In contrast to younger rats (6‐month of age), older rats (24‐month of age) exhibited severe cardiac ultrastructural defects, including deformed, fragmented mitochondria with high electron densities. Cardiomyocytes isolated from aged rats demonstrated increased reactive oxygen species (ROS), loss of mitochondrial membrane potential and altered mitochondrial dynamics, compared with younger controls. Moreover, mitochondrial defects were accompanied by mitochondrial and cytosolic Ca2+ ([Ca2+]i) overload, indicative of disrupted cellular Ca2+‐homeostasis. Interestingly, increased [Ca2+]i coincided with decreased phosphorylation of phospholamban (PLB) and contractility. Aged‐cardiomyocytes also displayed high Na+/Ca2+‐exchanger (NCX) activity and blood glucose levels compared with young‐controls. Interestingly, the protein level of SGLT2 was dramatically increased in the aged cardiomyocytes. Moreover, SGLT2 inhibition was sufficient to restore age‐associated defects in [Ca2+]i‐homeostasis, PLB phosphorylation, NCX activity and mitochondrial Ca2+‐loading. Hence, the present data suggest that deregulated SGLT2 during ageing disrupts mitochondrial function and cardiac contractility through a mechanism that impinges upon [Ca2+]i‐homeostasis. Our studies support the notion that interventions that modulate SGLT2‐activity can provide benefits in maintaining [Ca2+]i and cardiac function with advanced age.The prevalence of death from cardiovascular disease is significantly higher in elderly populations; the underlying factors that contribute to the age‐associated decline in cardiac performance are poorly understood. Herein, we identify the involvement of sodium/glucose co‐transporter gene (SGLT2) in disrupted cellular Ca Notably, the prevalence of death from cardiovascular disease is significantly higher in elderly populations.2+ transport and alteration in action potential duration.Mitochondrial function is essential for oxidative metabolism and ATP synthesis for maintaining tissue homeostasis and cell survival. Also, the mitochondrion serves as a platform for calcium regulation and programmed cell death by apoptosis and necrosis.+/H+ exchange‐1, (NHE‐1).+ and Ca2+ levels are early drivers of mitochondrial dysfunction, ROS production and cardiovascular morbidity and mortality.+ is an underlying cause of cardiac injury and metabolic dysfunction in diabetic cardiomyopathy, presumably from elevated SGLT2 activity.+ levels in cardiomyocytes of diabetic animals.2+ ([Ca2+]i), we explored the impact of SGLT2 on mitochondrial Ca2+ homeostasis in young and aged cardiac myocytes. Our data provide new important information that SGLT2 disrupts mitochondrial/SR Ca2+‐homeostasis and thereby promotes cardiac dysfunction by impairing mitochondrial energy metabolism.Notably, we recently demonstrated that sodium/glucose co‐transporter 2 (SGLT2), which is a member of the solute carrier family (SLC5A2) and is important in facilitating sodium‐dependent glucose‐transport and in the regulation of blood glucose levels, was significantly activated in the hearts of insulin‐resistant animals with metabolic syndrome (MetS). Furthermore, we demonstrated that pharmacological inhibition of SGLT2 improved glucose tolerance and cardiac function.22.1Our experimental protocols and all animal procedures are in coincidence with the guidelines of the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and are approved by Ankara University (reference # 2016‐18‐165).Wistar male rats were grouped as 24‐month‐old (Aged group) vs. 6‐month‐old (Young group) and parameters such as blood glucose levels were analysed as described previously.2.2Cardiomyocytes isolation was performed from the left ventricles of hearts, as described previously.2.3Mit), isolated cardiomyocytes were incubated with 2.5‐µmol/L MitoSOX Red for 30‐minutes at room temperature and cells were washed twice in PBS. Fluorescent intensity was measured by flow cytometry, and data were plotted as dot plots.To determine the mitochondrial superoxide level ([SOX]2+ level ([Ca2+]Mit) was measured by incubating cardiomyocytes with 4‐µmol/L Fluo4‐AM Ca2+ dye for 45‐minutes at room temperature in extracellular solution contains (in mmol/L) 156 NaCl, 3 KCl, 2 MgSO4, 1.25 KH2PO4, 10 D‐glucose, 2 CaCl2 and 10 HEPES, at pH 7.35. Cardiomyocytes are washed with PBS twice and incubated with intracellular solution contains (in mmol/L); NaCl 6, KCl 130, MgCl2 7.8, KH2PO4 1, CaCl2 0.4, EGTA 2, EDTA 10, malate 2, glutamate 2, ADP 2 and HEPES 20, at pH 7.1 solution including digitonin (25 mg/mL) and thapsigargin (10‐µmol/L) for 10‐minutes at room temperature. Real‐time measurement of the fluorescence emission from each sample is reported as dot plots measuring the percentage of stained cells with Fluo‐4AM fluorescence.The mitochondrial Ca2+ level but not in the mitochondrial intermembrane space, all experiments were performed in cells under same experimental conditions  through the determination of mitochondrial Ca2+ level and mitochondrial membrane potential (MMP), simultaneously by using confocal microscopy  levels  were measured in resting cardiomyocytes loaded with ion‐specific fluorescence dyes . Fluorescence values were recorded by the use of a ratio‐metric micro‐spectrofluorometer  for Ca2+ or a laser scanning microscope  for Na+, as described previously.The basal levels of intracellular free Ca2+‐changes (Ca2+‐transients) in cardiomyocytes , an electric‐field stimulation  was applied. We took into consideration the potential influences of a temperature‐dependent effect on lowered amplitude of intracellular Ca2+‐changes without changes in the time course of measurements,2+‐sensitive fluorescent probes,To determine the intracellular transient Ca2.6+‐channel currents, IK) or current‐clamp mode  to measure either ionic fluxes or membrane voltage changes, respectively, as described previously.Electrophysiological parameters of cardiomyocytes were recorded by using the Axoclamp patch‐clamp amplifier  at room temperature (23 ± 2°C). Cardiomyocytes were sampled and digitized at 5 kHz using an analog‐to‐digital converter and a software . The amplifier has been used for either voltage‐clamp mode , the resting membrane potential, and the maximum amplitude of action potentials were calculated from original records (at least 15‐20 records/cell).Action potential duration from the repolarization phase at 25, 50, 75, 90% (APD+/K+‐pump (NaK) current (INaK) was measured in freshly isolated left ventricular cardiomyocytes, as described previously.+ solution: NaCl 136, BaCl2 2, MgCl2 1, glucose 10, HEPES 5, NiCl2 5 ve TrisCl 4 (mmol/L) (at pH = 7.4). To activate NaK, cardiomyocytes were reperfused with normal 4 K+ solution: . The holding potential was −30 mV during the experiment.The Na+/Ca2+‐exchanger (NCX) current was measured as described elsewhere.NCX, was obtained from protocol composed of descending ramp from +80 to −120 mV at 0.1 mV/ms while holding potential was −40 mV. The external solution consisted of NaCl 130, TEA‐Cl 10, Na‐Hepes 11.8, MgCl2 0.5, CaCl2 1.8, ryanodine 0.005, nifedipine 0.02, glucose 10; pH 7.4 and the pipette solution CsCl 65, CaCl2 10.92, EGTA 20, HEPES 10, MgATP 5, MgCl2 0.5, TEA‐Cl 20 and at pH 7.2.The Na2.7The cellular level of ATP in isolated left ventricular cardiomyocytes was measured using a colorimetric ATP assay kit  with some modification, as described previously.2.8Electron microscopy was performed in cardiomyocytes, as described previously.To quantify the results, the mitochondrial aspect ratio as the ratio of length to width was calculated by using Image SP, the randomly selected five longitudinally arranged micrographs, as described previously.2.916 , phospholamban , SGLT2 . β‐actin  and GAPDH  were used as housekeeping proteins to detect equal protein loading. Immunoreactive bands were detected by a chemiluminescent reaction . The densities of the bands are analysed using ImageJ software, and the results were presented as fold changes.The protein samples were prepared from the cells and tissues as previously described.2.101. The fold changes in the genes were analysed based on the comparative (2−ΔΔCt) method. Primer sequence for SGLT2 is TCATTGCCGCGTATTTCCTG (forward) and AACACCACAAAGAGCGCATT (reverse).The mRNA levels were measured as described previously2.11P < 0.05 significance level.Chemicals were obtained from Sigma‐Aldrich  unless otherwise stated. Data were collected from different myocyte isolations. Data are presented as Mean ± SEM of at least three independent observations for Western blotting with GraphPad Prism 6.0 . To provide better presentation and distribution of Western blot data in terms of median and interquartile range, we used a box and whisker graph. Comparisons between quantitative variables were assessed by the unpaired two‐sided Student's t test at the 33.1To gain insight into the effects of ageing on cardiac dysfunction, we first determined whether the aged hearts (24 months) are hypertrophic and different from younger animals (6 months). As shown in Figure Also, we examined cardiac morphology by electron microscopy to examine ultrastructure and subcellular changes in the myocardium with ageing. Electron microscopy analysis revealed that in contrast to young animals, which displayed normal sarcomeric architecture and organized elongated mitochondria, severe ultrastructural defects characterized by damaged myofibrils, disorganized and fragmented mitochondria accompanied by a dilatation of SR T‐tubules were observed in the aged hearts, Figure Further, a significant decrease in mitochondrial aspect ratio was observed in the aged hearts, indicative of increased mitochondrial fragmentation, Figure 3.2As we observed a decline in mitochondrial ATP in aged animals, suggesting that mitochondrial perturbations may be associated with contractile dysfunction. We used mitochondria targeting antioxidant (MitoTEMPO) that we have previously shown to reduce ROS level in the ventricular cardiomyocytes,As our previous studies demonstrated a role for mitochondrial redox signalling (via increases in ROS level) in the aetiology of ageing‐associated organ dysfunction,Taking into consideration the essential processes of mitochondria fidelity such as mitochondrial dynamism (fusion and fission), we