The study aims to describe the evidence-based management and controversies in blunt splenic trauma.A shift from operative management to non-operative management (NOM) has occurred over the past decades where NOM has now become the standard of care in haemodynamically stable patients with blunt splenic injury. Splenic artery embolisation (SAE) is generally believed to increase the success rate of NOM. Not all the available evidence is that optimistic about SAE however. A morbidity specifically related to SAE of up to 47% has been reported. Although high-grade splenic injury is a prognostic factor for failure of NOM, an American research group has published a study in which NOM is performed in over half of haemodynamically stable patients with grade IV or V splenic injury without leading to an increased morbidity (in terms of complications) or mortality. Another area of current investigation in the literature is the exact indication for SAE. Although the generally accepted indication is the presence of vascular injury, a topic of current investigation is whether there might be a role for pre-emptive embolisation in patients with high-grade splenic injury. On the other hand, evidence is also emerging that not all blushes require an intervention . Lastly, the available evidence shows that splenic function is preserved after embolisation, and therefore, the routine administration of vaccinations seems not to be necessary. There might be a difference between proximal and distal embolisations; however, with regard to splenic function, in favour of distal embolisation.Nowadays, NOM is the standard of care in haemodynamically stable patients with blunt splenic injury. The available evidence  shows that splenic function is preserved after NOM, a major advantage compared to splenectomy. SAE is used as an adjunct to observation in order to increase the success rate of NOM. Operative management should be applied in case of haemodynamic instability or if associated intra-abdominal injuries requiring surgical treatment are present. Patient selection  is an ongoing subject of further research. Future studies should also focus on long-term outcomes of patients treated with embolisation . Nearly 199,800 people die from injury each year—1 person every 3 min . The preIn the past decades, the treatment of patients with blunt splenic injury has shifted from operative to non-operative management (NOM). Operative management was applied because of the belief that the spleen was an organ without function, that it could not heal on its own and could rupture in a later stage. Also, the mortality rate of patients who were not operated was unacceptably high, whereas patients who were operated had a change of survival . NowadayIn this review, evidence-based management and controversies in blunt splenic trauma are discussed and future perspectives are highlighted.NOM consists of close observation of the patient and can be supplemented with SAE if necessary. Observational management involves admission to a medium or intensive care unit with close monitoring of vital signs, bed rest, frequent monitoring of hemoglobin concentration, and serial abdominal examinations . TherefoIndications for SAE are the presence of vascular injury such as contrast extravasation (i.e. a blush), a cut-off, pseudoaneurysms, or arteriovenous fistula. Different types of embolisation exist: proximal  or distal  embolisation.Embolisation is generally believed to increase the success rate  of observation . HoweverMore recently, critical evidence appeared with regard to SAE. Chastang et al.  questionThe failure rate of NOM is around 10%. Early identification of patients at high risk for failure of NOM  SAE, or delayed splenectomy) is essential since delay in recognition and treatment of late splenic ruptures leads to increased resource use, morbidity, and mortality (preventable deaths) , 16. ManAlthough NOM is more likely to fail in patients with high-grade splenic injury, high-grade splenic injury  is not necessarily a contraindication for NOM. Scarborough et al. recently published data of 1516 patients (propensity-matched cohort) with high-grade splenic injury (grades IV and V) in which NOM was compared to immediate splenectomy. NOM was attempted in over half of the patients (52.2%). Interestingly, in approximately 20% of the patients with a NOM attempt, splenectomy was ultimately required. While increasing patient age, hypotension on admission, the presence of any underlying bleeding disorder, and grade V splenic injury (compared to grade IV) increased failure rate, the only protective factor against failure of NOM was the use of SAE. Delay in operative management  did not affect mortality (6.4% in failure of NOM group vs 16.4% in immediate splenectomy group) or the number of (non)infectious complications, but the hospital length of stay was significantly longer compared to patients undergoing immediate splenectomy •.An eventual further extension of SAE (not only in case of the presence of vascular injury) is currently being investigated by Gaarder et al. from Oslo. They are preparing a randomised controlled (multicentre) trial in which failure rate (due to splenic bleeding) of NOM is compared between haemodynamically stable patients with high-grade (IV or V) splenic injury undergoing pre-emptive SAE and patients not undergoing SAE.As important as recognising the risk of failure is being aware of the time interval between trauma and failure. Different research groups have investigated this topic. Peitzman et al.  reportedThe value of routine follow-up imaging has been debated in the literature. Some authors use routine follow-up CT scanning (at 48 h) to decrease the failure rate of NOM , 23 or iStreptococcus pneumoniae, Haemophilus influenzae type B, and Neisseria meningitides group C . Th. Th39]. The vast majority (60–90%) of the patients with splenic injury are treated non-operatively with success rates over 80%. A major advantage of NOM could be that splenic function is preserved as is suggested by the currently available literature . However, disadvantages of NOM are the  risk of delayed splenic rupture, the possibility of a re-bleed, and the fact that no view is obtained of other abdominal organs. Especially in patients with multiple organ injuries, the latter should be considered.Prognostic factors for failure of NOM include high-grade splenic injury grade (AAST grade 3 or higher), Injury Severity Score ≥25, and age ≥40 years. SAE can be used as an adjunct to observation in order to increase the success rate of NOM in patients with vascular injury  or maybe even pre-emptively in patients with high-grade splenic injury (currently under investigation). Operative management is still indicated in case of haemodynamic instability (not responding to transfusion) or when associated intra-abdominal injuries requiring surgical treatment are present. It should be noted that in hospitals without sophisticated interventional radiological facilities , NOM should not be chosen over surgical management.The challenge remains to stop the bleeding as quickly as possible by applying the most suitable or least invasive technique, reducing the number of unnecessary splenectomies in a safe manner , thereby maximising patients’ benefit. Patient selection should be an ongoing subject of further research. Future studies should also focus on long-term outcomes  of patients treated with embolisation.


To address this limitation, we provide data on excitation contraction coupling, cardiomyocyte contraction and relaxation, and Ca2+ handling with more spontaneous Ca2+ release events, increased diastolic Ca2+, lower Ca2+ amplitude, and prolonged time to diastolic Ca2+ removal in MI (P < 0.01). Significantly reduced transverse‐tubule density  and sarcoplasmic reticulum Ca2+ adenosine triphosphatase 2a (SERCA2a) function  in MI cardiomyocytes may explain the findings. Reduced protein phosphorylation of phospholamban (PLB) serine‐16 and threonine‐17 in MI provides further mechanisms to the reduced function.Nine MI patients and eight control patients without MI (non‐MI) were included. Biopsies were taken from the left ventricular myocardium and processed for further measurements with epifluorescence and confocal microscopy. Cardiomyocyte function was progressively impaired in MI cardiomyocytes compared with non‐MI cardiomyocytes when increasing electrical stimulation towards frequencies that simulate heart rates during physical activity (2 Hz); at 3 Hz, we observed almost total breakdown of function in MI. Concurrently, we observed impaired Ca2+ handling due to impaired SERCA2a activity caused by a combination of alteration in the PLB/SERCA2a ratio and chronic dephosphorylation of PLB as well as loss of transverse tubules, which disrupts normal intracellular Ca2+ homeostasis and handling. This is the first study that presents these mechanisms from viable and intact cardiomyocytes isolated from the left ventricle of human hearts at mid‐stage of post‐MI HF.Depressed cardiomyocyte contraction and relaxation were associated with impaired intracellular Ca Several mechanisms are responsible for this: disrupted cleft spacing between the L‐type Ca2+ channel and ryanodine receptors 2 (RyR2) by reduced density of transverse (T) tubules;2+ sensitivity leading to increased spontaneous Ca2+ release events from SR causing after‐depolarization and trigger arrhythmias;2+ adenosine triphosphatase 2a (SERCA2a) and increased Na+/Ca2+ exchanger activities.2+ uptake at increased stimulation frequencies, is especially important since this leads to a blunted frequency‐dependent acceleration of relaxation (FDAR), thereby impairing diastolic filling at high heart rates.The prevalence of heart failure (HF) in Western countries is at present known to be over 23 million worldwide.2+ handling after MI, data from patients with post‐MI HF are limited, and especially, data from earlier pre‐terminal stages are almost absent. Given that coronary artery disease and MI are the most common causes of HF, data from the heart of patients