accumulates in late endosomes, and the treatment with 2-hydroxypropyl-ß-cyclodextrin reduces cholesterol content (Sphingomyelin is able to inhibit Cachondria . Moreove release . Either  content . Here TR2+, and that these processes in turn sensitize cells to pro-apoptotic signals. In this regard, several reports suggest that lysosomal Ca2+ impairment is involved in the pathogenesis of neurodegenerative diseases. For this reason, the calcium channels expressed on lysosomes have been attracting a lot of attention lately, especially as potential new targets for fighting neurodegeneration. It is now well known that TPCs and TRPMLs are the two main calcium permeable receptor families expressed on lysosomes. However, the pharmacology of these receptors has not yet been well elucidated and still requires further studies. In addition, new findings are necessary to clarify if the Ca2+ efflux from lysosomes is helpful or not. In fact, contradictory data are present in the scientific literature. According to some researchers, the accumulation of calcium in lysosomes seems to be associated with lysosomal dysfunctions in neurodegenerative diseases (2+ mobilization is beneficial in some experimental models of neurodegenerative diseases. It is well established that the NAADP-induced lysosomal Ca2+ efflux is dependent on TPCs (MCOLN1–/– fibroblast model, NAADP action is abolished, an observation that suggests that NAADP-TRPML1 signaling plays a significant role (Neurodegenerative diseases are serious health problems. Numerous efforts have been made to identify neuropathological, biochemical and genetic biomarkers for them. Mitochondrial function and resistance to oxidative stress are compromised during the aging phase, and this is a starting point for the onset of neurodegenerative diseases . Other fdiseases . Others, on TPCs . However on TPCs . In factant role .2+ concentrations in each cellular compartment is essential for the maintenance of normal cellular functions and for neuronal plasticity (2+ buffering is controlled by the interplay between ER, mitochondria and lysosomes that express Ca2+ transport mechanisms such as TRPML channels. Moreover, Ca2+ mobilization is regulated by several cation channels expressed in the plasma membrane involved in the cation exchange with the microenvironment. It is definitively clear that the imbalance of Ca2+ concentrations is strongly involved in the pathogenesis of neurodegenerative diseases, as in these pathologies there is often an evident defect in intracellular calcium storage. In fact, in many different experimental models of neurodegeneration, Ca2+ mobilization from organelles to cytoplasm or vice versa is impaired. However, it is still difficult to clarify whether Ca2+ plays the same role in the different neuronal disorders, especially because it functions as a messenger in an intricate network regulated by the ER/mitochondria/lysosome axis involving both pro-survival and death pathways (In neurons, the regulation of Caasticity . Ca2+ bupathways . TherefoAs shown in this review, calcium imbalance, lysosomes and oxidative stress, as well as the function of TRPML1 seem to be highly significant in the neurodegenerative diseases described. Unfortunately, to date little data is available linking TRPML channels and neurodegeneration, and more studies are needed in order to clarify the role of these channels. In conclusion, a deeper understanding of the exact mechanism of neurodegeneration will offer a valid starting point for the development of new therapeutic strategies, and in this regard TRPML1 is turning out to be a key candidate.GS and MM contributed to the conception and design. MM, FM, and CA drafted the manuscript. OM, MN, and GS contributed to the critical revision of the manuscript. MM and CA re-examined and revised 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.