determined the expression levels of the mitochondrial fusion and fission proteins in the aged cardiomyocytes compared with young hearts in the absence and presence of MitoTEMPO. As shown by Western blot analysis in Figure 3.32+ level ([Ca2+]Mit) has been identified as a major activator of mitochondrial permeability transition pore (mPTP) opening.2+]i is considered to be a major determinant of mPTP opening. As ageing alters [Ca2+]i handling in cardiomyocytes,2+, [Ca2+]Mit is elevated in the aged cardiomyocytes. As shown in Figure 2+]Mit was increased by 50% in aged cardiomyocytes compared with those of young cardiomyocytes. To verify the detrimental effect of ageing on depressed contractile activity, we mimicked the action potentials (APs) of age with electrical‐field stimulation of cardiomyocytes and determined the transient changes of [Ca2+]i  in Fura‐2AM loaded aged cardiomyocytes. As shown in Figure 340/380) of [Ca2+]i transients were significantly smaller in the aged group compared with the younger group  could be altered with ageing, we performed further experiments using caffeine to assess SR Ca2+ extrusion. The responses to acute caffeine (10‐mmol/L) were significantly lower in the aged group compared with the corresponding young group , which modulates the SR Ca2+‐ATPase (SERCA) function. As shown in Figure To test whether the i) and H+ ([H+]i), measured as SNARF‐1 intensity changes, in the aged cardiomyocytes, Figure Next, we tested the resting levels of cytosolic CaWe previously have shown in a rat model of insulin‐resistance (MetS) that ionic balance was disrupted in an SGLT2 dependent manner.To validate the possible correlation with SGLT2 activation and insulin resistance in left ventricular aged cardiomyocytes, we performed an oral glucose tolerance test (OGTT) in aged and young rats. As shown in Figure 3.52+ homeostasis were driven by SGLT2 activation. We monitored the effect of SGLT2 inhibition  on critical proteins involved in SR function. For these studies, we determined the expression level of SR Ca2+ ATPase (SERCA2a), Na+/Ca2+‐exchanger (NCX), PLB and phospho‐PLB (pPLB) in young and aged rats in the absence and presence of SGLT2 inhibitor. As shown in Figure 2+ handling defects in aged cardiomyocytes were consistent with the down‐regulation of phosphorylated PLB (pPLB), a protein responsible for SERCA function, which was markedly recovered by inhibition of SGLT2, Figure 2+ ([Ca2+]Mit) load in aged cardiomyocytes  or NCX activity. Interestingly, we observed a twofold increase in NaK current (INaK) in the aged cardiomyocytes compared with corresponding young hearts, Figure NCXin and INCXou, respectively) in the aged cardiac myocytes, Figure NaK and INCX currents, respectively, in the aged cardiomyocytes highlighting the importance of SGLT2 in the regulation of intracellular ion‐exchange currents, Figure As significantly higher i under electrical‐field stimulation in aged cardiomyocytes  prolongation in aged cardiac myocytes. As shown in Figure s Figure  panel D.42+ dysfunction have been identified as important contributors to the metabolic derangements with the ageing process in muscle cells.2+]i homeostasis and age‐associated cardiac dysfunction. Our data demonstrate that ventricular cardiomyocytes isolated from aged‐rat hearts display mitochondrial abnormalities such as increased production of mitochondrial ROS and [Ca2+]Mit, reduced mitochondrial membrane potential (MMP) and altered expression of mitochondrial quality control proteins. The underlying mechanism for ageing‐associated increased ROS production is unclear. However, it may have resulted via an electron‐transport leak from mitochondrial complex I or complex III associated with [Ca2+]i‐overload. Furthermore, carbonylation of respiratory electron transport chain proteins resulting in monoamine oxidase activity and mPTP opening from excess mitochondrial Ca2+ (increased [Ca2+]Mit) may also underline the ageing‐associated increases in ROS production.2+]Mit overload disrupted normal cardiac function.2+ signalling defects were not restricted to mitochondria. Our data also demonstrated a marked increase in cytosolic Ca2+ level . This observation suggests that defects in intracellular Ca2+ signalling up‐stream of mitochondria likely account for the observed increased [Ca2+]Mit: This view is concordant with a report suggesting that under conditions where [Ca2+]i is high, the tendency of mitochondrial Ca2+‐uptake can increase by as much as 10‐ to 1,000‐fold, resulting an overload in [Ca2+]Mit.Ageing is a major factor that predisposes cardiac myocytes to metabolic and contractile abnormalities that leads to heart failure. Lipid peroxidation, oxidative stress and mitochondrial injury are central features of insulin resistance and obesity associated with advanced age.2+]i, we also observed a marked increase in basal [Na+]i in aged cardiomyocytes. The increased basal [Ca2+]i, in this case, maybe in part because of the depressed activity of PLB or leaky‐RyR2.2+‐sparks with high gating properties can contribute to the high [Ca2+]i in aged cardiomyocytes. Furthermore, we observed high basal levels of [Na+]i in aged cardiomyocytes with higher NCX activities detected both in forward and reverse directions. If NCX is operationally higher in reverse mode, this would permit high Na+–influx into cells, whereas in the forward mode would provide a major Ca2+–efflux mechanism.+]i could be because of the activation of SGLT2 in aged cardiomyocytes as we observed in our previous study in insulin‐resistant MetS rats.2+]i and [Na+]i in aged cardiomyocytes, as the SGLT2 inhibitor (dapagliflozin) normalized the age‐induced defects on intracellular [Ca2+]i and [Na+]i. As SGLT2 inhibition could confer significant protection against age‐associated defects in intracellular ion levels, we believe that SGLT2, when activated during ageing, contributes to aberrant alterations in [Ca2+]i and [Na+]i in cardiac myocytes. Our present data are in line with recent studies highlighting a role for SGLT2 inhibitors, on cardiometabolic, bioenergetics as well as the cardiac structure and NHE‐1 activity.+ level and Ca2+ level are a potential early drivers of death from cardiovascular diseases including heart failure, via induction of increased oxidative stress.+ can trigger mitochondrial ROS production in the diabetic heart through activation of SGLT2.+, Ca2+ and H+ in aged cardiomyocytes is concordant with previous studies which demonstrated normalization of the activated NHE‐1 and increased intracellular Na+ in cardiomyocytes under pathological conditions.In addition to i homeostasis. Our studies support the notion that interventions that modulate SGLT2 activity may provide benefits in maintaining [Ca2+]i levels and cardiac function with advanced age. More importantly, our data demonstrate for the first time that ageing increases SGLT2 expression which negatively impacts mitochondrial/SR Ca2+‐homeostasis and cardiac function. In aggregate, our data provide novel evidence that SGLT2 is an important regulator of cardiac dysfunction with advanced age. Our studies further supported the notion that interventions that modulate SGLT2 activity may provide benefits in maintaining [Ca2+]i levels and cardiac function with advanced age. Hence, targeted therapy for SGL2 inhibition may prove beneficial in protection against age‐associated contractile dysfunction.Hence, our data suggest that deregulated SGLT2 during ageing disrupts mitochondrial function and cardiac contractility. Those effects seem to be associated with a mechanism that impinges upon deregulated [CaNo conflicts of interest, financial or otherwise, are declared by the authors.Yusuf Olgar: Formal analysis ; Investigation . Erkan Tuncay: Data curation ; Investigation ; Methodology . Sinan Degirmenci: Investigation . Deniz Billur: Investigation . Rimpy Dhingra: Visualization . Lorrie Kirshenbaum: Supervision ; Writing‐review & editing . Belma Turan: Conceptualization ; Funding acquisition ; Project administration ; Writing‐original draft . App S1Click here for additional data file.


While it is now well established that mitochondrial dysfunction plays an important role in the pathophysiology of heart failure, this has been less investigated in atrial myocytes in AF. Considering the high prevalence of AF, investigating the role of mitochondria in this disease may guide the path towards new therapeutic targets. In this review, we discuss the importance of mitochondrial Ca2+ handling in regulating ATP production and mitochondrial ROS emission and how alterations, particularly in these aspects of mitochondrial activity, may play a role in AF. In addition to describing research advances, we highlight areas in which further studies are required to elucidate the role of mitochondria in AF.The molecular mechanisms underlying atrial fibrillation (AF), the most common form of arrhythmia, are poorly understood and therefore target-specific treatment options remain an unmet clinical need. Excitation–contraction coupling in cardiac myocytes requires high amounts of adenosine triphosphate (ATP), which is replenished by oxidative phosphorylation in mitochondria. Calcium (CaThe online version of this article (10.1007/s00395-020-00827-7) contains supplementary material, which is available to authorized users. Atrial fibrillation (AF) is the most common sustained arrhythmia and causes substantial morbidity and mortality, particularly due to embolic stroke , 88. Cur2+) handling abnormalities and oxidative stress are thought to play central roles in the pathophysiology of AF. Mitochondria are the main producers of cellular adenosine triphosphate (ATP) in cardiac myocytes, and both Ca2+ and adenosine diphosphate (ADP) are key regulators of respiratory flux to match energy supply to the constantly varying demands in the heart i) allows diastolic relaxation and cardiac filling. Main Ca2+ removal mechanisms are the SR Ca2+-ATPase (SERCA2a), which pumps Ca2+ back into the SR, and the sodium-calcium exchanger (NCX1), which brings 3 Na+ ions into the cell per extruded Ca2+ ion. The extent to which mitochondrial Ca2+ uptake shapes the systolic Ca2+ transient remains somewhat controversial; however, the current consensus is that it has little effect on the free cytosolic Ca2+ concentration i with Ca2+-dependent ion channels and transporters such as L-type Ca2+ channel and NCX1. [Ca2+]i-driven alternans is enhanced by factors increasing SR Ca2+ release and by factors reducing Ca2+ sequestration from the cytosol, such as increased SR Ca2+ leak and reduced SERCA expression or activity (for example due to reduced ATP levels) m) for regulation of mitochondrial function was suggested as far back as 30 years ago [2+ cycling and contribution to cytosolic [Ca2+] remain highly controversial, it is well accepted that Ca2+ regulates ATP production and ROS signalling and plays a crucial role in mitochondrial-determined cell death m threshold for Ca2+ uptake and preventing mitochondrial Ca2+ overload. Low Ca2+ affinity (Km ≈ 10–20 µM) is a critical property of the MCU, making mitochondrial proximity to microdomains of high [Ca2+]i (e.g. RyR2) a requirement for effective mitochondrial Ca2+ uptake. The importance of this proximity has been demonstrated by Lu and colleagues in rabbit ventricular myocytes transfected with a genetically encoded mitochondrial Ca2+ sensor (Mitycam); it was found that mitochondrial Ca2+ uptake is greater and faster near SR Ca2+ release sites (Z-line of sarcomere), compared to the middle of the sarcomere (M-line), where SR Ca2+ release is limited and MCU density is lower i changes in cardiac myocytes because of its slow recovery from inactivation. In contrast, mRyR1 allows Ca2+ sequestration at low cytosolic Ca2+ concentrations and therefore it could contribute to physiological beat-to-beat Ca2+ uptake in the heart ) is suggested to be the predominant Ca2+ extrusion mechanism i. This also acts to increase [Ca2+]m. [Ca2+]m stimulates substrate flow through the Krebs cycle by directly activating key enzymes, thereby increasing production of NADH and FADH2, fuelling oxidative phosphorylation and increasing ATP production i (10-20 µM), explaining why mitochondrial Ca2+ uptake through MCU is so rapid, despite its relatively low Ca2+ affinity [2+ uptake efficiency is maximal when Ca2+ is released from the SR, compared to Ca2+ entering the cell via sarcolemmal NCX [2+ for the mitochondria and suggests that SR and mitochondria must have close proximity. Molecular tethers such as Mfn2 likely maintain the close structural and functional association between SR and mitochondria. Suppression of Mfn2 in murine embryonic fibroblasts and HeLa cells increases the separation between SR and mitochondria, although this has spurred some controversies [2+ uptake is decreased in ventricular myocytes from Mfn2-deficient mice [Subcellular anatomy is thought to be crucial for effective mitochondrial Caaffinity . Thus, ammal NCX . This pooversies , 92, 93.ent mice , 107.atrial myocytes can also be damaged in response to different cardiac and metabolic diseases, potentially impacting on RyR-mitochondrion interaction [2+ from the SR to the mitochondrial matrix, at least in ventricular cardiac myocytes [3R is thought to play a greater role in the physiology of the atrium than the ventricle and has been shown to contribute to endothelin-induced arrhythmic activity in a rat model [3R-related pathological mechanisms induce or involve mitochondrial dysfunction remains unknown.The spatial organisation of mitochondria is disrupted in heart failure , 72 and eraction . These feraction . Furthereraction . There imyocytes , 86. Theat model , 76. Whe2+ release sites and mitochondria, to what extent they are altered in AF and the effects of such changes on mitochondrial Ca2+ uptake remain unclear and therefore represent major targets for future investigation. Previous studies on mitochondrial activity in ventricular cells/tissue will undoubtedly serve as an invaluable basis for the design of atrial studies [2+ at high pacing frequencies, as well as MCU function, is likely to be a crucial step in ascertaining the roles of Ca2+ handling dynamics between cytosolic and mitochondrial compartments and whether any alterations occur during—and contribute to—AF pathophysiology.The various interactions between intracellular Ca studies , 69. Mea-complex assembly in patients with diabetes and AF, compared with diabetes alone [F1/Fo ATP-synthase activity in a canine model of AF [As mentioned previously, the majority of cellular ATP is produced by oxidative phosphorylation in the mitochondria. Emelyanova and colleagues provided evidence for impaired activity of Complexes I and II in right atrial tissue from patients with AF . These fes alone . Down-rees alone . There ies alone , 87. Aniel of AF . Therefo+ overload via sarcolemmal Na+-H+ exchanger (NHE) and subsequent Ca2+ overload (via reverse-mode NCX), leading to SCaEs, as illustrated in Fig. 2+ handling are thought to play a central role in the initiation and maintenance of arrhythmic activity, and, therefore, the findings of the Blatter group provide an attractive theory as to how altered mitochondrial function could provide a pro-arrhythmic substrate.The Blatter group used pharmacological tools to investigate the effect of impaired mitochondrial ATP synthesis in cat atrial myocytes ; cytosol2+ signalling could also play a role. The initial phase of AF has been described as a short period of “cellular Ca2+ overload” on account of increased atrial activation rate, preceding a longer phase involving adaptation and remodelling of electrophysiology and Ca2+ dynamics, coined “Ca2+ silencing”, as reviewed by Greiser and Schotten [2+, after the initial overload period, may contribute to the mismatch of ATP supply to demand in AF, considering the important role of Ca2+ in stimulating mitochondrial ATP production. It may not be the amount of Ca2+ per se entering the mitochondria which is important, but the amount relative to what is required for an increase in ATP production, for the energetic demands of the myocyte to be met.Due to the high stimulation frequency endured during AF, the energy consumption and, therefore, energy requirement of atrial tissue is increased, likely activating the aforementioned “pull” condition at the ETC . It is bSchotten . It is t2 [2+ handling [Of course, many factors determine ATP production and there is increasing evidence that metabolic alterations play a crucial role in cardiac remodelling leading to arrhythmia, including AF, as comprehensively discussed by others , 45, 99.2 , 45. Fur2 . Importahandling , 46, 66.2+-controlled regulation, as discussed earlier. We suggest that the “limiting factor” may depend on the extent of electrophysiological and structural remodelling and, therefore, the stage of AF. In response to pathological elevations of cardiac workload in the mouse, the mitochondrial transhydrogenase (NNT) is able to support ATP production by reversing its direction, i.e. converting NADPH to NADH, the conformational aspects of which have recently been defined [2+ overload conditions alter the potential, thereby leading to impaired ATP production [It will be important in future investigations to identify the weakest link(s), i.e. the “limiting factor”, in matching ATP supply to demand in long-term AF. Energetic substrate- and oxygen supply, metabolism-related enzyme activity and ETC activity all represent potential culprits, with aspects of the latter two being partially under Ca defined , 97. Thioduction .Fig. 6Hy2O2, and an increase in its activity may be a potential predictive marker for poAF [2+ overload and chronic high oxidative stress levels in fibrillating atria may explain the rapid damage of mtDNA.An association between oxidative damage and AF in human tissue was first provided by Mihm and colleagues and it is now widely accepted that AF is associated with increased oxidative stress , 67, 85.for poAF . A decrefor poAF . ROS pro2+ leak or increased AF susceptibility, despite the RyR mutation, thus supporting evidence already found in ventricle, that RyR oxidation leads to pro-arrhythmic activity [2+ handling, whereby increased SR Ca2+ leak could impair mitochondrial function, leading to increased ROS production which triggers further Ca2+ leak [Atrial fibrillation occurs increasingly with age , and ageactivity , 124. Th2+ dynamics would impair mitochondrial function has yet to be fully resolved. However, we hypothesise that large alterations of cytosolic Ca2+, caused by increased Ca2+ leak from the SR, or upon defibrillation or during reperfusion after an ischaemic period, may result in a “push” condition at the ETC, as opposed to the aforementioned “pull”. This could increase the production of ∙O2−, thereby changing the redox environment to a state where rate of mitochondrial ROS production exceeds the rate of ROS scavenging, thus resulting in net mitochondrial ROS emission , as reviewed by Zorov et al. ; ROS actting ∆Ψm , 130. Thchondria , 127. Onchondria . At timechondria . Permane2+ handling and ROS dynamics all represent major targets for future research, as it is conceivable that the redox environment plays a crucial role during AF. In the current review, we discussed a hypothesis that remodelling and increased energy demand during AF lead to oxidative stress, shifting the redox environment to a state of energy deficit and compromised ROS scavenging capacity (reverse NNT hypothesis), a similar scenario as has been suggested for heart failure [2+ overload”, as may occur upon increased intracellular leak, or upon ischaemia reperfusion or defibrillation, mitochondria may experience the other extreme of redox environment, which could increase net ROS emission when ROS production exceeds mitochondrial scavenging capacity  and how such changes relate to altered mitochondrial ROS emission. Furthermore, the implementation of the correct experimental environment will be crucial for such investigations, as parameters such as excitation frequency, energy substrate supply and oxygen levels will influence mitochondrial energetics and oxidative stress.The limited understanding of cellular and molecular mechanisms governing AF pathophysiology impedes the development of effective target-specific therapeutic strategies. In the present review, we focussed on the potential role that mitochondria may play in AF. Mitochondrial energetics, Ca failure  Fig. 6)2+ handliSupplimentary file (DOCX 15 KB)Below is the link to the electronic supplementary material.