with post‐MI HF at earlier stages of the disease are needed. When a targeted treatment focused on intracellular molecules and physiologic processes that directly cause dysfunction in HF is aimed for, it will be necessary to confirm mechanisms of failure as well as improvement in human cardiomyocytes at stages of HF where rescue may still be possible. Therefore, in order to determine cardiomyocyte contraction and relaxation, Ca2+ handling, and T‐tubule density in addition to key protein regulations involved in the progression towards heart failure, we analysed cardiomyocytes freshly isolated from the intact left ventricular myocardium of post‐MI patients at mid‐stages of HF.However, all the presented findings are only predicted from experimental animal models and investigated in explanted human hearts at the terminal end stage of HF, a condition in which all interventions have failed to restore viable pump function. Despite research efforts on mechanisms of deteriorated cardiomyocyte contraction and relaxation and CaTableNine post‐MI patients with reduced left ventricle ejection fraction (EF < 35%) and eight patients without previous MI and normal EF (EF > 60%) (hereafter designated as control patients) scheduled for elective coronary artery bypass grafting (CABG) at the Department of Cardiothoracic Surgery, St. Olavs University Hospital, Trondheim, Norway, took part in the study. All patients included were on optimal treatment Table. The stuAll biopsies were sampled after sternotomy and pericardiotomy during CABG surgery. Biopsies were taken before aortic cross‐clamping from non‐fibrotic and viable left ventricle mid‐myocardium in the remote non‐infarcted area between the apex and base using a BioPince™ needle from Angiotech Pharmaceuticals, Inc . All perioperative procedures were performed according to standard routines of the department. Biopsies were put into an ice‐cold stabilizing physiological solution for further processing of cells and fibres within 20–30 min, whereas biopsies for biochemical analyses were snap‐frozen in liquid nitrogen within 30 s.2+‐free HEPES‐based solution for free dissection of fibres by fine forceps under a microscope. Samples were then transferred to 95% O2 and 5% CO2 gassed Ca2+‐free Krebs–Henseleit solution with collagenase Type 2  and essential fatty‐acid‐free bovine serum albumin (Sigma) that was shaken for 20 min at 37°C. Next, the biopsies were filtered through a 150 μm masked nylon mesh and thereafter centrifuged at 60 g at 20°C for 30 s, whereafter the supernatant was removed and the pellet resuspended in a HEPES‐based solution with a stepwise increase in Ca2+ concentration (0.1–1.8 mmol/L) with centrifugation between each step.The biopsies were put into a Ca2+ concentration was measured by counting 510 nm emission with a photomultiplier tube  after exciting with alternating 340 and 380 nm wavelengths (F340/380 ratio) . During the stimulation protocol, cells were continuously perfused with a normal physiological HEPES‐based solution (1.8 mmol/L Ca2+) in room temperature of 22 ± 0.5°C.Fura‐2/AM‐loaded  cardiomyocytes were stimulated by bipolar electrical pulses with increasing frequency (1–3 Hz) on an inverted epifluorescence microscope , whereupon cell shortening was recorded by video‐based myocyte sarcomere spacing . Intracellular CaIsolated quiescent cardiomyocytes loaded with the membrane‐specific Di‐8‐ANEPPS dye  were confocal imaged to study T‐tubules throughout the cell. The relative density of T‐tubules normalized to cell size was obtained from five images per cell, captured from the middle of each cell and analysed using a custom‐made application in IDL 6.0 , by counting pixels stained with the dye relative to the total number of pixels after removing pixels associated with the non‐T‐tubular sarcolemma.2+, oxalate (10 mM) to stabilize intra‐SR Ca2+, and ruthenium red (3 μM) to inhibit SR Ca2+ efflux and mitochondrial partitioning. The experiment was initiated by stirring tissue samples in a 150 μL cuvette while monitoring extra‐SR Ca2+ using the optical system described above and adding 50 μM Ca2+. Ca2+ loading induced an immediate increase in Ca2+ concentration, followed by a decline that was attributed to SERCA2a Ca2+ uptake. The ratio signal was converted to total Ca2+ concentration by measuring Rmin and Rmax in each tissue sample, and the rate of SR Ca2+ uptake was subsequently plotted against the corresponding free Ca2+. A logistic curve was fitted to estimate maximal Ca2+ uptake rate (Vmax).Biopsy samples were excised and permeabilized by saponin , before being transferred to an adenosine triphosphate solution containing (mmol/L) 0.05 egtazic acid, 5 adenosine triphosphate, 10 CrP, 25 HEPES, 100 KCl, and 5.5 MgCl. We added Fura‐2  to probe Cag at 4°C, whereupon the supernatant was removed. The lysates were then loaded onto 8% SDS‐PAGE for detection of phospho‐Thr‐286‐CaMKII  and SERCA2a  and on 12% SDS‐PAGE for total phospholamban (PLB), phospho‐Thr‐17‐PLB, and phospho‐Ser16‐PLB . α‐Actin  was used to normalize protein levels. Proteins were transferred onto nitrocellulose membranes , and membranes were blocked with TBS‐T/milk for 1 h at room temperature followed by overnight incubation with the mentioned antibodies. For protein detection, goat antirabbit or antimouse horseradish‐peroxidase‐conjugated secondary antibodies  and enhanced chemiluminescence  were used. Densitometric analysis was performed using ImageJ software .Biopsies were homogenized with radioimmunoprecipitation assay buffer  and centrifuged for 10 min at 20 000 P < 0.05 was considered statistically significant.Data are shown as mean ± standard deviation unless otherwise stated. The Mann–Whitney U test was used to identify statistical differences between the groups. P < 0.001, FigureP < 0.01, Figure2+ transient amplitude was significantly impaired at 0.5–2 Hz stimulation in MI patients, with as much as a ~45% reduction at 2 Hz  was markedly slower, with a difference of 15% (not significant) at 0.5 Hz, increasing to 20% and 21% at 1 and 2 Hz, respectively , but in contrast to cardiomyocytes from non‐MI, we found a negative shortening–frequency relationship in MI cardiomyocytes: at a 2 Hz stimulation rate, cardiomyocyte shortening was significantly depressed , where the most marked reduction was found in the mid‐regions of the cardiomyocyte .Because rapid and controlled activation of the CaP < 0.01, FigureA).The time from peak cardiomyocyte contraction to re‐lengthening provides a measure of diastolic cardiomyocyte relaxation. At 2 Hz stimulation, time to 50% re‐lengthening was 41% longer in MI cardiomyocytes, indicating severe diastolic dysfunction , suggesting impaired adaptability at higher heart rates in these patients.Under normal conditions, negative FDAR with increasing heart rates accommodates adequate ventricular filling with shorter diastolic periods. We found negative FDAR in both non‐MI and MI cardiomyocytes at slow stimulation rates . When stimulation frequency is increased further , a marked shift occurred; whereas non‐MI cardiomyocytes further reduced time to 50% re‐lengthening, the MI cardiomyocytes did not Figure3A, su2+. Time to 50% Ca2+ decay was 33% (P < 0.01), 30% (not significant), and 40% (P < 0.01) longer at 0.5, 1, and 2 Hz, respectively, in MI cardiomyocytes . Moreover, the effect of increasing stimulation frequencies on frequency‐dependent acceleration of Ca2+ decay was similar to those observed for FDAR, suggesting that observed differences between groups are due to abnormal Ca2+ handling, which was further evidenced by significantly higher diastolic Ca2+ levels in MI cardiomyocytes .Cardiomyocyte relaxation during diastole is controlled by the removal of cytoplasmic Ca2+ transient decay during diastole is SERCA2a.2+ removal via SERCA2a was 26% lower in MI samples , while Ca2+ sensitivity of SERCA2a was 24% reduced . Cardiomyocyte cytosolic Ca2+ concentrations during diastole were significantly higher in MI hearts; 12% (P < 0.05), 16% (P < 0.01), and 20% (P < 0.05) at 0.5, 1, and 2 Hz, respectively .In human cardiomyocytes, the main contributor to CaP = 0.11 and 0.055, respectively)  Figure5. Nota2+ releases between regular electrical stimulated contractions; as much as 90% of the total number of cells had spontaneous Ca2+ release events between regular twitch stimulations at 0.5 Hz, in contrast to only 20% in non‐MI cardiomyocytes ; only 20% of MI cardiomyocytes followed stimulation at 3 Hz, in contrast to 80% in non‐MI  substantiates deterioration of contractile and electrical properties in MI.The corresponding changes of reduced contractility and intracellular Ca2+ uptake and are therefore integral for treatment to succeed. One key mechanism for the regulation of SERCA2a is PLB that upon inhibitory binding to SERCA2a inhibits the Ca2+ transport of the pump; therefore, we analysed expression levels of PLB and determined the ratio between the two. This comparison did not strengthen our explanation of reduced SERCA2a function as PLB was unchanged between the groups and the ratio between PLB/SERCA2a only revealed a strong tendency (P = 0.055). PLB interaction with SERCA2a relies, however, on the phosphorylation status of PLB. We found a significant lower expression of phosphorylated PLB at both Ser‐16 and Thr‐17 in MI hearts. Phosphorylation of both the protein kinase A (PKA) site Ser‐16 and the CaMKII site Thr‐17 relieves the PLB inhibition of SERCA2 and thus increases its capacity to remove cytosolic Ca2+. Although reduced PLB phosphorylation is regarded as an important mechanism of lower SERCA2a activity in HF, conflicting results exist from both animal models and the limited work done in human HF; human data from explanted dilated cardiomyopathy hearts with terminal HF found both Ser‐16 and Thr‐17 phosphorylations of PLB to be reduced,Despite the direct recordings of impaired SERCA2a function, we did, however, only observe a tendency towards reduced SERCA2a protein expression in the left ventricle from MI patients. Limited data exist on the regulation of SERCA2a from