Acute myeloid leukemia (AML) is an aggressive blood cancer resulting in accumulation of immature, dysfunctional blood cells in the bone marrow. Changes in cell metabolism are features of many cancers, including AML and this may be exploited as a therapeutic target. In this study we investigated the in vitro antileukemic effects of seven metabolic inhibitors that target different metabolic pathways. The metabolic inhibitors were tested on AML cells derived from 81 patients using proliferation and viability assays; we also compared global gene expression and proteomic profiles for various patient subsets. Metformin, 2DG, 6AN, BPTES and ST1326 had strong antiproliferative and proapoptotic effects for most patients, whereas lonidamine and AZD3965 had an effect only for a minority. Antiproliferative effects on AML cells were additive when combined with the chemotherapeutic agent AraC. Using unsupervised hierarchical clustering, we identified a strong antiproliferative effect on AML cells after treatment with metabolic inhibitors for a subset of 29 patients. Gene expression and proteomic studies suggested that this subset was characterized by altered metabolic and transcriptional regulation. In addition, the Bcl-2 inhibitor venetoclax, in combination with 2DG or 6AN, increased the antiproliferative effects of these metabolic inhibitors on AML cells. Therapeutic targeting of cellular metabolism may have potential in AML, but the optimal strategy will likely differ between patients. Acute myeloid leukemia (AML) is the most common leukemia in adults. It is an aggressive hematologic malignancy characterized by accumulation and enhanced cell proliferation of immature myeloid precursors in the bone marrow, leading to marrow failure . AML is Changes in cellular metabolism have been described as hallmarks of cancer and metabolic targeting is considered as a possible therapeutic strategy in various cancers, including AML . Cancer d-Glucose (2DG), lonidamine and AZD3965 which can inhibit various steps in or influencing glycolysis; bis-2- ethyl sulfide (BPTES) that inhibits glutaminolysis; 6-aminonicotinamide (6AN) that inhibits the pentose phosphate pathway and ST1326 that inhibits fatty acid metabolism , and included >90% AML cells and contaminating cells were mainly small lymphocytes -thymidine incorporation assay -thymidine  was added and cultures were incubated for an additional 22 hours before nuclear incorporation was determined by liquid scintillation counting. Proliferation (uptake of thymidine) was estimated by comparing values in treated cultures with untreated control cultures  and detectable proliferation was defined as at least 1000 counts per minute (cpm). The median of triplicate cultures was used in all calculations.Proliferation was analyzed using a -thymidine incorporation assay after three days of coculture, as described in detail previously -thymidine incorporation assay was used for the initial dose-response studies, that is we evaluated the ability of AML cells to survive seven days and still be able to proliferate after inhibitor treatment. Six concentrations of each inhibitor were initially tested . For these patients with detectable cell proliferation, inhibitor treatment resulted in a highly significant antiproliferative effect compared to untreated controls , for all inhibitors except lonidamine and AZD3965. The latter two inhibitors did not show any significant inhibitory effects on proliferation, though proliferation was decreased after inhibitor treatment for certain patients (FLT3 or NPM1 mutations or occurrence of previous disease (versus de novo AML). A few associations of borderline significance were detected  whereas the others died from resistant/relapsed AML. The antiproliferative effects observed with the metabolic inhibitors did not differ between patients when comparing long-term survivors and patients dying from resistant leukemia .p-value < 0.05, Kruskal Wallis test, Dunn’s post-hoc test).The antiproliferative effects of metabolic inhibitors were also tested alone or in combination with AraC 0.0125 µM . The following concentrations of metabolic inhibitors were then used, metformin 1.25 mM and 2.5 mM, 2DG 0.3 and 0.6 mM, 6AN 50 and 100 µM, BPTES 10 and 20 µM, ST1326 50µM, lonidamine 300 µM and AZD3965 200 nM. Metabolic inhibitors combined with AraC had significantly greater antiproliferative effects compared to AraC alone, when testing metformin 2.5 mM, 2DG 0.6 mM, 6AN 50 and 100 µM and ST1326 50 µM (de novo), AML cell differentiation , karyotype, FLT3-ITD nor NPM1 mutations, age, gender or survival of the younger patients receiving intensive therapy , AML cell differentiation , karyotype, FLT3-ITD, NPM1 mutations or survival of the younger patients receiving intensive therapy  inhibitors that can affect glucose metabolism  A and iiB, did noAdditional molecular genetic analyses were available for 35 patients  and are summarized in Finally, we performed an unsupervised hierarchical cluster analysis based on the effect of inhibitors on cell proliferation after values were