Immunomodulatory drugs (IMiDs) are a class of molecules composed of thalidomide and its analogs. There is a growing interest in IMiDs on the treatment of acute myeloid leukemia (AML) patients. As AML has a worse prognosis, especially in elderly patients, novel drugs such as IMiDs, as monotherapy or combination with standard induction chemotherapy or hypomethylating agents, are urgently needed. The effect of IMiDs on AML immunosurveillance seems to be the clue to understanding their clinical application in this setting. We describe the several IMiDs currently available and also future directions. Clinical trials have to find the optimal dosing and schedule and combination agents to improve response rates with the use of IMiDs and, finally, survival in patients with AML.Immunomodulatory drugs (IMiDs) are analogs of thalidomide. They have immunomodulatory, antiangiogenic and proapoptotic properties and exert a role in regulating the tumor microenvironment. Recently IMiDs have been investigated for their pleiotropic properties and their therapeutic applications in both solid tumors  and hematological malignancies. Nowadays, they are applied in de novo and relapsed/refractory multiple myeloma, in myelodysplastic syndrome, in del5q syndrome with specific use of lenalidomide and B-cell lymphoma. Several studies have been conducted in the last few years to explore IMiDs possible use in acute myeloid leukemia treatment. Here we report the mechanisms of action of IMiDs in acute myeloid leukemia and their potential future therapeutic application in this disease. Immunomodulatory drugs (IMiDs) are a class of molecules composed of thalidomide and its analogs—lenalidomide, pomalidomide and the new molecules CC-122, CC-220, CC-885, CC-9009. They have immunomodulatory and anti-proliferative properties and are applied in the treatment of several hematologic and solid malignancies as well as in autoimmune diseases ,4,5,6. CFinally, they enhance NK cell degranulation and the production of cytokines (IFN-γ and TNF-α) and promote NK-cell mediated antibody-dependent cell-mediated cytotoxicity against tumor cells (ADCC) . It is wThe first molecule in the IMiDs family is thalidomide. Thalidomide has different mechanisms of action and has been tested in untreated and R/R AML patients. This drug has a tremendous antiangiogenic activity in AML, due to its direct inhibition of endothelial cell proliferation, reduction of fibroblast growth factor (FGF) and downregulation of a VEGF receptor, neuropilin-1, which is overexpressed in AML and whose high levels are correlated with lower survival . Aguayo In the last few years, lenalidomide has been employed as an essential anticancer drug. It was approved by the Food and Drug Administration (FDA) to treat multiple myeloma, del5q MDS and mantle cell lymphoma. It has several mechanisms of action, including a direct anti-tumor effect, inhibition of angiogenesis, anti-inflammatory activity and immunomodulation . In this way, lenalidomide promotes tumor cell apoptosis both directly and indirectly, through antiangiogenic and anti-osteoclastogenic effects, immunomodulatory activity and inhibition of bone marrow stromal cell support . In the The use of lenalidomide in studies reported in literature is generally in a particular subset of AML patients—untreated elderly patients, poor cytogenetic risk or R/R patients. A meta-analysis and systematic review by Chun-Hong Xie et al.  reported2 twice daily on days 1–6 with or without lenalidomide (20 mg/day 1–21). The enrolled patients were 222 previously untreated with newly diagnosed AML or high-risk MDS (aged 66 years or older). The study results showed that the addition of lenalidomide did not improve the therapeutic outcome of older AML patients. The CR/CRi rates in the two arms were not different (69 vs. 66%), the EFS at 36 months was 19% for the standard versus 21% for the lenalidomide arm and OS was 35% vs. 30%, respectively. The lenalidomide/cytarabine association was investigated in different AML settings, yielding controversial results. Griffiths et al. . . 61]. JäAs far as the use of pomalidomide and the new molecules in AML is concerned, studies are still few but promising. Several studies have highlighted the relevance of NK and T cells in AML development but it is necessary to clarify the role of these cells as therapeutic targets. The effect of IMiDs on AML immunosurveillance seems to be the clue to understanding their clinical application in this setting. The discovery that interactions with the cells of the microenvironment play an essential role in activating processes of the proliferation of leukemic cells is also of interest. The central role of CRBN binding in the wide range of mechanisms and targets of action of IMiDs has raised notable interest in their application in AML patients; certainly, further studies will be required to establish the therapeutic use of these drugs in AML. Since 2017, there has been an increase of newly approved AML treatment options, with the majority of new drugs targeting specific gene mutations  and pivotal cell survival pathways (such as venetoclax) . At pres


Tuberous Sclerosis Complex (TSC) is a genetic disorder, with renal manifestations like angiomyolipoma (AML) occurring in 70–80% of patients. AML usually cause more complications in TCS patients than in non-TSC patients. However, AML patients are not routinely investigated for TSC. Our aim was to retrospectively assess the correlation between radiologically diagnosed AML and TSC.All patients were stratified into AML related vs. unrelated to TSC. Correlations were calculated to determine the association between age, AML, and TSC.p <  0.01). 37 (7%) of the 521 patients were within the age-range of 18–40 years, in which TSC occurred in 6 cases, 4 (66.7%) of which presented with multiple, bilateral renal AML (p <  0.05), and 2 (33.3%) of which with a single, unilateral AML (p <  0.05). In patients with AML but without TSC, unilateral AML was found in 83.9% and bilateral AML in 16.1% (p <  0.05). Simple binary logistic regression analysis revealed bilateral AML  ) to be a risk factor for TSC.Complete data were available for 521 patients with renal AML, in 7 of which the concurrent diagnosis of TSC was found. Younger age significantly positively correlated with the prevalence of TSC in AML patients (The presence of bilateral AML in patients within the age-range of 18–40 years should raise suspicion for TSC as the underlying cause. Therefore, our advice is to refer patients with multiple bilateral renal AML for further investigations regarding TSC. Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic disorder with a birth incidence of 1:6000 and an estimated 1 million affected individuals worldwide . These pAML are mostly diagnosed by computed tomography (CT), magnetic resonance imaging (MRI), or sonography , generalTherefore, the aim of this study was to investigate whether the radiologic diagnosis of renal AML can lead to the identification of TSC patients and thus increase disease awareness for TSC.We performed an observational study at the departments of Urology and Radiology of the University Hospital Basel – a tertiary care centre in Switzerland. We retrospectively analysed all CT, MRI, and sonography reports performed between 2010 and 2016.The study protocol was approved by the Ethics Committee of Northwestern and Central Switzerland, with a waiver for individual informed consent (No. 2018–00037). We followed the ‘Strengthening the Reporting of Observational Studies in Epidemiology’ guidelines.Between January 2010 and December 2016, all CT, MRI, and sonography reports provided by the radiological reporting system were screened using the search terms “AML” and “angiomyolipoma”, in order to identify patients suffering from AML. The occurrence of renal AML was confirmed by manually reviewing the radiologic reports. In case of repeated exams, the exam with the first diagnosis was chosen for analysis. Only patients with completed datasets and patients aged 18 years and older were included.We extracted all relevant information from in-house electronic medical records. The medical history was reviewed and patients with previously diagnosed, concurrent TSC (TSC+) were identified by clinical diagnosis in the medical history, independent of the method of initial testing for TSC .Correlations were calculated using a non-normality Spearman’s rank correlation coefficient to determine the association between age, AML, and TSC. Correlation of nominal parameters  were calculated using the phi-coefficient. All tests were performed at a significance level of α = 0.05. Continuous data are shown as median with interquartile range (IQR). Simple binary logistic regression analysis was performed to predict a relationship between bilateral or unilateral AML and TSC. All analyses were performed with SPSS Statistics 19  and Graph Pad Prism Version 6.0 .The search terms were identified in 1122 (1.06%) of the overall 105,694 radiological reports, and the manual review of these reports led to the identification of 599 (53.4%) patients. Complete data were available for 547 (91.3%) of these patients, in 521  females and 164 (31.5%) males) of which renal AML was present (AML+). The concurrent diagnosis of TSC was confirmed in 7 (1.3%) of these 521 patients (3 (0.6%) females and 4 (0.8%) males; Fig. Median (IQR) AML+ patients’ age was 67.5 (19–84) years, and median (IQR) AML+/TSC+ patients’ age was 28.3 (19–42) years.AML diagnosis was primarily confirmed by CT , followed by sonography 172; 33.0%) and MRI  . Younger age, however, significantly correlated with the occurrence of TSC (p <  0.01). 37 (7.1%) of the 521 AML+ patients had an age range of 18–40 years, 15 (2.9%) had an age range of 40–45 years, and 475 (91.2%) were older than 45 years . TSC+ patients with bilateral renal AML all had multiple AML lesions, whereas all TSC+ patients with unilateral AML had only a single lesion , respectively. Among the TSC- group, unilateral AML presented predominantly  and bilateral AML uniquely  as a single lesion  and 2 (33.3%) of the 6 patients presented with bilateral and unilateral renal AML, respectively (p = 0.028). Simple binary logistic regression analysis revealed bilateral AML  ) to be a risk factor for the presence of TSC . Accordingly, 0.6% of the male and 0.8% of the female AML+ patients had concurrent TSC. These findings are in line with the literature: while the prevalence of TSC was similar in female and male patients, the clinical manifestation – i.e., the prevalence of AML – differed between genders. The higher prevalence of AML in females has been reported previously [We found a significant positive correlation between younger age and TSC prevalence in AML+ patients. Epidemiological studies regarding TSC patients show an increase of renal AML manifestation during childhood and adolescence . In our A previous longitudinal study demonstrated that 55% of paediatric TSC patients with a mean age of 6.9 years had some type of renal abnormality, and that this proportion even rose to 80% at a mean age of 10.5 years . Renal Ap < 0.05). Multiple lesions were identified in all TSC+ patients with bilateral AML, whereas primarily single lesions were found in both uni- and bilateral AML+/TSC- patients. The distribution of uni- and bilateral AML in TSC patients has already been investigated in the 1980s: 29% of the AML were unilateral, and 71% occurred on both sides [In our cohort, 66.7% of the TCS patients with an age range of 18–40 year presented with bilateral renal AML and 33.3% with unilateral renal AML , should also be considered. These include von Hippel-Lindau syndrome  and neurIn our study, the majority of AML (91.2%) was identified in patients under the age of 45 years. A previously published study investigating the AML incidence in TSC patients demonstrated a significant association between increasing age and the incidence of AML . HoweverIn summary, bilateral AML could be identified to be a risk factor for the occurrence of TSC. A previous study on histological findings in AML also concluded that pathologists should recognize that the presence of multiple AML is presumptive evidence for the diagnosis of TSC .The risk for haemorrhage from renal AML in patients with TSC is estimated between 25 and 50% , and up The presence of renal AML in patients with an age-range of 18–40 years should raise suspicion for concurrent TSC as the underlying cause. Therefore, particularly patients with bilateral renal AML should be referred for further testing for TSC, especially in light of the recent changes in the management of TSC and the option of a disease targeted therapy with mTOR-Inhibitors .