human sources, and all data are from explanted hearts at terminal end‐stage HF, that is, a substantially different cohort than the current patients. Notwithstanding this, controversies exist regarding regulation of SERCA2a proteins, where several reports show no change of SERCA2a protein in HF,2+ concentration. Our data display significantly reduced pCaMKII in post‐MI HF that may further explain the dephosphorylated Thr‐17 PLB in these patients. The reduced CaMKII phosphorylation in mid‐phase stage HF patients in the present study is not easy to reconcile with earlier studies from both experimental animal models  dissociation from the RyR, with subsequent diastolic SR Ca2+ leak. This initial observation was followed up by a series of reports supporting the hypothesis,2+ release events, given that pPLB Ser‐16 (which is the PKA phosphosite of PLB) was almost undetectable in biopsies from MI patients. Other candidates therefore include accessory protein‐mediated effects; for example, S100A and sorcin are both Ca2+ binding proteins known to modulate RyR open probability independent of phosphorylation status.To further determine the upstream signalling of reduced PLB phosphorylation at Thr‐17, we analysed the activity of CaMKII by measuring phosphorylated CaMKII at the Thr‐286 site. pCaMKII‐Thr‐286 increases the affinity of the CaMKII complex and traps CaMKII on the autophosphorylated subunit. As a result, the kinase retains close to full activity as long as CaM is trapped, regardless of the Ca2+ release during systole,2+ cycling and contractile failure.2+ transient may indicate reduced coupling between membrane excitation and SR Ca2+ release, because of disrupted cleft spacing between the L‐type Ca2+ channels and RyRs.The presence of a dense network of T‐tubules connected to the plasma membrane ensures a rapid and coordinated RyR CaIn conclusion, for the first time, viable and intact cardiomyocytes have successfully been isolated from the mid‐myocardium in remote, non‐infarct areas of the left ventricle of human hearts at a stage of post‐MI and mid‐phase of HF, before the condition has decompensated and reached end‐stage HF. This is an obvious departure from previously published studies of HF in human patients2+ handling dysfunction. Depressed cardiomyocyte function was associated with impaired systolic and diastolic intracellular and SR Ca2+ handling, probably due to impaired SERCA2a activity. This results from a combination of alteration in PLB/SERCA2a ratio and chronic dephosphorylation of PLB as well as loss of T‐tubules, which disrupts normal intracellular Ca2+ homeostasis and handling. To our knowledge, these mechanisms are not previously explored in human left ventricle myocardial samples from MI patients at mid‐stage of HF. The controversies existing on the cellular mechanisms explored in the present paper further highlight the importance of considering both the stages and the aetiology of the disease.Functional assessments of the human left ventricle cardiomyocytes in the current study reveal contractile and intracellular CaNone declaredK.G. Jebsen Family Foundation; Norwegian Council of Cardiovascular Disease, the Norwegian Research Council (Young Outstanding Investigators); Funds for Cardiovascular and Medical Research at St Olav's University Hospital, Trondheim; Simon Fougner Hartmanns Family Foundation; The British Heart Foundation; Fondation Leducq Award to the Alliance for Calmodulin Kinase Signaling in Heart Disease; The Liaison Committee between the Central Norway Regional Health Authority (RHA); the Norwegian University of Science and Technology (NTNU).


We review a recently discovered developmental mechanism. Anterior-posterior positional information for the vertebrate trunk is generated by sequential interactions between a timer in the early nonorganizer mesoderm (NOM) and the Spemann organizer (SO). The timer is characterized by temporally collinear activation of a series of Hox genes in the early ventral and lateral mesoderm  of the Xenopus gastrula. This early Hox gene expression is transient, unless it is stabilized by signals from the SO. The NOM and the SO undergo timed interactions due to morphogenetic movements during gastrulation, which lead to the formation of an anterior-posterior axial pattern and stable Hox gene expression. When separated from each other, neither the NOM nor the SO is able to induce anterior-posterior pattern formation of the trunk. We present a model describing that the NOM acquires transiently stable hox codes and spatial collinearity, and that morphogenetic movements then continually bring new cells from the NOM within the range of SO signals that cause transfer of the mesodermal pattern to a stable pattern in neurectoderm and, thereby, create patterned axial structures. In doing so, the age of the NOM, but not the age of the SO, defines positional values along the anterior-posterior axis. We postulate that the temporal information from the NOM is linked to mesodermal Hox expression. The role of the SO for trunk patterning turns out to be the induction of neural tissue as prerequisite for neural hox patterning. Apparently, development of a stable anterior-posterior pattern requires neural hox patterning. We believe that this mechanism represents a developmental principle.


MT). In comparison to the branches of cerebral arteries (CA) showing prominent MT, the third branches of mesenteric arteries (MA) with similar diameters show weaker MT. Here, we aimed to analyze the electrophysiological differences responsible for the weaker MT in MA (MAMT) than MT in CA (CAMT). We measured ionic current using patch clamp in isolated MA smooth muscle cells (MASMCs) and CA smooth muscle cells (CASMCs) of rats. MT was analyzed using video analysis of pressurized small arteries. Quantitative‐PCR (q‐PCR) and immunofluorescence confocal microscopy were used to compare the mRNA and protein expression level of big‐conductance Ca2+‐activated K+ channel (CBKa) subunits (Slo1α and Sloβ1). Whole‐cell patch clamp study revealed higher density of voltage‐operated Ca2+ channel current (CIaV) in the MASMCs than in CASMCs. Although voltage‐gated K+ channel current (KIv) was also higher in MASMCs, treatment with Kv inhibitor (4‐aminopyridine) did not affect MAMT. Interestingly, CBKa current density and the frequency of spontaneous transient outward currents (STOCs) were consistently higher in MASMCs than in CASMCs. Inside‐out patch clamp showed that the Ca2+‐sensitivity of CBKa is higher in MASMCs than in CASMCs. Iberiotoxin, a selective CBKa inhibitor, augmented MAMT by a larger extent than CAMT. Although q‐PCR analysis did not reveal a significant difference of mRNAs for Slo1α and Sloβ1, immunofluorescence image suggested higher expression of Slo1α in MASMCs than in CASMCs. Despite the large CIaV density, the high activities of CBKa including the more frequent STOCs in MASMCs veils the potentially strong MTMA.Arterioles and small arteries change their tone in response to transmural pressure changes, called myogenic tone ( Plum). Since the contraction occurs independent of neurohumoral influences, it is named as myogenic tone (MT) of arteries. MT contributes to the peripheral resistance of vascular system, and thus blood pressure  is less consistent in the absence of agonists  and of skeletal arteries  has indicated that MAs are not simple conduit but have active control of resistance Lundgren . In factMA might be due to weak mechanosensation or effector functions in the mesenteric arterial smooth muscle cells (MASMCs). Since the wall tension changes induced by the increased luminal pressure (Plum) is accompanied by membrane depolarization and activation of voltage‐gated Ca2+ channels (CaV) in the smooth muscle cells  and big‐conductance Ca2+‐activated K+ channel (BKCa) are commonly expressed in the most types of arterial myocytes (Standen and Quayle + channels (Kir) also play important roles in the fine regulation of resistance arteries including CA. However, the expression of Kir is not significant in MASMCs  are generally higher in smooth muscle cells than other cell types (Hu and Zhang Ca are concertedly activated by increased cytosolic Ca2+ concentration ([Ca2+]c) and membrane depolarization. BKCa activity not only reflects the overall [Ca2+]c, but also the dynamic changes of subsarcolemmal concentration [Ca2+]sl. From the early period of patch clamp studies in both visceral and vascular smooth muscle cells, spontaneous transient outward currents (STOCs) lasting about 100 msec have been observed under physiological intracellular Ca2+ buffering conditions. The STOCs are thought to represent the simultaneous opening of up to a hundred BKCa  than skeletal arterial smooth muscle cells (SkASMCs)  rats (8 weeks old) were fully anesthetized with pentobarbital sodium (100 mg/kg ip) and immediately sacrificed by decapitation. And their brain and gastrointestinal tract with mesentery arcade were moved into a normal Tyrode's (NT) solution. The study protocol was in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , and also conforms to the Institutional Animal Care and Use Committee (IACUC) in Seoul National University μm) were carefully dissected under the stereomicroscope. The arterial segments were placed in a glass‐bottomed vessel chamber . The vessel chamber contained NT and its composition was as follows (in mmol/L): 141.4 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 Glucose, and 10 HEPES (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid), adjusted to pH 7.4 with NaOH. The temperature was set at 37–38°C using a controller . The vessels were cannulated using glass micropipettes and secured with 12–0 nylon suture. Inner diameters (Din) of pressurized vessels were measured using a UBS CCD camera  and DMTVAS 6.2 software . The value of luminal pressure (Plum) was set to 60 mmHg for at least an additional 40 min; spontaneous tone  developed within this time frame and reached a steady state. Arterial segments without stable tone were discarded. After confirming the generation of