normalized to their median values, when all metabolic inhibitors and all concentrations were included. Based on this analysis, the patients could be divided into three patient clusters , showingp < 0.05) and thirty-one of these also had a significant fold change (p < 0.05)  . Among t   . The highest concentrations of inhibitors that were tested in the proliferation assay, were also tested in the apoptosis flow cytometric assay, that is metformin 2.5 mM, 2DG 0.6 mM, 6AN 100 µM, BPTES 20 µM, ST1326 50 µM, lonidamine 300 µM and AZD3965 200 nM . The gat < 0.05) A. The pe < 0.05) B.To further study the patient heterogeneity, we did a hierarchical cluster analysis based on AML cell viability after treatment with or without metabolic inhibitors A, where p < 0.0005) and 6AN (p = 0.013) compared to the other patient cluster (de novo), cytogenetics, AML cell differentiation , karyotype, FLT3-ITD or NPM1 mutations. Finally, we could not detect any correlations between the antiproliferative and proapoptotic effects of individual inhibitors on patient samples except for 2DG, which showed a highly significant though weak correlation .We next did an unsupervised hierarchical cluster analysis based on the relative effect of the metabolic inhibitors on cell viability, that is percent viability of cell cultures treated with metabolic inhibitors compared to control cultures  and the clustering analysis then revealed two clusters B. An upp cluster . FurtherWe analyzed the concentration of 19 soluble mediators in culture supernatants collected after 48 hours in vitro culture of AML cells derived from 72 patients. All patient samples analyzed had more than 5% viable cells in the control cultures after 48 hours of incubation (see above). The release profiles varied considerably between patients, however based on these profiles, patients could be clustered into three main subsets using hierarchical cluster analysis . Still, U-test, p = 0.0016). The constitutive release of soluble mediators was also measured in cocultures with and without 2DG treatment. Only minor changes in mediator concentrations were found in 2DG-treated cocultures compared to control cocultures  and overall the mediator release profile seemed to remain similar in the absence and presence of the glycolytic inhibitor 2DG  in untreated control cultures. Treatment with metabolic inhibitors had a similar antiproliferative effect on UCB cells as seen for primary AML cells .The effects of metabolic inhibitors on the proliferation of human UCB-derived mononuclear cells were evaluated using the -thymidine incorporation assay to evaluate cell proliferation after treatment with metabolic inhibitors [3H]-thymidine is then added after six days of culture and nuclear incorporation is assayed one day later, that is we investigated the AML cell subset that is able to survive and still proliferate after six/seven days of culture. We selected the concentrations in our studies based on (i) concentration-response curves, (ii) no effect of the DMSO concentration (≤ 0.2%) used for dissolving inhibitors; (iii) previous experimental studies and (iv) clinical tolerated levels for those inhibitors that have so far been used in patient treatment. Metformin, 2DG, 6AN, BPTES and ST1326 all showed significant antiproliferative and proapoptotic effects, while lonidamine and AZD3965 only showed effects for a minority of patients  or long-term AML-free survival.Patients with cytogenetically normal AML cells  have been reported to have a distinct glucose metabolism  and the Metabolic plasticity allows cancer cells, including leukemia cells, to reprogram their metabolism to survive during changing environments. This highly diverse and flexible metabolism of AML cells most likely not only contributes to the aggressiveness of the disease and the high percentage of refractory disease, but it can also make metabolic targeting challenging since blocking one metabolic pathway can simply make the cells shift to use another pathway to gain energy. Simultaneously targeting multiple metabolic pathways may therefore be an approach to remove their ability to survive under stressful conditions and render them vulnerable to anti-cancer treatment. Our hierarchical cluster analyses identified a patient subset with high susceptibility to five metabolic inhibitors with overall significant effects (The two patient subsets identified in our hierarchical cluster analysis based upon the antiproliferative effects after treatment with five metabolic inhibitors B, did noTreatment with metabolic inhibitors decreased AML cell viability and had proapoptotic effects and we could identify a subset of patients with increased susceptibility to the inhibitors (MSCs have a cytokine-mediated supporting effect on AML cell growth and survival during in vitro culture  and MSCsRecent studies suggest that AML cells have considerably metabolic plasticity  and our Venetoclax, a Bcl-2 inhibitor, is now used especially in the treatment of human AML for elderly and unfit patients in combination with either a hypomethylating agent or