Grb7 is a signalling adapter protein that engages activated receptor tyrosine kinases at cellular membranes to effect downstream pathways of cell migration, proliferation and survival. Grb7’s cellular location was shown to be regulated by the small calcium binding protein calmodulin (CaM). While evidence for a Grb7/CaM interaction is compelling, a direct interaction between CaM and purified Grb7 has not been demonstrated and quantitated. In this study we sought to determine this, and prepared pure full-length Grb7, as well as its RA-PH and SH2 subdomains, and tested for CaM binding using surface plasmon resonance. We report a direct interaction between full-length Grb7 and CaM that occurs in a calcium dependent manner. While no binding was observed to the SH2 domain alone, we observed a high micromolar affinity interaction between the Grb7 RA-PH domain and CaM, suggesting that the Grb7/CaM interaction is mediated through this region of Grb7. Together, our data support the model of a CaM interaction with Grb7 via its RA-PH domain. Caenorhabditis elegans Mig-10  and a C-terminal Src-homology 2 (SH2) domain [Growth receptor bound 7 (Grb7) protein is a cytoplasmic adapter protein that couples activated tyrosine kinase receptors to a nexus of downstream proliferative, migratory and survival signalling pathways ,2. Grb7 ) domain . The GM ) domain , the N-t) domain ,6, and tThe PH domain was also reported to bind the small, ubiquitously expressed protein calmodulin (CaM) in a calcium dependent manner . The VilTogether, these experiments show compelling evidence for a Grb7/CaM interaction. However, a direct Grb7/CaM interaction has never been verified with pure full-length Grb7 protein nor quantitated. Furthermore, it has been established that Grb7 can be phosphorylated on the central GM region, specifically Y188 and Y338, and this phosphorylation is required for ErbB2 mediated signalling via Grb7 ,15. WhetEscherichia coli and analyzed their interaction using surface plasmon resonance (SPR) that detects molecular interactions with high sensitivity. We also produced the RA-PH domain of Grb7 in isolation, as well as the SH2 alone, in order to determine the requirement of the RA-PH domain for the Grb7/CaM interaction. We demonstrated that CaM is able to interact with full-length Grb7 in a calcium dependent manner, and that this interaction is not mediated through the SH2 domain. In contrast, we observed high micromolar affinity binding between the Grb7 RA-PH domain and CaM that is also dependent on the presence of calcium. Thus, we are able to confirm that Grb7 and CaM do indeed directly interact, although whether or not additional factors are required to augment the interaction in vivo is still open for investigation.The current study was therefore undertaken to determine whether direct interactions between CaM and purified Grb7 could be detected in vitro and in the absence of post-translational modifications or additional cellular factors. To do this we expressed and purified recombinant CaM and full-length Grb7 from Escherichia coli using a previously established expression and purification protocol [In order to verify a direct interaction between CaM and Grb7 in vitro, high purity CaM and Grb7 protein were prepared. Recombinant CaM was overexpressed in protocol . Figure Escherichia coli and purified as previously described, generating protein to > 95% purity . Multi-cycle kinetics was utilized to allow a regeneration step with a buffer containing 5 mM EGTA to disrupt the Grb7/CaM interaction. All sensorgrams were double referenced by subtracting the GST alone from reference flow cell as well as buffer only injections.The purified GST-tagged Grb7 constructs were immobilised on a CM5 sensor chip surface using GST antibody capture, as previously described for Grb7-SH2 domain  Figure C and purIn the presence of calcium we observed a direct interaction between full-length Grb7 and CaM A and theka) of 275 M−1s−1 and off-rate (kd) of 0.066 s−1. The slowed dissociation phase of the Grb7 RA-PH/CaM interaction suggests a higher affinity interaction compared with the full-length Grb7/CaM interaction. The determined on-rate and off-rates also permitted the direct determination of an affinity of the Grb7 RA-PH/CaM interaction with a KD of 240 μM. This micromolar affinity is consistent with the CaM interaction with Grb7 occurring through the RA-PH domain. These data are summarised in A calcium dependent interaction was also observed for CaM and the Grb7-RA-PH domain construct, with binding observed in the presence of calcium and no binding observed in the presence of EGTA B. The shFinally, no interaction between CaM and the Grb7-SH2 domain was observed in either the presence or absence of calcium C, supporIt should be noted that there was a small amount of non-specific binding detected on the GST control reference flow cell when CaM was injected at high concentrations. This subtraction of non-specific binding is evident in the Grb7-SH2 sensorgrams with negative values detected upon injection of the analyte. Nevertheless, it can be concluded that CaM binds to Grb7 via the RA-PH region in a calcium dependent manner.In this study, we investigated the relationship between Grb7 and the calcium sensor protein CaM. Using SPR we demonstrated that CaM binds to full-length Grb7 and that this interaction is calcium dependent. To further characterize the interaction, a construct that spans the RA-PH unit of Grb7 (and includes the postulated CaM binding motif) was cloned, expressed and purified. Using the same SPR experimental approach, we demonstrated that CaM does indeed bind to this RA-PH region of Grb7, also in a calcium dependent manner. Furthermore, no binding was detected to the Grb7 SH2 domain, supporting a RA-PH mediated interaction. This was important to ascertain as it was previously demonstrated that both apo CaM and calcium bound CaM were able to bind to the SH2 domain of Src .The finding of a calcium dependent direct interaction between CaM and Grb7 is consistent with reports by the Villalobo group ,9,10. WeKD = 100 nM) [The structure of the RA-PH domain of Grb7 is not yet known; however, the Grb10 RA-PH unit was solved using X-ray crystallography and is likely a homologous structure to that of Grb7 RA-PH domain . In this 100 nM) . In this15N-1H-HSQC spectra of the Grb7-SH2 domain upon titration with the Grb7-RA-PH domain [Although the affinities of interaction in this study are estimates, it is of interest to compare the interactions of CaM with full-length Grb7 compared with Grb7-RA-PH domain. It is particularly noticeable that the sensorgram shapes greatly differ between binding of CaM to full-length Grb7 and binding to the Grb7 RA-PH domain. The slower off-rate seen for the Grb-RA-PH domain reflects a higher affinity interaction compared to that of the full-length Grb7. Such an occurrence could be due to a difference in the available Grb7 binding surface between the two constructs. It was previously postulated by the Lyons group that Grb7 is regulated through an intra-molecular association whereby the SH2 domain interacts with the RA-PH domain. This interaction was demonstrated by measuring chemical shift changes in H domain . Such anFinally, the current study demonstrates a low affinity interaction between CaM and the Grb7 RA-PH domain. Due to the high level of homology between Grb7 and family members Grb10 and Grb14, it could be predicted that CaM may also interact with these proteins in a similar manner. Indeed, initial work has revealed that Grb10 and Grb14 are also bound by CaM and that the interaction site may be similar to that proposed for Grb7 . WhetherEscherichia coli BL21 (DE3) pLysS cell line. The cells were lysed by sonication in buffer comprising PBS (pH 7.4), 2 mM EDTA, 0.5% (v/v) Triton X-100 and 1 mM dithiothreitol (DTT), before the Triton X-100 concentration was increased to 1% (v/v) and stirred for 1 h at 4 °C. The cleared lysate was subsequently purified by glutathione affinity chromatography (including a 2 M NaCl wash step to remove bound nucleic acid) and size exclusion chromatography  in a buffer comprising 50 mM NaPO43− (pH 7.0), 300 mM NaCl, 10% (v/v) glycerol and 5 mM DTT.Glutathione-S-transferase (GST) tagged Grb7 SH2 domain (encoding residues 415–532) and a GST alone control were expressed and purified as previously described . Grb7 fuEscherichia coli BL21 (DE3) plysS Rosetta2 cell line, and purified as per Grb7 full-length, with the exclusion of the 2 M NaCl wash during the glutathione affinity chromatography step.The Grb7 RA-PH construct (encoding residues 98–353) was cloned into the pGex6p2 plasmid, and two point-mutations introduced (C113S and C207S) using QuikChange site-directed mutagenesis  to preveEscherichia coli BL21 (DE3) pLysS cell line. Similar to previously described [2, the soluble fraction purified by hydrophobic interaction chromatography . The bound CaM was eluted in a buffer comprising 50 mM Tris-HCl (pH 7.5), 1 mM EGTA and further purified by size exclusion chromatography  in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2. For binding experiments, CaM was dialysed overnight into the appropriate analyte running below (see below).The pET14b plasmid containing CaM was kindly provided by Antonio Villalobo, and expressed in the escribed , in a bu43− (pH 7.4), 150 mM NaCl and 1 mM DTT, polyclonal anti-GST antibody  was immobilised on a BIAcore CM5 series S sensor chip  via amine coupling with bound responses between 934 and 1081 response units (RU). GST-alone was immobilised on the reference flow-cell, and GST-Grb7-SH2, GST-Grb7 RA-PH and GST-Grb7 full-length immobilised on the active flow-cells with capture levels of 212 RU, 266 RU, 391 RU and 368 RU, respectively. In duplicates, at 30 μL/min, CaM at various concentrations (1.56–200 μM) was injected over the chip surface for 60 s, followed by a dissociation time of 180–360 s. The running buffer comprised 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM DTT, and either 5 mM CaCl2 or 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). When the running buffer contained CaCl2, a regeneration step was incorporated comprised of an 80 s injection of running buffer including 5 mM EGTA. The responses on the active flow cells were double referenced by subtracting the GST-alone reference flow cell as well as buffer only injections. The data were analysed using Scrubber2.0 .SPR experiments were performed on a BIAcore T100 at 25 °C similar to previously described ,20,21,22