myogenic tone, Plum was lowered to 30 mmHg and incubated for equilibrium, the increase to 60 and 100 mmHg evaluate the MT. On the step‐like increase in Plum, Din initially increased and then slowly decreased. To quantify MT of arteries, the maximal passive diameter  was measured in Ca2+‐free NT solution (0Ca‐NT) with 1 mmol/L EGTA (ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid). The maximal increase in Din (ΔDmax) by changing to the 0Ca‐NT was divided by Dmax,0Ca , resulting the MT (%) at each Plum.The third and fourth‐order branches of mesenteric arteries and the first‐branches of posterior and cerebellar arteries  for 10–15 min and changed to the digestion medium II (0Ca‐NT solution containing 3 mg/mL collagenase) for another 10–15 min. Both digestion media contained bovine serum albumin (1 mg/mL) and dithiothreitol (1 mg/mL). The enzyme‐treated arteries were then gently agitated using a fire‐polished glass pipette and in a KIsolated cells were transferred to a small chamber (0.2 mL) on the stage of an inverted microscope . The conventional whole‐cell or inside‐out configuration was performed with a patch‐clamp amplifier  and voltage‐clamp experiments were performed at room temperature (22–25°C). Membrane currents were recorded using the glass microelectrode with a resistance of 2–2.5 MΩ and 7–8 MΩ for whole‐cell and inside‐out patch clamp recordings, respectively. The signals were filtered at 5.0 kHz and sampled at a rate of 2.0 kHz and 10.0 kHz for whole‐cell and inside‐out patch clamp recordings, respectively. The pCLAMP software v.9.2 and Digidata‐1440A (Axon Instruments) were used to acquire data and apply command pulses.2, 10 tetraethylammonium chloride (TEA‐Cl), 0.5 MgCl2, adjusted to pH 7.4 with NaOH. The pipette solution for recording the CaV current contained (in mmol/L): 20 CsCl, 10 HEPES, 1 MgCl2, 5 EGTA, 120 Aspartic acid (Asp), 3 Mg‐ATP, adjusted to pH 7.2 with CsOH. To record the Kv current (whole‐cell), the NT solution contained 1 mmol/L TEA was used as bath solution. The pipette solution for recording the Kv contained the following (in mmol/L): 20 KCl, 10 HEPES, 1 MgCl2, 5 EGTA, 120 Asp, 3 Mg‐ATP, adjusted to pH 7.2 with KOH. To investigate the BKCa current (whole‐cell), we used the NT as bath solution. The pipette solution contained (in mmol/L): 20 KCl, 10 HEPES, 1 MgCl2, 120 Asp, 3 Mg‐ATP; pH was adjusted to 7.2 with KOH. And free Ca2+ activity in the pipette solution was clamped to 1 μmol/L using 10 mmol/L EGTA and CaCl2 with WinMAX program . For recording STOCs (whole‐cell), we used the NT as bath solution. The pipette solution contained (in mmol/L): 20 KCl, 10 HEPES, 1 MgCl2, 0.1 EGTA, 120 Asp, 3 Mg‐ATP; pH was adjusted to 7.2 with KOH. To analyze the single‐channel conductance of BKCa (inside‐out), the bath solution contained the following (in mmol/L): 140 KCl, 10 HEPES, 10 Glucose, 1 MgCl2; pH was adjusted to 7.2 with KOH. And we added 10 mmol/L EGTA and CaCl2 in the bath solution to fix at 1 μmol/L free Ca2+. The pipette solution contained the following (in mmol/L): 140 KCl, 10 HEPES, 10 Glucose, 1 MgCl2; pH was adjusted to 7.4 with KOH. To investigate Ca2+‐dependent activation of BKCa (inside‐out), the bath solution contained (in mmol/L): 140 KCl, 10 HEPES, 10 Glucose, 1 MgCl2, adjusted pH to 7.2 with KOH; variable amounts of CaCl2 were added to the bath solution to obtain 3, 10, and 30 μmol/L free Ca2+. We made 3 and 10 μmol/L free Ca2+ solution using 10 mmol/L of N‐(2‐Hydroxyethyl) ethylenediamine‐N,N′,N′‐triacetic acid (HEDTA) and CaCl2 calculated with the WinMAX program. For making 30 μmol/L free Ca2+ solution, we simply added CaCl2 to the bath solution. We used NT as pipette solution.The name and composition of the experimental solutions used in the electrophysiological recording are as below. The bath solution for recording CaV (whole‐cell) current contained the following (in mmol/L): 120 NaCl, 5 CsCl, 10 HEPES, 10 Glucose, 10 BaClCaα‐subunit ; BKCaβ1‐subunit . cDNAs were amplified with SYBR Green  using a real‐time PCR system  under the conditions 95°C for 10 min, 40 cycles at 95°C for 10 sec, 55°C for 15 sec, and 72°C for 20 sec. After the final PCR cycle, a melting curve analysis was performed to analyze the specificity of the reaction. Relative gene expression was calculated using the comparative threshold (Ct) method (2−∆∆Ct).Total RNA was extracted using TRizol reagent (Invitrogen), and its purified concentration was determined NanoDrop at 260 nm. The extracted RNA was reverse transcripted into cDNA using the PrimeScript RT Master Mix Perfect Real Time (Takara) according to the manufacturer's instructions. The cDNA was amplified using the primers as follows: GAPDH  or anti‐Sloβ1‐subunit  overnight at 4°C. The channels expression levels were detected using Alexa Fluor 488‐conjugated goat anti‐rabbit secondary antibody . Imaged were acquired with Olympus FV1000 confocal microscope with a ×100 oil immersion objective.Fresh isolated MASCMs and CASMCs were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X‐100 for 15 min at room temperature. Then cells were blocked with 10% FBS for 1 h at room temperature, and incubated with primary antibodies of anti‐Slo1All drugs and chemicals used in this study were purchased from Sigma Chemical Co.  excluding iberiotoxin. Iberiotoxin was purchased from Tocris .n. Student's unpaired t‐test was used to test for significance at the level of 0.05.Data are shown as means ± SEM with the number of tested arteries indicated as in during the step‐like increase Plum from 30 to 60 and 100 mmHg showed rebound contractions following initial passive dilation, consistent with the presence of MT . We also compared MTMA with the MT of coronary and deep femoral arteries; MTMA was consistently lower than the MT from the other types of arteries . Throughout the tested voltages, the density of ICaV (pA/pF) was larger in MASMCs than CASMCs  was about twofold higher in MASMCs than CASMCs  and normalized to the maximum conductance at +80 mV (g/gmax). The conductance to voltage relations were fitted to Boltzmann equation to obtain the half‐activation voltages, that were not different between MASMCs and CASMCs  was applied. The amplitudes of IKv was decreased to about 50% of control by 1 mmol/L of 4‐AP, respectively , and MT levels with or without 1 mmol/L TEA and 100 nmol/L iberiotoxin were summarized as bar graphs  seem to be same between MASMCs and CASMCs (Xia et al. The higher density of MCs Fig. C and D. ent Fig. B, the α‐Ca activity in rat CASMCs is higher than skeletal arterial myocytes that was ascribed to the higher ratio of β1‐subunit over α‐subunit of Slo1 (Yang et al. α and β1 subunits between MASMCs and CASMCs (Fig. Ca subunits. Although the confocal images require careful interpretation in terms of the protein expression levels, the experimental results suggest that Slo1α expression appeared higher in MASMCs than CASMCs (Fig. β1 signal was more prominently observed than the Slo1α, while no significant difference between MASMCs and CASMCs (Fig. Ca current density might be ascribed to the differential expression of Slo1α. Also, an unidentified difference in channel modulatory factors might underlie the higher Ca2+‐sensitivity of BKCa in MASMCs, which requires further investigation.According to Yang et al., BKMCs Fig. E and F. MCs Fig. A. In botMCs Fig. B. Taken 2+‐sensitivity of BKCa and more frequent generation of Ca2+ sparks in MASMCs. Because the Ca2+ sparks can be triggered by flickering activation of CaV, the higher density of CaV in MASMCs might also contribute to the more frequent STOCs. In contrast to the global [Ca2+]c increase, Ca2+ sparks negatively regulate contraction of arterial smooth muscle (Jaggar et al. 2+ spark coupling in the arterial myocytes of diabetic rat model (Rueda et al. 2+ sparks using confocal microscopy combined with patch‐clamp study might provide more clear evidence of the putative differential STOCs/Ca2+ spark coupling between different arteries.The more prominent STOCs in MASMCs Fig.  might beMA would be physiologically relevant, and the higher activity of BKCa would be a compensatory property to keep the low MTMA despite the higher ICaV. The weak MTMA also implies intrinsically low resistance without vasoactive signals. On the other hand, the low resistance of MA might be more beneficial to effectively redirect the intestinal blood flow for the recruitment under ‘fight and flight’ conditions. Under such conditions, the higher density of IKv might play a role to counterbalance the excessive vasoconstriction of MA (Fig. A previous study of intestinal microcirculation has shown that the microvilli blood flow is effectively autoregulated while less effective in the muscular layer (Davis and Gore  MA Fig. A and B.CA coupled with their parallel arrangement would promote regional regulation of flow with minimal overall changes in blood volume. Also, according to LaPlace's law, the myogenic constriction would effectively decrease the wall tension and thus reduces the stimulus for further constriction. Thus, the more sensitive MTCA might enable to maintain a lower wall tension in CA than the similar sizes of MA. Previously, the mechanism underlying such difference may be related to a higher ICaV in CASMCs (Asano et al. + channels in VSMC might be more critical factor to determine the level of MT.In comparison with the other systemic arteries, sympathetic regulation is minimized in the cerebral resistance arteries, and the MT‐dependent autoregulation seems to be the critical mechanism for the constant cerebral blood flow. The marked MTCa activities. The differential patterns of ion channel currents may provide variability in MT and underlying membrane voltages in the arteries suppling heterogeneous tissues and organs. Physiological and pharmacological modulation of BKCa would be an effective manner to regulate the intestinal blood flow.In summary, our study indicate that MA contain an intrinsic capacity of potent MT that is veiled by higher BKNo conflicts of interest, financial or otherwise, are declared by the authors.