low-dose cytarabine ,51,52. ASeveral previous studies have shown that metabolic targeting of AML cells can increase their sensitivity against other antileukemic agents. Firstly, targeting of hexokinase can result in increased sensitivity toward conventional antileukemic treatment, including both cytarabine and anthracyclines ,54. GlucThe metabolic status of AML cells can also be influenced by the amount of nutrients and metabolites in their microenvironment ,10,46,60Some of the metabolic inhibitors in our present studies could also reduce the proliferation of normal UCB cells, and 2DG also decreased the proliferation of normal bone marrow MSCs. Especially the risk of hematological toxicity will be important when using metabolic inhibitors in AML therapy, because these patients have leukemia-induced bone marrow failure and additional treatment-induced hematological toxicity is common. Five of the agents in our study have been or are included in ongoing registered clinical trials according to the ClinicalTrials or PubMed databases, though, as far as we know, only clinical trials with metformin have included AML patients. With regard to side effects, the most common side effects of metformin are metabolic and gastrointestinal toxicity ,64, whicWe investigated the effects of seven metabolic inhibitors on AML cells derived from 81 patients, which target various metabolic pathways including glycolysis, the pentose phosphate pathway, glutaminolysis and fatty acid oxidation. Several of these inhibitors also indirectly target OXPHOS. The metabolic inhibitors had antiproliferative and proapoptotic effects on leukemia cells for most AML patients. The inhibitor with the strongest effect varied between individual patients, although inhibition of glycolysis had the strongest effect for the majority of patients. Our overall results suggest that several strategies for metabolic inhibition have antileukemic effects, but the treatment should possibly be individualized because the optimal inhibitor seems to vary between patients. Thus, the various strategies for metabolic targeting in AML should be further characterized, especially in patient-derived xenograft models, but also in clinical studies and there should be a focus on patient heterogeneity in these studies. Further studies should also evaluate effects on the leukemic stem cell population that are thought to contribute to relapsed/refractory disease. In our opinion, the clinical studies should include both AML stabilizing studies and combination with chemotherapy and potentially curative treatments.


Acute leukemia (AL) is a group of highly heterogeneous hematological malignancies. Circular RNAs (circRNAs) are covalently closed circRNA molecules implicated in the development of many diseases. However, the role of circRNAs in AL remains largely unknown. Therefore, this study aimed to identify new classification diagnostic biomarkers for subgroups of AL. The circRNA expression signatures discriminating acute lymphoblastic leukemia (ALL) from acute myeloid leukemia (AML) were identified by microarray, followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) validation. Receiver operating characteristic curve analysis was used to evaluate the diagnostic efficiencies of hsa_circ_0001857 and hsa_circ_0012152, and hsa_circ_0012152 was selected for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The results showed that the circRNA expression profiles, hsa_circ_0001857, and hsa_circ_0012152 could clearly discriminate ALL from AML. The target genes of hsa_circ_0012152 might be involved in biological processes, such as myeloid cell differentiation, covalent chromatin modification, histone modification, and rat sarcoma (Ras) protein signal transduction, and participate in pathways such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3′-kinase (PI3K)-Akt signaling pathway. Hsa_circ_0012152 might be involved in the initiation and development of AML through miR-491-5p/epidermal growth factor receptor (EGFR)/MAPK1 or miR-512-3p/EGFR/MAPK1 axis. Our results showed that circRNA expression profiles and specifically expressed circRNAs were promising classification biomarkers to designate AL into ALL or AML. Acute leukemia (AL) is a diverse group of hematological malignancies, characterized by aberrant proliferation and accumulation of malignantly transformed hematopoietic stem cells in the bone marrow and other hematopoietic tissues . Acute lPML-RARα, MLL-AF9, and MLL-AF4, respectively, and might be involved in the initiation and development of AL. Therefore, they were potential biomarkers and therapeutic targets for acute leukemia. CircMYBL2 (FLT3-ITD) mutations, could enhance the translational efficiency of FLT3 kinase by recruiting PTBP1 to promote the development and confer drug resistance of FLT3-ITD-positive AML. Circ-RPL15  are a class of covalently closed single-stranded RNA molecules whose 3′ and 5′ ends are linked by a back-splice event . Some ofircMYBL2 , which wrc-RPL15 , which wIn this study, the circRNA expression profiles of AML, ALL, and healthy individuals