However, the physiological role and the molecular identity of the mitochondrial Ca2+ uptake mechanisms remained elusive for a long time. The identification of the individual components of the mitochondrial calcium uniporter complex (MCUC) in the inner mitochondrial membrane in 2011 started a new era of research on mitochondrial Ca2+ uptake. Today, many studies investigate mitochondrial Ca2+ uptake with a strong focus on function, regulation, and localization of the MCUC. However, on its way into mitochondria Ca2+ has to pass two membranes, and the first barrier before even reaching the MCUC is the outer mitochondrial membrane (OMM). The common opinion is that the OMM is freely permeable to Ca2+. This idea is supported by the presence of a high density of voltage-dependent anion channels (VDACs) in the OMM, forming large Ca2+ permeable pores. However, several reports challenge this idea and describe VDAC as a regulated Ca2+ channel. In line with this idea is the notion that its Ca2+ selectivity depends on the open state of the channel, and its gating behavior can be modified by interaction with partner proteins, metabolites, or small synthetic molecules. Furthermore, mitochondrial Ca2+ uptake is controlled by the localization of VDAC through scaffolding proteins, which anchor VDAC to ER/SR calcium release channels. This review will discuss the possibility that VDAC serves as a physiological regulator of mitochondrial Ca2+ uptake in the OMM.Already in the early 1960s, researchers noted the potential of mitochondria to take up large amounts of Ca Many proteins including RyR, LetM1, or UCP2/3 had been suggested to mediate mitochondrial Ca2+ uptake, before the molecular identity of the MCU complex (MCUC) in the inner mitochondrial membrane was identified as the main route of Ca2+ uptake in the early 2010s. Subsequently, research on mitochondrial Ca2+ uptake experienced a revival when molecular structures and knock-out models of the individual MCUC components became available. However, on its way into mitochondria Ca2+ has to pass two membranes, and the first barrier before Ca2+ even reaches the MCUC is the outer mitochondrial membrane (OMM), a fact that is still often neglected in current literature, where the OMM is described as freely permeable to ions. Indeed, the term mitochondrial Ca2+ uniport is often used synonymously for mitochondrial Ca2+ uptake, and some textbook representations do not even show the OMM but depict the MCUC such that it is directly facing the cytosol.Mitochondria are known as the cell’s powerhouses as they produce ATP by oxidative phosphorylation. However, already in the 1950s and 1960s, researchers noted the potential of mitochondria to sequester vast amounts of CaThe OMM is widely occupied by large pore-forming structures, the voltage-dependent anion channels (VDACs), which mediate the flux of ions and metabolites over the OMM. While bacteria express only one form of VDAC, higher eukaryotes express multiple isoforms. In yeast, two isoforms are described, while plants and animals express three isoforms (VDAC1-3).2+ over the OMM [2+, and VDAC1 reconstituted into liposomes mediated Ca2+ efflux from these vesicles. These in situ results are supported by the observations that VDAC1 overexpression enhanced Ca2+ uptake into mitochondria [2+ uptake [2+ flux through VDAC could be blocked by ruthenium red (RuR) and La3+ and that VDAC possesses a Ca2+ binding site, raising the intriguing hypothesis that VDAC is not freely permeable to Ca2+ but might present a regulated barrier for Ca2+ flux over the OMM.In a ground-setting publication in 2001, Shoshan-Barmatz and colleagues provided several lines of evidence that the voltage-dependent anion channel (VDAC) is the main carrier for Ca the OMM : when inchondria , and kno+ uptake  in vitro2+ uptake at the outer mitochondrial membrane physiologically relevant?However, considering that VDAC forms a pore large enough to allow the passage of metabolites such as ATP, two questions arise: how can such a big pore be regulated at the molecular level and is the regulation of mitochondrial Ca2+ uptake at the OMM: (i) the tight connection between VDAC1 and the IP3 receptor (IP3R) was reported to be a key regulatory mechanism to either promote enhanced cellular metabolism or to induce apoptosis by modulating mitochondrial Ca2+ signaling [2+ uptake through channel monoubiquitinylation [2+ uptake approx. 3-fold in cardiomyocytes [Several recent reports have argued in favor of a regulation of mitochondrial Caignaling ,6, (ii) nylation  was demonylation , and iiimyocytes ,10. This2+ uptake through VDAC and then review their putative roles in cell physiology and pathophysiology.In this review, we will first discuss possible regulatory mechanisms of mitochondrial Ca2 as the charge carrier, the channel was shown to be permeable for Ca2+. Interestingly, the ion selectivity of the channel changes upon gating from a higher anion selectivity in the high-conductance state to a lower selectivity and, thus, higher proportional conductance for Ca2+ in the gated states [2 used or the composition of the bilayer making an estimation of VDACs ion selectivity in the native environment in the cell difficult. Nevertheless, the relative permeability of cations over anions in VDAC1 was identified experimentally and by molecular simulations to be in the range of 0.05 to 0.4 for the high-conductance state, while the relative permeability for cations over anions was almost 1 for the gated states [2+ conductance of VDAC3.The common perception that VDAC creates pores in the OMM that are freely permeable to ions is closely related to its structure. VDACs are 30–35 kD transmembrane proteins consisting of approximately 280–300 amino acids, depending on isoform and species. They form barrel-like pores consisting of 19 β-sheets aligned in an antiparallel orientation. On the N-terminal end, an α-helix lines the channel pore inside the barrel ,12. The d states ,21. Thoud states ,21 for Vd states . Ambiguid states , this isd states . However2+ flux over the OMM. However, gating of the channel was experimentally induced by voltage, and although several mechanisms to create and regulate a membrane potential over the OMM were postulated [2+ [2+ conductance of the channel.The transition between high-conductance and gated states induces drastic changes in the permeability and selectivity. Although, lipid bilayer experiments are performed in an artificial and rather unphysiological system and care must be taken when transferring them directly to the in vivo situation, it is conceivable that gating of the channel presents a mechanism to regulate not only ATP, but also Castulated ,25,26, iated [2+ , metabolated [2+ , the lipated [2+ ,30,31,32ated [2+ , small mated [2+ ,34,35, iated [2+ ,37, and ated [2+ ,39 were E. coli and, subsequently, purified and refolded from inclusion bodies forms functional channels in lipid bilayers, which are completely devoid of any regulatory partners that might be associated with the channel in its native environment [Evidence for the ability of the channel to undergo intrinsic conformational changes for gating was provided by lipid bilayer experiments on purified channels ,16. VDACironment ,11. Thesironment ,40 and mironment ,41. Howeironment . This fa2+ conductance, hence gating would be an intriguing possibility to regulate VDAC Ca2+ flow. However, the existence of voltage-dependent gating in the native environment remains controversial. Several extrinsic factors were described to influence and modulate voltage-gating of the channel and are discussed later.As described earlier, gating of the channel was demonstrated to directly affect its Ca2+ specifically, the occluded state was also demonstrated to make the channel more cation selective [2+ permeability.In addition to the intrinsic gating of the channel, another intriguing hypothesis that was more recently developed is a regulation of the channel by occlusion. Given the large pore size of the channel, which is wide enough for the passage of metabolites, it appears conceivable that metabolites or domains of interacting proteins enter the channel to serve as “channel plugs”. Among those suggested to serve as molecular plugs is NADH: while it was known for long that NADH closes VDAC ,43, it welective , thus su2+ conducting state.Interestingly, a recent study using NADH measurements combined with mathematical modeling has proposed that in cardiac cells 98% of all VDACs reside in a state that is impermeable for ATP, but it remains speculative whether this is the gated or occluded state . It raisIn the following, we will summarize factors that were discussed as possible physiological triggers that control gating or occlusion of the channel.The most straight-forward idea when bearing the lipid bilayer experiments in mind is a regulation of the channel by voltage. Since VDAC resides in its high-conductance state at 0 mV and only starts gating at potentials above 20 to 30 or below −20 to −30 mV, a physiologically relevant voltage-dependent gating of the apo-form of the channel would depend on a membrane potential across the OMM. Although the generation of a membrane potential has often been considered impossible due to the high abundance and high conductance of VDACs in the OMM, a series of modeling studies by Victor and Sergy Lemeshko provides reasonable evidence for a membrane potential over the OMM. This potential was suggested to be created by a spatial separation of VDAC from the electron transport chain and to be regulated through the local concentration of ADP within the contact sites and the Gibbs free energy of the hexokinase reaction bound to VDAC . Most st2+ uptake  a Ca2+ induced shift in electrophoretic mobility of VDAC1, (ii) a blockade of the channel by La3+, which could be reversed by the addition of EGTA, and (iii) an inhibition of the channel by ruthenium derivatives such as RuR, Ru360 [2+. More recently, a combination of NMR and single-molecule force spectroscopy revealed that VDAC barrels are highly flexible, but a significant reduction in conformational variability is induced when Ca2+ is bound to the channel [2+, but starts gating and mainly resides in the high-conductance state as the concentration of Ca2+ increases [2+. Indeed, the relationship between the external Ca2+ concentration and the uptake rate of Ca2+ into VDAC liposomes follows a non-linear relation, indicating the presence of a Ca2+-regulated Ca2+ flux through VDAC [Besides conducting CaR, Ru360  that was channel . In a vencreases , indicatugh VDAC .2+ binding site, it was observed that the addition of an excess amount of Ca2+ prevented the blocking effect of RuR, which was reinstalled after the addition of EGTA. This suggests that ruthenium derivatives compete with Ca2+ for the same binding site [2+-mediated control. However, E73 is located on the outside of the barrel facing the lipidic environment [2+ can enter the lipidic environment of the membrane. Alternatively, the mere presence of E73 could stabilize the channel in a conformation that is required for Ca2+-mediated channel regulation.In an effort to localize the Caing site . Mutatioironment ,12, and ironment , it rema2+ transport activity by E73 was recently confirmed in physiological experiments, where Ca2+ transfer from lysosomes into mitochondria after lysosomal Ca2+ release through TRPML1 was shown to depend on E73 [2+ transfer from the SR into mitochondria in cardiomyocytes is also regulated through E73 [2+ regulated uptake of Ca2+ through VDAC.Nevertheless, a regulation of the channels Cad on E73 . In agreough E73 . These f2+ conductance [2+ accumulation [A common way to regulate ion channels are posttranslational modifications. Phosphorylation of all three VDAC isoforms was repeatedly reported, and several studies showed that PKA phosphorylation results in altered gating properties of VDAC. Interestingly, PKA phosphorylation affected channel gating asymmetrically with no influence at positive potentials but a reduction in both single channel conductance and open probability at negative potentials ,62. Thisductance . In lateductance  and phosmulation .2+ flux, and the bilayer experiments are, thus, not in line with the physiological observations of a limited Ca2+ uptake after PKA phosphorylation [These experiments are difficult to interpret. Bilayer experiments indicate that phosphorylation of VDAC-induced voltage-dependent closure of the channel and in vitro phosphorylation of VDAC by either glycogen synthase kinase-3β (GSK3β) or cAMP-dependent protein kinase A (PKA) increased tubulin binding and, thereby, channel occlusion . Both thrylation . This mi2+ conductance in vivo was established in a recent study where PINK1-dependent monoubiquitinylation of VDAC1 was directly shown to limit mitochondrial Ca2+ uptake. Abolishing VDAC1 monoubiquitinylation by introducing a K247R mutation in VDAC1 induced a Parkinson disease phenotype in fruitflies was associated with excessive apoptosis and could be relieved by MCU knock-out [2+ flux are missing in this report, these data directly indicate that reducing mitochondrial Ca2+ uptake through VDAC1 monoubiquitinylation is a critical mechanism to suppress apoptosis.A critical link between posttranslational modifications and CaFinally, the lipidic environment was shown to influence ion channels properties, and corresponding observations were also made for VDAC. When inserted into lipid bilayers, the addition of