In the failing heart a small population of cardiac ryanodine receptors (RyR2) displays sub-conductance-state gating leading to Ca2+ leakage from sarcoplasmic reticulum (SR) stores, which impairs cardiac contractility. Previous evidence suggests contribution of RyR2-independent Ca2+ leakage through an uncharacterized mechanism. We sought to examine the role of Zn2+ in shaping intracellular Ca2+ release in cardiac muscle. Cardiac SR vesicles prepared from sheep or mouse ventricular tissue were incorporated into phospholipid bilayers under voltage-clamp conditions, and the direct action of Zn2+ on RyR2 channel function was examined. Under diastolic conditions, the addition of pathophysiological concentrations of Zn2+ (≥2 nm) caused dysregulated RyR2-channel openings. Our data also revealed that RyR2 channels are not the only SR Ca2+-permeable channels regulated by Zn2+. Elevating the cytosolic Zn2+ concentration to 1 nm increased the activity of the transmembrane protein mitsugumin 23 (MG23). The current amplitude of the MG23 full-open state was consistent with that previously reported for RyR2 sub-conductance gating, suggesting that in heart failure in which Zn2+ levels are elevated, RyR2 channels do not gate in a sub-conductance state, but rather MG23-gating becomes more apparent. We also show that in H9C2 cells exposed to ischemic conditions, intracellular Zn2+ levels are elevated, coinciding with increased MG23 expression. In conclusion, these data suggest that dysregulated Zn2+ homeostasis alters the function of both RyR2 and MG23 and that both ion channels play a key role in diastolic SR Ca2+ leakage.Aberrant Zn Relaxation of cardiac muscle follows when the concentration of Ca2+ is sufficiently reduced due to the combined action of RyR2 channel closure, extrusion of Ca2+ from the cell, and uptake of Ca2+ back into the SR Ca2+ stores. In addition to Ca2+, the activity of RyR2 is in part regulated by Mg2+, which helps prevent inappropriate channel openings during diastole  is a recently identified voltage-dependent non-selective cation-conducting channel shown to have particular abundance on the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane , 8. Verya2+ leak .2+ homeostasis is associated with dysregulated intracellular Ca2+ release, reduced cardiac contractility, and significantly prolonged elevations of systolic Ca2+ . Interestingly, at this concentration, cardiac RyR2 channels incorporated into planar bilayers respond poorly to systolic concentrations of Ca2+  was studied  from 0.26 ± 0.13 to 0.09 ± 0.01 . In the continued presence of 1 mm Mg2+ the subsequent addition of 0.1 nm Zn2+ to the cytosolic face of the channel caused channels to gate with a comparable Po to Mg2+-free Ca2+-activated control channels . This suggests that Zn2+ plays a key role in regulating channel function enabling RyR2 to operate under conditions of systole. When Zn2+ levels were further incremented in the range 0.1-100 nm, channel activity was significantly increased from Mg2+-treated control channels (A and B). However, there was no significant difference between individual Zn2+ treatments .To determine if Zn studied . Using C studies , 17, the2+ are altered in heart failure and can be chronically elevated by as much as 30-fold (2+ could modulate RyR2 function in the presence of diastolic concentrations of Ca2+ (100 nm) and in the continued presence of 1 mm Mg2+. Using Ca2+ as the permeant ion and holding at a command potential of 0 mV, when the cytosolic free Ca2+ was reduced from 5 μm to 100 nm, as expected, channel Po was reduced (A). Consistent with the role of Mg2+ as an endogenous regulator of channel activity, the addition of 1 mm Mg2+ to the cytosolic face of the channel resulted in a further reduction in channel Po (A). Under these conditions nearly all channel openings were abolished (B). The addition of physiological concentrations of Zn2+ (0.1 nm) did not significantly increase channel activity (A and B). However, elevation of free Zn2+ to pathophysiological levels (∼2 nm) resulted in a significant increase in channel Po (B).In heart failure RyR2 channels become inappropriately activated during diastole. Because levels of Zn 30-fold , we next reduced A. Consisannel Po A. Under bolished B. The adactivity , A and Bannel Po B.2+ under diastolic conditions. Channel gating after the addition of Zn2+ ≤0.2 nm was not significantly different compared with openings from Ca2+-activated control channels (C), suggesting that in the presence of physiological levels of free Zn2+, channels are still regulated by Ca2+. Importantly, elevating the free Zn2+ concentration to 2 nm caused channels to gate to a comparable Po to those activated by systolic levels of Ca2+ , but the mode of gating under these conditions was altered (C). Under these conditions, channels gated in significantly longer-lived open states compared with control. This type of gating is consistent with Zn2+-dependent openings.We next looked at the mean open time of RyR2 gating after the addition of Znchannels C, sugges altered C. Under 2+ currents were observed, 38% (15/39) of our bilayers displayed openings that were not characteristic of RyR2 gating (A). Using Ca2+ as the permeant ion, construction of a current-voltage relationship revealed that this other SR Ca2+-permeable channel displayed a Ca2+ conductance of 51 ± 1.5 picosiemens (A). This is consistent with the single channel Ca2+ conductance reported previously for the SR ion-channel MG23 . In these experiments, reducing the level of Ca2+ to a subactivating concentration (4 nm) by the addition of 1 mm 2,2′-ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid (BAPTA) abolished RyR2 openings as expected, but MG23 openings were still apparent. In the presence of activating levels of Ca2+ (5 μm), the addition of 10 mm caffeine, which is a known activator of RyR2 (C). It has been shown previously that MG23 openings are very fast and frequent and that multiple channels often gate together in the bilayer in a coordinated manner (m caffeine had no effect on channel activity (D). This reveals that the regulation of MG23 and RyR2 is markedly different.In experiments where Ca2 gating A. Using osiemens A. This iand MG23 B. In the of RyR2 , signifi of RyR2 C. It hasactivity D. This r2+ leak in heart failure is widely accepted. Here we investigate if MG23 also contributes to the release of Ca2+ from SR stores alongside RyR2. In heart failure, cardiac cells encounter hypoxic conditions (A). Using the Zn2+-specific fluorescent indicator ZnAF-2 diacetate (DA), we measured the relative cumulative fluorescent intensity and show that intracellular Zn2+ levels in H9C2 cells are elevated when exposed to ischemic conditions (B). This is important as dysregulated Zn2+ homeostasis is linked with ischemia and chronic heart failure . Using Ca2+ as the permeant ion, in the presence of physiological levels of Mg2+ (1 mm) and holding at a command potential of 0 mV, the addition of 0.1 nm Zn2+ to the cytosolic face of MG23 had no significant effect on channel activity (C and D). Interestingly a 10-fold increase in the concentration of Zn2+ from 0.1 nm to 1 nm had a significant effect on MG23 channel activity (D).We next assessed if Zn methods . Construactivity , C and Dactivity D.2+ current in response to elevated Zn2+ levels when gating together with RyR2. In control recordings where both RyR2 and MG23 were gating, using Ca2+ as the permeant ion and holding at a command potential of 0 mV, construction of an all-points histogram from our single-channel data displayed two clear peaks . In control recordings, amplitude histograms were best fit with a mixture of two Gaussian distributions displaying mean values of −0.47 ± 0.16 pA and −4.03 ± 0.23 pA . Amplitude histograms were now best fit with a mixture of three Gaussian distributions (A) and others (m Zn2+ increased RyR2 activity and more events resided in the open state (B). However, only two peaks were ever observed, and data were best fit with a mixture of two Gaussian distributions .We next wanted to investigate if native MG23 channels provide a leak Caar peaks A, left.  0.23 pA . These vird peak A, right.ibutions . The smaibutions A and othd others  report fen state B. Howeveibutions , as theributions B, right.2+ altered the phosphorylation status of RyR2 at site serine 2809. Incubation of isolated SR vesicles with 1 nm Zn2+ had no effect on the level of phosphorylation at site serine 2809 (C).Previous studies have suggested that in heart failure RyR2 becomes hyperphosphorylated at serine 2808/2809, which leads to the dissociation of the FK-506-binding protein 1B (FKBP12.6), resulting in sub-state gating . In contrast, MG23-mediated Ca2+ currents were observed in 22% (n = 5/23) of our bilayer experiments when we used SR vesicles prepared from wild-type mice . In these experiments MG23 channel activity was significantly increased after the addition of 1 nm Zn2+ to the cis chamber . SR vesicles prepared from Mg23 knock-out mice displayed functional RyR2 channel gating , and consistent with our previous findings the addition of 1 nm Zn2+ to the cytosolic face of RyR2 significantly increased channel Po . Construction of all-points-amplitude histograms from our single-channel data showed that the addition of 1 nm Zn2+ to the cis chamber significantly increased RyR2 activity . There was no evidence of RyR2 sub-conductance state gating. All data were best fit with a mixture of two Gaussian distributions representing the