were found to be markedly different. Hsa_circ_0012152 and hsa_circ_0001857, upregulated in AML and ALL, respectively, could clearly discriminate ALL from AML, and their combination could improve the diagnostic efficiency. Hsa_circ_0012152 might be involved in the progression of AML through miR-491-5p/epidermal growth factor receptor (EGFR)/MAPK1 or miR-512-3p/EGFR/MAPK1 axis.Bone marrow (BM) samples from 49 newly diagnosed patients with AML, 43 newly diagnosed patients with ALL, and 8 healthy individuals were used in this study. Five AML samples, three ALL samples, and three healthy individual samples were used for the microarray analysis of circRNA expression profiles, and the other 89 samples were used for RT-qPCR amplification assay. Mononuclear cells from BM aspirates were separated by Ficoll–Paque Plus  density gradient centrifugation. Written informed consents were obtained from all participants involved in this study before using the clinical samples for research purpose. The protocol of this study was approved by the ethics committee of the Ningbo Hospital of Zhejiang University and carried out in accordance with the Declaration of Helsinki. The diagnoses of AML and ALL were made in accordance with the 2016 World Health Organization Classification of Tumors of Haematopoietic and Lymphoid Tissues . The sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols by Shanghai KangCheng Bio-tech Co. Ltd. Total RNAs were digested with RNase R  to remove linear RNAs and enrich circRNAs. Then, enriched circRNAs were amplified and transcribed into fluorescent complementary RNA (cRNA) by a random priming method . The labeled cRNAs were hybridized onto an Arraystar Human circRNA Array V2 . After washing the slides, the arrays were scanned using an Agilent Scanner G2505C.2FC| ≥ 2 and false discovery rate (FDR) <0.05 filtering. Differentially expressed circRNAs with statistical significance were presented with a heatmap and volcano plot using heatmap R package and ggplot R package. Hierarchical cluster analysis  was used to analyze acquired circRNA array images. Quantile normalization and differentially expressed circRNAs were performed by R software . Differanalysis  was perf–ΔΔCT) method was used to analyze the gene expression level, and three technological replicates were used to ensure the reliability of the analysis. The RT-qPCR reaction system with 20 μl of volume consisted of 1 μl of cDNA, 1.6 μl of primer, 10 μl of SYBR Green, 0.4 μl of ROX, and 7 μl of RNase-free H2O. The amplification of RT-qPCR reaction was carried out at 95°C for 1 min, followed by 40 cycles at 95°C for 15 s, 64°C for 30 s, and 72°C for 32 s to record fluorescence, and finally followed by the melting program at 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, and 60°C for 15 s. The Primer sequences are shown in Total RNA was extracted with TRIzol reagent  following the manufacturer’s protocols. The purity and concentration of RNA samples were detected with a NanoDrop ND-1000 spectrophotometer . The cDNAs were synthesized using a PrimeScript RT Master Mix  following the manufacturer’s protocol. The RT-qPCR was performed in the Stepone Real-Time PCR System  using an SYBR Green PCR kit . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as the endogenous control. A comparative cycle threshold (21. A protein–protein interaction (PPI) network was established using the STRING database2 and presented as a graph using Cytoscape software (Version 3.6.1). Hub proteins were the proteins having an extremely high connection with other proteins in the PPI network and were vital in many biological processes  is a gene function classification system widely used in the field of bioinformatics. It simply annotates genes from the cellular component, molecular function, and biological process. Kyoto Encyclopedia of Genes and Genome (KEGG)P value. Analysis of variance, Student t test, or Kruskal–Wallis H tests were used as appropriate to determine the differences between groups. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic efficiencies of different circRNAs. Youden’s index was used to identify the best cutoff value and calculate the specificity, sensitivity, positive predictive value, and negative predictive value. The diagnostic efficiency for the combination of biomarkers was assessed using serial test, parallel test, or calculating weight coefficients for every biomarker, obtaining the largest possible AUC in ROC analysis. Weight coefficients were valuated using a multivariable-adjusted logistic regression model. A P < 0.05 was considered statistically significant.All data were presented as the median or mean ± standard error of the mean (SEM) and analyzed using SPSS 23.0 software or GraphPad Prism 7.0 software with a two-tailed 2FC| > 2 and FDR < 0.05, including 25 upregulated circRNAs and 11 downregulated circRNAs in ALL compared with AML  or healthy individuals , and hsa_circ_0012152 was markedly upregulated in AML compared with ALL  or healthy individuals . These results indicated that hsa_circ_0001857 and hsa_circ_0012152 