nonlamellar lipids such as phosphatidylethanolamine (PE) or cardiolipin induced asymmetry in voltage gating, evident by increased channel closure at negative potentials . A similAlthough lipid bilayer experiments and molecular dynamics simulations might not directly recapitulate channel function in vivo due to the simplicity of the system and the lack of additional regulatory factors present in its native environment, a regulation of the channel by membrane lipids might fine-tune channel activity in response to cellular stimuli also there. Indeed, a change in the membrane composition was reported, for example for the induction of apoptosis .2+ homeostasis or regulated by Ca2+.Many of the aforementioned mechanisms might be regulated by partner proteins. VDAC was shown to interact with many cellular proteins ranging from small signaling molecules to large enzymes and other ion channels, and these interactions are often associated with Ca2+ uptake by binding to VDAC, presumably at a structure formed by the N-terminal helix and the loop connecting beta sheets 14 and 15 [2+ uptake, when Bcl-xL is associated with the channel [2+ into mitochondria [2+ uptake into mitochondria [2+ uptake is triggered by local IP3Rs in Ca2+ microdomains within contact sites of mitochondria and the SR and permeabilized cells in which mitochondrial Ca2+ uptake is triggered by a global rise in Ca2+ , are a key player in Ca2+ shuttling. MAMs cover approximately 5–20 percent of the mitochondrial surface in mammalian cells [2+ release sites, where local cytosolic Ca2+ can reach particularly high levels [2+ release channel in the ER membrane of non-excitable cells is tethered to VDAC1 but not VDAC2 or VDAC3 [2+ into mitochondria, the SR-OMM contact points align with contact points of OMM and IMM [Most eminently, mitochondria intimately and dynamically interact with the internal Caan cells . Interesh levels ,82,83,84or VDAC3 , throughor VDAC3 ,86. Moreor VDAC3 ,77. For  and IMM , and VDA and IMM .2+ release (IP3R/RyR2), Ca2+ shuttling (VDACs), and Ca2+ uptake sites (MCUC) provide an efficient route for direct Ca2+ shuttling from high Ca2+ microdomains of the SR into mitochondria. They were shown to be dynamic, as they can change in response to cytosolic or extracellular triggers to regulate mitochondrial Ca2+ uptake [These couplons of Ca+ uptake ,86 and a+ uptake ,87 , and a block of mitochondrial Ca2+ uptake was shown to be beneficial for the survival of cardiac tissue [2+ handling proteins has been shown to be significant during I/R. Resveratrol, a polyphenolic compound in red wine, prevents VDAC1 upregulation in I/R, leading to a reduced infarct size in vivo [2+ shuttling by the IP3R-Grp75-VDAC1 complex [2+ shuttling between IP3R and VDAC1, limits the Ca2+ transfer from SR/ER to mitochondria and prevents mitochondrial Ca2+ overload [Abnormal mitochondrial Cal damage . Likewismulation . Anothermulation . Strikinmulation . Similarc tissue ,112. The in vivo . Liproxs complex . In aduloverload .2+ overload is a key player in heart failure and I/R. However, contrarily to these results, other studies propose heart failure to be associated with a reduced ER–mitochondria coupling: in diabetes type 2, where a reduced Ca2+ shuttling in MAM regions was repeatedly demonstrated for many tissues [2+ transfer to mitochondria was observed for cardiomyocytes. The impaired SR–mitochondrial Ca2+ coupling resulted in an impairment of mitochondrial energy supply, reduced cell contraction, and thus, cardiomyopathy [All these data indicate that mitochondrial dysfunction through Ca tissues ,116,117,myopathy .2+ uptake can be protective in pressure overload induced heart failure: TSPO closely interacts with VDAC1, acts as a negative regulator, and decreases mitochondrial Ca2+ uptake by inhibiting VDAC1 expression and mediating PKA phosphorylation [2+ transfer; however, further research is needed to clarify opposing results of a diminished or increased ER/SR–mitochondria coupling.These data are in line with the idea that an enhanced mitochondrial Carylation ,119. Micrylation . Taken t2+ uptake in the heart, it is, however, tempting to investigate the potential of drugs that interfere with this process. Indeed, our lab has recently described a very prominent role of the small molecular VDAC2 modulator efsevin. In lipid bilayer experiments, efsevin induced gating of VDAC2 and promoted the closed state of the channel [2+ transfer from the SR into mitochondria and, thereby, spatially and temporally restricted diastolic Ca2+ sparks and prevented the formation of arrhythmogenic Ca2+ waves [Considering the important role of VDAC in physiological and especially pathophysiological modulation of mitochondrial Ca channel . In card2+ waves . Most im2+ homeostasis was shown to be associated with diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS) [2+ uptake and cells expressing PD-associated DJ-1 mutants showed impaired mitochondrial function [2+ uptake that seems to be beneficial for axon regeneration [Additionally, for neurodegenerative diseases disruption of functional ER–mitochondrial Cais (ALS) . One prois (ALS) ,122. DJ-function ,123. A rneration .2+ uptake, in particular, though its close interaction with the ER/SR calcium release sites, is an important modulator of physiology and pathophysiology of the cell. However, discrepancies exist between results obtained from lipid bilayer experiments and experiments performed in cells, in particular concerning the flux of Ca2+ upon channel closure, indicating that in the native environment other or additional levels of VDAC regulation exist and lipid bilayer experiments should not be overinterpreted. VDAC-mediated mitochondrial Ca2+ uptake represents a critical component to decipher cellular Ca2+ signals and to mediate either enhanced energy production or induction of apoptosis but also to fine-tune intracellular Ca2+ signals. Additionally, at least in cardiomyocytes, it further shapes cytosolic Ca2+ signals and acts as a buffer to blunt cytosolic Ca2+ spikes. VDAC-mediated Ca2+ uptake is regulated at different levels, ranging from expression to protein partners as well as intrinsic properties of VDAC, which can adapt multiple states of conductance. However, the specific molecular mechanisms leading to these different conductance states have not been fully resolved and care should be taken when using the term “channel closure”, since voltage-induced closure, as well as occlusion, prevents the flux of metabolites but increases Ca2+ flux. Future studies are needed to resolve open questions such as the role of voltage gating in vivo the relevance of VDAC gating cellular physiology and the selective contribution of the regulation of mitochondrial Ca2+ uptake at the OMM and IMM, respectively.Taken together, the compiled data indicate that VDAC-mediated mitochondrial Ca


Dysfunction of the tumor suppressor p53 occurs in most human cancers. Mdm2 and MdmX are homologous proteins from the Mdm (Murine Double Minute) protein family, which play a critical role in p53 inactivation and degradation. The two proteins interact with one another via the intrinsic RING  domains to achieve the negative regulation of p53. The downregulation of p53 is accomplished by Mdm2-mediated p53 ubiquitination and proteasomal degradation through the ubiquitin proteolytic system and by Mdm2 and MdmX mediated inhibition of p53 transactivation. To investigate the role of the RING domain of Mdm2 and MdmX, an analysis of the distinct functionalities of individual RING domains of the Mdm proteins on p53 regulation was conducted in human osteosarcoma (U2OS) cell line. Mdm2 RING domain was observed mainly localized in the cell nucleus, contrasting the localization of MdmX RING domain in the cytoplasm. Mdm2 RING was found to possess an endogenous E3 ligase activity, whereas MdmX RING did not. Both Mdm2 and MdmX RING domains were able to dimerize with endogenous full-length Mdm2 and MdmX protein and affect their cellular function. The results showed that overexpression of the Mdm2 or MdmX RING domains interfered with the endogenous full-length Mdm2 and MdmX activity and resulted in p53 stabilization and p53 target gene activation. However, both Mdm RING domains showed oncogenic activity in a colony formation assay, suggesting that the Mdm RING domains possess p53-independent oncogenic properties. This study highlights the distinct structural and functional traits of the RING domain of Mdm2 and MdmX and characterized their role in cellular responses through interfering with p53 dependent signaling pathway. MDM2 or MDMX resulted in embryonic lethality that could be rescued by concomitant deletion of TP53. Phenotypically, MDM2 knockout showed prevalent apoptosis, whereas MDMX knockout caused primarily cell cycle arrest in these genetic studies [The tumor suppressor protein p53, known as the “guardian of the genome”, is dysfunctional in most of human cancers. As a key transcriptional factor, p53 orchestrates cellular responses to DNA damage, nutrient depletion, and abnormal oncogenic events by regulating diverse signaling pathways ,2,3. Los studies ,16,17. TMDMX RING or mutation that impairs Mdm2/MdmX dimerization caused embryonic lethality, which could only be rescued by concomitant TP53 knockout, despite Mdm2 E3 ligase activity and the ability to interact with p53 were maintained for the mutants [Further genetic knock-in studies revealed an interconnected and dependent nature of Mdm2 and MdmX function in vivo. Importantly, deletion of the  mutants ,18,19. T mutants . In vitr mutants ,22. In a mutants ,24,25. M mutants .MDM2 and MDMX genes have been identified from various aggressive forms of cancers. However, the functions of these splice variants remain poorly understood. For instance, MDM2-A and HDM2ALT1 are the MDM2 gene products characterized as lacking the N-terminal p53 binding domain, however, containing the complete C-terminal RING domain [MDMX gene splice variant, HDMX211, misses an N-terminal p53-binding domain, but possesses an intact C-terminal RING domain [Aberrant splice variants of the G domain ,28,29,30G domain . These sTo better understand the functions of Mdm2 and MdmX RING domains, we ectopically expressed the minimal RING domain regions of Mdm2  and MdmX  as YFP fusion proteins in U2OS cells A. FluoreALT1 and HDMX211 [Mdm2 and MdmX RING domains have been identified as the functional domain in several aberrant splice variants of Mdm2 and MdmX identified from cancers, such as HDM2 HDMX211 ,31,32. TTo explore the functions of the isolated Mdm2 and MdmX RING domains, we overexpressed Mdm2 or MdmX RING, and examined their individual effect on the endogenous Mdm2 and MdmX proteins. Overexpression of Mdm2 RING decreased the cellular level of Mdm2 . InteresAs both Mdm2 and MdmX are actively involved in the DNA repair-signaling pathway through p53, the isolated Mdm2 and MdmX RING domains are expected to affect p53 signaling through the interactions with endogenous Mdm2 and MdmX. To study functions of the Mdm2 and MdmX RING domains in the p53 response pathway, FLAG-tagged Mdm2 and MdmX RING were ectopically expressed in U2OS cells, and full-length FLAG-tagged Mdm2 and MdmX were included as positive controls . The effConsistent with the results obtained using YFP-fusion proteins, the MdmX RING domain stabilized both endogenous Mdm2 and MdmX. However, the effect of the Mdm2 RING domain on endogenous Mdm2 and MdmX was not obvious A. The diThe effect of ectopic expression of the Mdm2 and MdmX FL and RING on the level of total p53 as well as the activated p53  was examined next. In the absence of DOX, both total and activated p53 levels were stabilized in the presence of the ectopically expressed Mdm2 RING and MdmX FL, whereas MdmX RING stabilized p53 to a lesser extent B. A visiCDKN1A mRNA levels, suggesting that p21 regulation occurs at both the transcriptional and the protein level. However, no changes were observed in Bax protein and mRNA levels as a result of overexpression