closed and fully open state of RyR2 (To confirm that Zn of RyR2 .2+ concentration (100 nm) and the presence of millimolar levels of Mg2+ that prevent unwanted RyR2 channel openings. This is important as even a small increase in the open probability of RyR2 during diastole will lead to a leak of Ca2+ into the cytoplasm, and this will have major consequences for normal cardiac function. Irregular RyR2 activity is linked to various forms of heart failure, but the underlying molecular mechanisms that cause altered RyR2 channel function remain unclear. In this study we reveal that in the presence of diastolic Ca2+ levels (100 nm) and 1 mm Mg2+, small elevations in the level of cytosolic free Zn2+ from resting concentrations (∼0.1 nm) to ≥1 nm results in irregular RyR2 channel openings. This provides the first evidence that dysregulated Zn2+ homeostasis is directly linked with leaky RyR2 channels. We also reveal that in H9C2 cardiac cells the level of intracellular Zn2+ becomes elevated after exposure to hypoxic conditions and that this coincides with increased protein expression of the ER/SR located non-selective cation channel MG23. Furthermore, the activity of MG23 is significantly enhanced when cytosolic Zn2+ is elevated from 0.1 nm to ≥1 nm. MG23 displays Ca2+-handling properties, suggesting that increased channel activity alongside increased channel expression may contribute to SR Ca2+ leak alongside RyR2.During cardiac relaxation, it is the combined effect of both a lowered Ca2+ result in a large release of Ca2+ from SR stores, and this controls contractility. In bilayer studies, however, the presence of millimolar levels of Mg2+ causes RyR2 channels to respond poorly to systolic Ca2+ levels . We recently showed that this mode of gating is consistent with Zn2+-dependent openings and that RyR2 is likely to have multiple Zn2+-binding sites  that move Zn2+ into organelles and out of the cell and ZIPs (Zrt/Irt-like proteins) that mediate the flux of Zn2+ into the cell or from organelles into the cytosol. Recent evidence links increased Zip12 expression in pulmonary vascular smooth muscle to elevated intracellular Zn2+ levels as a result of hypoxia, suggesting a fundamental role for Zn2+ in pulmonary vascular responses  and ZIPs  where RyR2 and MG23 gate together, construction of an all-points amplitude histogram revealed that channel openings resided in two open states, one with a mean current amplitude consistent with the full open state of RyR2 and another with a mean current amplitude that is consistent with the unitary current amplitude of the reported RyR2 sub-conductance state. However, this smaller open state is also consistent with the mean current amplitude that we (A) and others report for MG23 at 0 mV  had no effect on MG23 function, but increasing the Zn2+ concentration to 1 nm significantly increased channel activity. Importantly we also show that intracellular Zn2+ levels are elevated when cardiac H9C2 cells are exposed to ischemic conditions and that this elevation in the level of Zn2+ coincides with an increase in the protein expression of MG23. Importantly, changes in MG23 expression in H9C2 cells after hypoxia are comparable with the reported changes in protein expression of key Ca2+-regulatory proteins including SERCA, phospholamban, and the sarcolemmal Na+/Ca2+ exchanger that occur in heart failure  or Sigma. All solutions were made in de-ionized water, and those for use in bilayer experiments were filtered through a Millipore membrane filter (0.45-μm pore). BAPTA, ATP, and the catalytic subunits of PKA were obtained from Sigma.Mg23 knock-out mice were produced using J1 embryonic stem (ES) cells derived from 129/Sv mice as described previously  used for this study were obtained. To confirm the mouse genotypes, PCR analysis was conducted using the MG23F and MG23R primers as described previously  using previously described techniques (trans chamber was perfused with 250 mm glutamic acid and 10 mm HEPES (pH 7.2) with Ca(OH)2 (concentration of free Ca2+ ∼50 mm). The identity of RyR2 was confirmed by the single-channel conductance, and the number of channels gating in the bilayer was assessed by the application of 500 μm suramin at the end of the experiment. The average capacitance of our bilayers was 89 ± 8 picofarads, and this was consistent throughout the entire recording. Experiments were carried out at room temperature (22 ± 2 °C). The concentration of free Ca2+ and pH of all of our solutions was determined using a Ca2+ electrode and a pH electrode  as previously described (2+ was added as ZnCl2 to the cis chamber at the required concentration from an appropriate stock solution as described previously (Sheep cardiac heavy sarcoplasmic reticulum (HSR) membrane vesicles were prepared and fused with planar phosphatidylethanolamine lipid bilayers, as described previously . Sheep hchniques . The traescribed . Zn2+ waeviously .Po) was taken from 3 min of continuous recording. As reported previously, multiple MG23s often gated together in the bilayer . Channel events were detected by the 50% threshold method  using TA bilayer . When maeviously .m NaCl, 0.1 mm EGTA, 0.15 mm CaCl2, 25 mm PIPES, and 2.5 mm DTT. Insoluble material was removed by centrifugation for 30 min at 150,000 × g. The supernatant was incubated for 120 min with protein G-Sepharose  cross-linked with an αTMEM109 antibody (Sigma). The resin was washed in 20 mm Tris·HCl, 500 mm NaCl, 0.1% CHAPS, protease inhibitor mixture P8340 (Sigma) (1:500), 1 mm PMSF, 2.5 mm DTT (pH 7.4). MG23 protein was recovered by overnight incubation with an epitope peptide (Genscript) at 4 °C. Epitope peptide was removed by gel filtration using a HiTrap desalting column . Elution fractions were collected. Identification of MG23 within each fraction was determined by sodium dodecylsulfate (SDS)-PAGE followed by silver staining. MG23-positive fractions were snap-frozen and stored at −80 °C.MG23 channels were enriched and purified from sheep cardiac HSR vesicles. HSR vesicles were solubilized for 60 min at 4 °C at a protein concentration of 2 mg/ml in a CHAPS detergent solubilization buffer containing 1% w/v CHAPS, 1 2+ concentration of our recording solutions was reduced from ∼5 μm to 100 nm by the addition of 7.6 μm concentrations of the chelation agent BAPTA. The free Ca2+ concentration was measured using a Ca2+-selective electrode. BAPTA is known to bind other divalent cations including Zn2+ . Detection of free Zn2+ was performed using a pZn meter as described previously (m.The addition of ZnCleviously  and dete2 and sub-cultured between 60 and 70% confluence.H9C2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies) under 95% air, 5% CO2 in a Don Whitley H35 hypoxystation . Under hypoxic conditions cell lysates were collected in radioimmunoprecipitation assay buffer . Lysates were snap-frozen and stored at −80 °C before use.H9C2 cells were subjected to 0.5% Om NaCl, 16 mm KCl, 0.33 mm NaH2PO4, 10 mm PIPES, 20 mm deoxyglucose, 20 mm sodium lactate, 1 mm MgCl2, 2 mm CaCl2, and 0.2 mm 2,4-dinitrophenol, pH 6.8, with NaOH. After aspiration of ischemia, buffer cells were washed in phosphate-buffered saline (PBS) and incubated with 5 μm ZnAF-2  in PBS in the dark for 30 min at 37 °C. Cells were subsequently washed in fresh PBS followed by 100 μm EDTA in PBS before fixing in 4% paraformaldehyde for 10 min at room temperature. Cells were then washed three times in PBS. Cells were permeabilized in 0.2% Triton X-100 in PBS for 30 min before loading with 10 μm 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 10 min at room temperature. Basal Zn2+ fluorescence intensity  was determined by FLUOstar OPTIMA microplate reader (BMG Labtech) and was normalized to DAPI .To simulate ischemia, H9C2 cells were exposed to an ischemia buffer containing 108 mHSR proteins or H9C2 cell lysates were size-fractionated by SDS-PAGE on a 4–12% Bis-Tris precast gel (Life Technologies). After separation, proteins were transferred to a nitrocellulose membrane, and nonspecific binding sites were blocked for 1 h at room temperature using 5% dried milk and Tris-buffered saline . Membranes were probed overnight at 4 °C with primary antibodies specific for RyR2 phosphorylated at site serine 2809 , anti-MG23 , and anti-β-actin  in 5% dried milk and Tris-buffered saline. Secondary horseradish peroxidase-linked goat anti-rabbit or anti-mouse IgG  Sigma) antibodies were used in combination with an enhanced chemiluminescent detection system  and visualized using a Fujifilm LAS-3000 detection system. Densitometry was performed using ImageJ software .t test was used to assess the difference between mean values. Where multiple treatments were compared, a one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test was used to assess the difference between treatments. p values ≤0.05 were taken as significant.Data are shown as the mean ± S.D. Where appropriate, Student's B. R.-O. and G. B. R. designed and performed the experiments, analyzed the data, and contributed to writing the manuscript. A. K. and C. M. performed the experiments and analyzed the data. W. B. provided the theoretical calculations of free ions. H. T. and M. N. designed the experiments and provided key experimental tools. A. J. S. designed the experiments and contributed to writing the manuscript. S. J. P. designed the experiments, analyzed the data, wrote the manuscript, and supervised the project. All authors discussed results and commented on the manuscript at all stages.