were potential classification biomarkers to discriminate ALL from AML due to their differential expression patterns between ALL and AML. The association between hsa_circ_0012152 and hsa_circ_0001857 with several clinical features is shown in AML1-ETO, PML-RARα, FLT3-ITD, NPM, and other known driver genes. The expression of hsa_circ_0001857 in ALL was associated with age, and younger patients had lower expression than older patients. However, the expression of hsa_circ_0001857 was independent of sex or known mutations or fusion genes such as IKZF1 and BCR-ABL. These suggested that hsa_circ_0012152 and hsa_circ_0001857 functioned independently of these commonly known driver genes.The expression levels of five circRNAs  that were chosen from the top 10 differentially expressed circRNAs between ALL and AML were quantified by RT-qPCR using the same cohort of patients as the circRNA microarray to validate the results of circRNA microarray and identify potential biomarkers to distinguish ALL from AML . Their eP = 0.0005; P = 0.0896, P < 0.0001), a sensitivity of 0.775, and a specificity of 0.886 (P < 0.0001), a sensitivity of 0.875, and a specificity of 0.909 , the sensitivity increased to 0.925, and the specificity (0.909) reached the optimal level when hsa_circ_0012152 and hsa_circ_0001857 were diagnosed alone. These results indicated that hsa_circ_0012152 and hsa_circ_0001857 were promising potential classification biomarkers to distinguish ALL from AML, and their combination could better identify ALL from AML, although they showed an excellent performance in distinguishing ALL for AML when they were alone. Clinicians expect a clear separation of ALL from AML in the management of AL. That is, the biomarker was expected to have an excellent performance in specificity. Hence, the serial test and logistic regression predictive model might be a better choice.Serial and parallel tests were used to improve the efficiency of classification diagnosis to discriminate ALL from AML . The speTP53, MAPK1, PPP2R1A, CREBBP, CUL1, HIST2H2BE, POLR2C, PIK3R1, H2AFV, and EGFR . Although hsa_circ_0001857 was differentially expressed between patients with ALL and healthy individuals, it could not discriminate ALL from healthy individuals , which might be due to the small sample size. Hsa_circ_0012152 was upregulated in patients with AML compared with ALL or healthy individuals, could discriminate patients with AML from healthy individuals , and was a promising potential classification biomarker to discriminate ALL from AML . As early as in 2107, Li et al. (Hence, the initial purpose of this study was to identify new classification biomarkers to distinguish ALL from AML and explore the potential mechanism of circRNAs in AL. The results indicated that circRNA expression profiles could clearly distinguish ALL from AML. The hsa_circ_0001857 was upregulated in patients with ALL compared with AML or healthy individuals and was a promising classification biomarker to discriminate ALL from AML (AUC = 0.9091, i et al.  found thi et al. , circPANi et al. , and ciri et al. , were foi et al. . Even thi et al. . The lnci et al. . SimilarMAPK activation could inhibit the progression of AML cell cycle and promote the sensitivity of AML cells to chemotherapy (A possible mechanism for the different expression patterns of hsa_circ_0001857 and hsa_circ_0012152 between ALL and AML might be that they participated in the leukemogenesis of ALL and AML, respectively. For example, circ-ANAPC7 participated in the occurrence and development of AML through the miR-181 family , circPANotherapy . The EGFotherapy , 55. Mahotherapy  found thThis study showed that hsa_circ_0012152, which was upregulated in AML, was a promising classification biomarker to discriminate ALL from AML and might participate in the progression of AML through the miR-491-5p/EGFR/MAPK1 axis or the miR-512-3p/EGFR/MAPK1 axis. Hsa_circ_0001857, which was upregulated in ALL, could serve as a classification biomarker to discriminate ALL from AML. The diagnostic efficiency would be improved by combining hsa_circ_0012152 and hsa_circ_0001857 for the serial test and logistic regression predictive model. The study provided an additional tool to improve the clinical management of AML, including diagnosis, classification, and targeted therapy. However, the results need to be verified using a larger sample size. In addition, the relationship between hsa_circ_0012152 or hsa_circ_0001857 and gene mutation, prognosis, and chemoresistance, as well as their mechanism of action in AL, remains to be further explored.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ningbo First Hospital Ethics Committee. The patients/participants provided their written informed consent to participate in this study.HH, GO, and QM were the principal investigators. SG and BL performed most of the experiments. YC, YZ, and NW performed part of the experiments. DZ and SY performed data analysis. SG wrote the manuscript. LS, HH, GO, and QM revised the manuscript. 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.