of the exogenous proteins.External stress stimuli, such as DNA damage, stabilize and activate p53, which in turn regulate transcription of its target genes. To further elucidate the effects of the ectopically expressed Mdm2 and MdmX RING domains on the transcriptional activity of p53, the expression of its downstream target genes, p53-mediated expression of p21 involved in regulation of the cell cycle progression and a proapoptotic Bax protein was investigated B,C 33,3,333,34. To examine the effect of ectopic Mdm2 and MdmX RING domains on U2OS cell cycle progression, flow cytometry analysis was carried out following transfection of U2OS cells with the full-length or RING domains of either Mdm2 or MdmX. Cell DNA contents were stained with propidium iodide and analyzed. We did not observe any significant difference in cell cycle profiles as a result of overexpression of the exogenous proteins. The proportion of cells in G1, S, and G2/M was relatively similar for all the constructs A.p < 0.01). Despite the fact that overexpression of Mdm2 and MdmX RING led to p53 stabilization, ectopic expression of Mdm2 and MdmX RING led to increased colony formation by 2- fold and 3-fold, respectively, compared to the control group. This result suggested that both Mdm2 and MdmX RING promoted tumor growth, but MdmX RING was a more potent tumorigenic construct and comparable to Mdm2 and MdmX FL.To examine the effect of the ectopic Mdm2 and MdmX RING on U2OS cell growth, clonogenic assays were carried out B. As expRegulation of p53 by its negative regulators Mdm2 and MdmX is crucial for proper cellular functioning and prevention of tumorigenesis. Mdm2-mediated ubiquitination of p53 remains the main mechanism modulating p53 stability through proteasomal degradation. Mdm2 and MdmX share the most similarity in their RING domains. However, only Mdm2 possesses E3 ligase activity through its RING domain, whereas MdmX carries an inactive RING domain ,35,36,37Next, we examine the differential function of Mdm2 and MdmX RING in the regulation of endogenous Mdm2 and MdmX. We found that Mdm2 RING possessed an active E3 ligase activity, but showed opposite effects on the levels of endogenous Mdm2 and MdmX. On one hand, it promoted proteasome-mediated degradation of endogenous Mdm2 through its active RING domain. On the other hand, it formed a heterodimer with endogenous MdmX, which resulted in protection of MdmX from degradation by endogenous Mdm2 and subsequent MdmX stabilization. The net outcome may be dependent on the relative stoichiometric ratios of ectopically expressed Mdm2 RING domain and the endogenous Mdm proteins, which would influence the relative E3 ligase activity of the endogenous Mdm2/MdmX complex . We alsoNuclear localization of Mdm2 was previously reported, and transport between the nucleus and the cytoplasm was enabled due to its nuclear localization signal and nuclear export sequence . MdmX diThe effects of the ectopic expression of Mdm2 and MdmX RING on p53 and downstream p53 effects were examined in the absence or presence of DNA damage, induced by doxorubicin. Following a 24-hour transfection in human osteosarcoma cells, we observed the effects of the exogenous proteins on the levels of endogenous p53 and its main negative regulators Mdm2 and MdmX. Furthermore, by means of detecting levels of phosphorylated p53 and its downstream gene targets, namely, p21 and Bax, the gene transcription activity of p53 was examined. The effects of the exogenous proteins were compared between normal and stress conditions, triggered by the DNA-damaging agent doxorubicin.Following ectopic expression of Mdm2 FL under normal or stress conditions, an increased cellular Mdm2 level and decreased endogenous p53, phosphorylated p53, and p21 levels were found. There were no changes in the endogenous levels of MdmX and Bax. Low p53 levels could be explained by the Mdm2-mediated p53 polyubiquitination, which targeted it for the proteasomal degradation. Consequently, low levels of the phosphorylated p53 and p21 were observed. This was further supported by the fact that Mdm2 FL was primarily localized within the nucleus. In addition, several studies showed Mdm2-dependent destabilization of p21, which could also contribute to the observed low p21 level ,43.Overexpression of MdmX FL did not affect levels of endogenous Mdm2 and Bax regardless of doxorubicin administration. Interestingly, under normal conditions, we observed a slight accumulation of p53 and phosphorylated p53, and low levels of p21 expression. Following doxorubicin treatment these effects were not found. Different cell responses to MdmX overexpression indicated functional complexity and differential recruitment of MdmX in normal and stress conditions. An increase in the levels of p53 and phosphorylated p53 could occur as a result of inhibition of the Mdm2-mediated p53 degradation by means of complex formation between Mdm2 and MdmX. Although, p53 was stabilized in the cell, it was transcriptionally inactive as exemplified by the levels of p21 C. This eIn absence of doxorubicin treatment, ectopically expressed Mdm2 RING domain led to a slight stabilization of endogenous Mdm2 and no changes in MdmX, p53, phosphorylated p53, p21, and Bax levels. Following drug treatment, more efficient stabilization of endogenous Mdm2, an increase in phosphorylated p53 level, and p21 expression were observed. No significant changes in MdmX, p53, and Bax levels were found in presence of a stress signal. Co-immunoprecipitation experiments demonstrated the ability of the overexpressed Mdm2 RING to interact with endogenous full-length Mdm2 and MdmX. Dimerization with Mdm2 could explain nuclear translocation of the exogenous Mdm2 RING from the cytoplasm. Therefore, the observed Mdm2 stabilization in stress conditions could be explained by the dimer formation between endogenous Mdm2 and exogenous Mdm2 RING domain, which led to inhibition of Mdm2 autoubiquitination activity and its degradation. The cellular response to Mdm2 RING overexpression revealed that Mdm2 RING acted in a dominant negative fashion to suppress endogenous Mdm2 E3 ligase function and specifically in presence of a DNA-damaging signal prevented Mdm2-mediated inhibition of the p53 gene transcription activity.Under normal and stress conditions, stabilization of endogenous Mdm2 and MdmX as a result of MdmX RING overexpression was observed, whereas there was no effect on p53, phosphorylated p53, and Bax levels. The MdmX RING domain was shown to interact with endogenous full-length Mdm2 and MdmX. Stabilization of endogenous Mdm2 and MdmX could be a consequence of inhibition of the Mdm2 E3 ligase activity by means of complex formation between the ectopically expressed MdmX RING and endogenous Mdm2. These findings indicated that the exogenous MdmX RING domain, like Mdm2 RING, exhibited a dominant negative phenotype inhibiting Mdm2 ubiquitination activity, which in turn resulted in p53 stabilization.Note that despite the stabilization effect of p53 by overexpression of Mdm2 and MdmX RINGs, they enhanced the clonogenicity of tumor cells in a colony-forming assay. The molecular basis for p53-independent tumor-promoting activity of the Mdm2/X RING domains has not been well studied. However, increasing number of studies revealed p53-independent roles of Mdm2 and MdmX in tumor cells. Indeed, Mdm2 RING possesses nucleotide-binding activity . Acting Collectively, this study implies that Mdm2 RING and MdmX RING function differently but inter-connectively in cells depending on the presence or absence of stress stimuli . As expeHuman full-length Mdm2, full-length MdmX, Mdm2 RING (residues 417–491), and MdmX RING (residues 416–490) domains were cloned into pcDNA3.1/YFP, pcDNA3.1/CFP, and pc-DNA3.1/FLAG expression vectors using a standard PCR subcloning technique.Human osteosarcoma U2OS cells were grown in McCoy’s medium supplemented with 10% FBS. Cells were transfected with plasmids (10 µg of DNA per 10 cm tissue culture plate) using PolyJet transfection reagent .The antibodies for immunoblotting and co-immunoprecipitation were mouse monoclonal against Mdm2 , MdmX , p53 , actin , GAPDH , YFP-tag (Abm G163), FLAG-tag (Sigma F3165), rabbit monoclonal against phosphorylated p53 (serine 15) , p21 , Bax , and rabbit polyclonal against FLAG-tag (Bethyl Laboratories A190-102A). To detect proteins of interest HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch 115-035-166) and anti-rabbit IgG (Jackson ImmunoResearch 111-035-003) antibodies were used.U2OS cells were transfected with YFP (empty vector), CFP-Mdm2 FL, CFP-MdmX FL, YFP-Mdm2 RING, and YFP-MdmX RING or FLAG (empty vector), FLAG-Mdm2 FL, FLAG-MdmX FL, FLAG-Mdm2 RING, and FLAG-MdmX RING. Cells were harvested 24 h post-transfection and lysed in RIPA buffer ) followed by 2 sec sonication time with 10% amplitude. Supernatants were boiled in SDS sample buffer for 5 min at 95 °C. Samples were resolved on 12% or 7.5% SDS-PAGE, and immunoblotting was performed using antibodies described above.U2OS cells were transfected with CFP-Mdm2 FL, CFP-MdmX FL, YFP-Mdm2 RING, and YFP-MdmX RING and grown on coverslips. The 24-hour post-transfection cells were fixed in 1% paraformaldehyde in PBS and permeabilized with 0.5% Triton-X in PBS. Cells were washed with PBS two times. Images were obtained using Zeiss LSM 700 Laser Scanning microscope and LSM software ZEN 2010 .U2OS cells were transfected with CFP-Mdm2 FL and either FLAG-Mdm2 RING or FLAG-MdmX RING, CFP-MdmX FL, and either FLAG-Mdm2 RING or FLAG-MdmX RING to detect protein–protein interactions. Cells were harvested 24 hours post-transfection and lysed in RIPA buffer  followed by 2 sec sonication time with 10% amplitude. Cell lysates were incubated with a rabbit polyclonal anti-FLAG tag primary antibody for 2 hours followed by addition of pre-cleared protein A/G Plus-Agarose beads  for 1 hour. Immunoprecipitates were washed with RIPA buffer three times and then boiled in SDS sample buffer for 5 min at 95 °C to release protein complexes from the beads. Samples were resolved on 12% SDS-PAGE, and immunoblotting was performed using antibodies described above.U2OS cells were transfected with pcDNA3.1/FLAG empty vector, FLAG-Mdm2 FL, FLAG-Mdm2 RING, FLAG-MdmX FL, and FLAG-MdmX RING. Cells were harvested after 24- hour transfection and total RNA isolated with Trizol . cDNA was generated using Invitrogen SuperScript Reverse Transcriptase kit. SYBR green kit (SSoFast EveGreen Supermix) from Biorad  was used for qPCR. Relative mRNA expression levels were determined using ΔΔCt method. The following oligonucleotides were used:CDKN1A (5’TGAGCGATGGAACTTCGACT3’ and 5’TTAGGGCTTCCTCTTGGAGA3’), BAX (5’AGACAGGGGCCCTTTTGCTTC3’and 5’TGCAGCTCCATGTTACTGTCC3’).GAPDH (5’AAGGTCATCCCTGAGCTGAAC3’ and 5’ACGCCTGCTTCACCACCTTCT3’).U2OS cells were transfected with pcDNA3.1/FLAG empty vector, FLAG-Mdm2 FL, FLAG-Mdm2 RING, FLAG-MdmX FL, and FLAG-MdmX RING. Following a 24-hour transfection, cells were fixed in 70% ethanol. After fixing, cells were washed with a staining buffer . Cells were mixed with staining buffer and RNAseA (100 µg/mL). Cells were stained by propidium iodide  for 1 hour. Samples were analyzed in a BD FACScalibur. Analysis was performed in CellQuest Pro .t-test was used to compare the values between two groups and one-way analysis of variance (ANOVA), was used to compare the multiple groups. p < 0.05 was considered statistically significant.U2OS cells were transfected with pcDNA3.1/FLAG empty vector, FLAG-Mdm2 FL, FLAG-Mdm2 RING, FLAG-MdmX FL, and FLAG-MdmX RING. Following a 24-h transfection, 800 viable cells were seeded in 3-cm cell culture dishes in triplicate for each transfection event. After 10 days, the cells were fixed and stained by methylene blue dye . Colonies larger than 1 mm were counted. All the experiments were done in triplicate in each group and repeated. Statistical analyses were performed by using Sigma Stat software. All of the data was represented as Mean ± SEM. Student’s