The CBD1 and CBD2 domains form a regulatory tandem (CBD12), where Ca2+ binding to CBD1 activates and Ca2+ binding to CBD2 (bearing the splicing segment) alleviates the Na+-induced inactivation. Here, the NCX2-CBD12, NCX3-CBD12-B, and NCX3-CBD12-AC proteins were analyzed by small-angle X-ray scattering (SAXS) and hydrogen-deuterium exchange mass-spectrometry (HDX-MS) to resolve regulatory variances in the NCX2 and NCX3 variants. SAXS revealed the unified model, according to which the Ca2+ binding to CBD12 shifts a dynamic equilibrium without generating new conformational states, and where more rigid conformational states become more populated without any global conformational changes. HDX-MS revealed the differential effects of the B and AC exons on the folding stability of apo CBD1 in NCX3-CBD12, where the dynamic differences become less noticeable in the Ca2+-bound state. Therefore, the apo forms predefine incremental changes in backbone dynamics upon Ca2+ binding. These observations may account for slower inactivation (caused by slower dissociation of occluded Ca2+ from CBD12) in the skeletal vs the brain-expressed NCX2 and NCX3 variants. This may have physiological relevance, since NCX must extrude much higher amounts of Ca2+ from the skeletal cell than from the neuron.Mammalian Na The isoform-dependent and alternative splicing-dependent modification of Ca2+-binding regulatory domains are especially important for Ca2+-transporting proteins operating in excitable tissues3, since feedback interactions of Ca2+ with allosteric regulatory domains dynamically modulate the Ca2+-transport rates in accordance with dynamic changes in cellular Ca2+ oscillations 5. Despite the fundamental importance of this issue, the structure-dynamic determinants governing the isoform-dependent and alternative splicing-dependent modification of regulatiory specificity in Ca2+-binding domains remain poorly understood6.The properties of Ca+/Ca2+ exchanger (NCX) proteins serve as a major route for Ca2+ extrusion from cells4. In mammals, three gene isoforms  of NCX proteins  and their splice variants are expressed in a tissue-specific manner, while exhibiting distinct regulatory phenotypes  can be freely combined with cassette exons  , whereas exon-B-containing NCX1 splice variants are mainly expressed in non-excitable tissues   in the presence or absence of an additional exon C  Fig. 3, 4, 8, ey) Fig. 3, 4, 8, ing Fig. . NCX3 isn C Fig. 8, 10, 11ain Fig. 11.2+ or three Na+ ions and transports them in separate steps17. Eukaryotic (but not prokaryotic) NCX proteins contain a large cytosolic loop between TM5 and TM6, mainly composed of two Ca2+-binding domains, CBD1 and CBD218. The two CBDs form a head-to-tail attached two-domain tandem (termed CBD12) through a very short interdomain linker , where the ion-binding pocket selectively binds either one Ca–10, wher18 and X-ray crystallography24 studies revealed that CBD1 and CBD2 share a similar structure, exhibiting an immunoglobulin-like β-sandwich structure with seven antiparallel β-strands  in all NCX orthologs, isoforms, and splice variants26. The Ca2+ binding sites of CBD1 are located at its C-terminal tip, near the CBD1-CBD2 interface  takes place only at CBD2 and involves the Ca2+ binding sites21. Therefore, in A-exon-containing NCX1 variants, CBD2 binds two Ca2+ ions, whereas in B-exon-containing NCX1 variants, CBD2 does not bind Ca2+ ions  was shown to exhibit less Na+-dependent inactivation compared with NCX3-B (which does bind Ca2+ at CBD2) in electrophysiological studies11.NMRons Fig. 21, 25. Sity Fig. 21, 26. SBD2 Fig. 26. Intri2+-dependent regulation in splice variants of NCX1, thereby providing the insights into the mechanisms underlying CBD interactions in isolated CBD12 and full-size NCX132. These studies have shown that the isolated preparations of CBD12 largely represent the Ca2+ sensitivity and kinetics of Ca2+-dependent regulation in their matching of full-size NCX isoforms and splice variants30. Therefore, isolated CBD12 serves as an ideal model for investigating the structure-dynamic mechanisms underlying the allosteric regulation specificities in NCX isoforms and splice variants. The interactions between CBD1 and CBD2 in the context of CBD12 results in increased affinity for Ca2+ at CBD1 and slow dissociation of an “occluded” Ca2+ ion from CBD127. In addition, alternative splicing at CBD2 not only affects the Ca2+ binding affinity and capacity at CBD2—it also affects the Ca2+ interactions with CBD1, establishing up to 50-fold differences both in the Ca2+ binding affinity and Ca2+ off-rates in the cardiac (ACDEF), brain (AD), and kidney (BD) splice variants of NCX127. Using 45Ca2+ equilibrium binding and stopped-flow kinetic assays, we have recently demonstrated that NCX2 and NCX3 also exhibit alternative-splicing dependent synergistic interactions between the CBDs, similarly to NCX126. NCX3-CBD12 splice variants exhibit similar Ca2+ affinity at CBD1, but the dissociation rate of the occluded Ca2+ ion is ~10-fold slower in skeletal muscle (AC) than in the brain (B) variants26. These differences in Ca2+ binding affinity and the Ca2+ off-rates at the primary allosteric sensor (CBD1) may have physiological relevance for diversifying regulatory responses in full-size NCX variants expressed in a tissue-specific manner6.Recent studies using diverse biophysical, biochemical, and structural methods have identified synergistic interactions between the CBDs that shape the dynamic range and kinetic features of Ca2+ binding to CBD1 results in Ca2+ occlusion and tethering of CBD1 and CBD2 through the formation of a hydrogen-bonded salt-bridge network at the two-domain interface, yielding a more stable structure19. The crystallographic structures of CBD12 variants of NCX1, exhibiting different regulatory properties, show nearly identical interdomain angles between CBD1 and CBD2 and thus, global CBD alignment does not seem to shape regulatory specificities in NCX1 splice variants19. In line with this notion, small-angle X-ray scattering (SAXS) studies revealed that Ca2+ binding and occlusion at CBD1 of NCX1-CBD12 splice variants induces a shift towards narrowly distributed elongated conformations, as predicted by the population shift mechanism28. This population shift of conformational states is essential for regulation, thereby representing a common mechanism for NCX1 splice variants regardless of their regulatory specificity28. Finally, hydrogen-deuterium exchange mass-spectrometry (HDX-MS) analyses of NCX1-CBD12 splice variants have shown that Ca2+ binding to all tested variants mainly rigidifies the backbone dynamics of CBD2 (and not that of CBD1), where the alternative splicing of CBD2 secondarily modifies the strength and expansion of rigidification throughout CBD231. Importantly, the Ca2+-induced effects on CBD2 backbone dynamics correlate well with the regulatory specificity found in the matching splice variants of full-size NCX1s31.X-ray crystallography revealed that Ca2+ occlusion and tethering of CBDs , whereas alternative splicing of CBD2 controls the propagation and strength of Ca2+-dependent rigidification and thereby, diversifies the regulatory response31. Currently it is unclear to what extent  the above-described mechanisms are also valid for NCX2 and NCX3 splice variants and how the exons control the CBD dynamics as related to shaping the regulatory specificity in a given isoform/splice variant. To overcome this gap, here we analyzed the apo and Ca2+-bound forms of isolated NCX2-CBD12, NCX3-CBD12-AC, and NCX3-CBD12-B proteins with SAXS29 and HDX-MS31 in order to identify their conformational dynamics and the effect of alternative splicing. The present analysis reveals a general mechanism for regulatory coupling associated with Ca2+ interactions with regulatory CBD domains of NCX1, NCX2, and NCX3 isoforms and their splice variants, where the dynamic effects of alternative splicing on conformational dynamics of the two-domain regulatory tandem (CBD12) control the contributions of a given isoform/splice variant to dynamic handling of cytosolic Ca2+ oscillations in distinct cell types.Thus far, NCX1 splice variants share a common mechanism for the initial decoding of the regulatory signal upon Ca33. In all the examined proteins , the Ca2+-bound form exhibited similar Rg and Dmax values, with PDDF, which represents an elongated conformation  sensor of the CBD12 tandem