During Ca2+ transients, Ca2+-binding to troponin C shifts the “on–off” equilibrium of the thin filament state toward the “on” sate, promoting actomyosin interaction. Likewise, recent studies have revealed that the thin filament state is under the influence of temperature; viz., an increase in temperature increases active force production. In this short review, we discuss the effects of temperature on the contractile performance of mammalian striated muscle at/around body temperature, focusing especially on the temperature-dependent shift of the “on–off” equilibrium of the thin filament state.In skeletal and cardiac muscles, contraction is triggered by an increase in the intracellular Ca Under physiological conditions, striated muscle generates force and heat. Skeletal muscle plays a critical role in maintaining body temperature which increases during exercise. Human body temperature is maintained at ∼37 ± 1°C throughout the day . In huma2+ from the sarcoplasmic reticulum (SR) via ryanodine receptors; however, in cardiac muscle, it is the Ca2+ entry from the extracellular fluid through voltage-dependent L-type Ca2+ channels that triggers the Ca2+ release, a mechanism known as Ca2+-induced Ca2+ release (2+concentration ([Ca2+]i) promotes Ca2+ binding to troponin C (TnC) on thin filaments (2+]i is maintained relatively low (∼10–6 M), even at the peak of systole  and tropomyosin (Tm), which form a complex on actin filaments. At rest, the Tn–Tm complex prevents/weakens actomyosin interaction. At this “off” state, the carboxyl-terminal domain of TnI strongly binds to actin, and Tm blocks myosin binding to actin and/or force production of bound myosin. When [Ca2+]i is increased, Ca2+-bound TnC interacts with TnI, and the carboxyl-terminal domain of TnI is dissociated from actin. The Tn conformational changes result in displacement of Tm on actin, which subsequently induces myosin binding to actin and force generation   is widely used to express Ca2+ sensitivity; an increase in the pCa50 value indicates an increase in Ca2+ sensitivity and vice versa. Ca2+ sensitivity is influenced by various factors, such as the intracellular concentrations of Mg2+   : see eceptors , causingnotropy)   , hence, 2+ transients is likely to underlie the inhibited active force production.In contrast, an increase in temperature to ∼40–42°C has been reported to decrease end-systolic pressure in canine  and rat Physiologists have realized for nearly a century that despite being under resting conditions, the warming of muscles increases active force, known as “heat contraction” or “heat rigor”. For instance, 2+-independent manner, and instead, it is regulated at the sarcomere level. Indeed, intracellular Ca2+ imaging with fluo-4 revealed little or no increase in [Ca2+]i upon infrared laser irradiation, and heating-induced contraction was blocked by the myosin II inhibitor blebbistatin. A similar phenomenon was observed in C2C12 myotubes (from mouse) when temperature was increased from 36.5 to 41.5°C using gold nanoshells in combination with near-infrared laser irradiation  induces transient and reversible shortening in isolated intact rat ventricular myocytes . In thisadiation . These s fashion .2+ sensitivity is increased with increasing temperature above 37°C   see , and intat ∼50°C .in vitro motility assay, recent studies confirmed heating-induced activation of thin filaments by measuring the sliding velocity of reconstituted thin filaments. 2+ (+EGTA). We performed a rapid-heating experiment using infrared laser irradiation and found that thin filaments that had been reconstituted with bovine cTn and human Tm exhibited sliding movements at >∼35°C in the absence of Ca2+  and Tm, modulate CaM or HCM . A generutations . Becauset manner , local hIt is worthwhile noting that in previous studies discussed, thus far, different mammals were used that have different body temperatures cf. , thus, f2+]i. The Ca2+-dependent structural changes of thin filaments allow for myosin binding to actin and thereby facilitate active force production. Cooling increases the contractility of striated muscle via Ca2+-dependent activation: first, a rapid decrease in temperature triggers a release of Ca2+ from the SR, and second, long-term cooling increases the amplitude as well as the period of intracellular Ca2+ transients. Contrary to these cooling effects, heating increases myofibrillar active force (and ATPase activity) and Ca2+ sensitivity; the latter is coupled with an increase in the affinity of TnC for Ca2+. Moreover, heating induces structural changes of thin filaments , thereby shifting the “on–off” equilibrium of the thin filament state toward the “on” state at a given [Ca2+]i (Ca2+-independent activation). The characteristics of heating-induced, Ca2+-independent activation may be useful to augment the heart’s contractility in patients with DCM in future clinical settings.In striated muscle, sarcolemmal depolarization causes an increase in [CaSIs, KO, and NF wrote the first version of the manuscript. SS, FK-S, and S’Is contributed comments and suggestions. All authors approved the final version of 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.