either in NCX1, NCX2, or NCX3 results in a Ca2+-induced population shift of preexisting conformational states, where more elongated conformational states become more populated  or 639–648 (B)], adjacent to the CBD1 Caate Fig. . In addiate Fig. . This fi2+ binding.Although a direct comparison of deuterium uptake levels between NCX2-CBD12 and NCX3-CBD12 variants is not feasible, we can compare the regions affected by Ca2+-dependent changes in backbone dynamics, we plotted the difference in deuterium uptake as a function of residue at different deuterium uptake durations, and the Ca2+-induced deuterium uptake changes were also color-coded onto the CBD12 model structures  upon Ca2+ binding, meaning that all NCX1, NCX2, and NCX3 isoform/splice variants, tested until now, share a common mechanism for the initial decoding of regulatory Ca2+ signals.The interdomain CBD1-CBD2 linker (peptide 513–525 in NCX2 and 524–533 in NCX3) exhibits lower deuterium uptake upon Caere Figs . This ev2+-bound state  and NCX3  splice variants expressed in the brain, as well as NCX2, exhibit faster Ca2+ dissociation kinetics from CBD12 compared with NCX1 and NCX3 splice variants expressed in muscles 26. This is in line with the prolonged Ca2+ transients and action potential durations in muscles compared with neurons26. These hallmark features, assigned to Ca2+ interactions with CBD1 and CBD2, strictly correlate with characteristic regulatory modes ascribed to matching (full-size) NCX variants26. The equilibrium and kinetic parameters measured in isolated NCX1-CBD12-ACDEF preparations25 were successfully applied to computer-aided dynamic modeling of allosteric activation of the cardiac NCX1 (ACDEF) during the action and resting potentials, thereby providing indispensable information on the dynamic contributions of NCX in handling the Ca2+-dependent Ca2+-release from the sarcoplasmic reticulum, the Ca2+-spark appearance, and duration within local sub-cellular compartments and the fine-tuning of excitation-contraction coupling in cardiomyocytes35. Similar applications for computer-aided modeling are straightaway required for clarifying the partial contributions of multiple NCX isoforms and their splice variants to the dynamic handling of dynamic Ca2+ swings in neuronal tissues. The emerging working hypothesis is that specific conformational variances, raised by exons, diversify the affinity/off-rates of Ca2+ at CBD1  and the affinity/capacity of Ca2+ binding to CBD2 26.First and foremost, CBD1 and CBD2 act as Ca2+ at CBD2, to alleviate Na+-induced inactivation. The ability to counteract Na+-induced inactivation is of major importance in excitable tissues where large Na+ fluxes (through the voltage-sensitive Na+-channels) occur at the initial stage of cell depolarization. NCX2 (expressed in the brain) lacks the Na+-induced inactivation, whereas in NCX3 the Na+-induced inactivation can be counteracted by increased levels of cytosolic Ca2+, but to varying degrees11. It was previously proposed that an electrostatic switch at CBD2 underlies the alleviation of Na+-induced inactivation upon Ca2+ binding, but this proposal cannot account for the regulatory properties of NCX2 (where the Ca2+ binding affinity at CBD2 is extremely low) or for regulatory features of NCX3-AC26. However, the data presented here may resolve the principal structural platform for the dynamic regulation of NCXs, as well as it can pinpoint the exon-governed fine shaping in the backbone dynamics that may account for regulatory diversity.In NCX1, both the brain (AD) and cardiac muscle (ACDEF) splice variants bind Ca42. The accumulating evidence raises several important points. Protein rigidity seems to serve as a common communication route between distant sites in proteins, resulting in structural coupling between the regulatory regions  and the effector site 42. Moreover, in some instances, allosteric interactions are mediated only by dynamic changes, without having any gross effects on the protein conformation39. In other cases, the population shift mechanism describes a shift in the protein ensemble of conformations towards active states, where no obvious global conformational change is observed37. These observations were recently formulated into a unified model of allostery, based on thermodynamics, the structure, and the free energy landscape of the population shift41. According to this general model, the allosteric mechanism in a specific protein requires three elements41, allowing us to formulate a general scheme for NCX isoforms and regulation of splice variants.In recent years, a growing body of evidence has suggested an updated view of allosteric regulation in proteins. More specifically, a greater importance is given to changes in protein dynamics and conformational distributions rather than to global conformational transitions41. This conformation is provided by the crystal structure of NCX1-CBD12-AD-E454K19, which also allows us to model NCX2-CBD12 and NCX3-CBD12 in the Ca2+-bound conformation. The molecular envelopes calculated here  does not create any new conformational states but instead, only shifts the population among the existing states, towards more stable elongated conformations , the hydrophobic core , and salt bridge elements  tethering CBDs upon Ca2+ binding29. For example, the structural evidence revealed by HDX-MS demonstrates the reduced Ca2+-dependent CBD2 rigidification in the F450G mutant, thereby confirming the general model for Ca2+-dependent tethering of CBDs and the rigidification of the two-domain interface29. Consistent with this statement, this residue is highly conserved among NCX orthologs19 and F472 in NCX2-CBD12 and F474 in NCX3-CBD12 (corresponding to F450 in NCX1-CBD12) show reduced deuterium uptake upon Ca2+ binding  the mechanism by which the ligand binding modulates the activity of NCX proteins.Finally, one should consider whether ligand binding at the regulatory site stabilizes the active conformation, destabilizes the inactive conformation, or does both simultaneously2+ binding ” display some clear and highly conserved interdomain interactions between residues, although the complete dynamic response in CBD2 is different among distinct isoforms and splice variants31. The present  CALX1.1 responds to the regulatory Ca2+ binding in a negative mode, thereby exhibiting inhibition rather than activation29.Having this general mechanism in mind, it can be concluded that different NCX isoforms and splice variants share a similar “active” conformation, which becomes highly populated upon Caing Figs 19, 28, 2ent Figs  and 5 an2+-bound states, meaning that Ca2+ binding results in greater rigidification of NCX3-CBD12-AC and therefore the apo form predefines the incremental effect of Ca2+ binding. The greater dynamic effect may explain the reduced Na+-dependent inactivation. Similarly, in NCX1-CBD12 proteins, A-exon-containing variants in which Ca2+ can alleviate Na+-dependent inactivation exhibit greater rigidification of CBD2 (aided by Ca2+-binding). Therefore, the observed deviations in the conformational dynamics of the NCX1 and NCX3 splice variants  and on the other side, neighbors the splicing segment at CBD2  stabilizes Ca2+ occlusion at high-affinity sites of CBD1, thereby allowing a further transformation and propagation of the allosteric signal. Similar conclusions were drawn for Ca2+-dependent rigidification of the α-helix in NCX1-CBD12 variants29. Therefore, accumulating evidence strongly supports a general mechanism for the examined NCX1, NCX2, and NCX3 variants, according to which Ca2+-binding stabilizes the interdomain linker, the hydrophobic interface between the CBDs, and the nearby α-helix/FG-loop segment on CBD2 next to the splicing segment31. It is quite obvious that the Ca2+-dependent rigidification of the two-domain interface detected by HDX-MS and SAXS techniques is coupled with the Ca2+ occlusion and two-domain tethering of CBDs, which further stabilizes the active conformation in all tested isoform/splice variants of CBD12 proteins44. The experimental methods described here may be used in future studies to resolve the role of alternative splicing in shaping the allosteric regulation of many protein families.In general, the Carms Fig. . The Ca2E. coli Rosetta2 (DE3) competent cells (Novagen) as described26. Overexpressed proteins were purified on a TALON-superflow  column followed by size exclusion chromatography on FPLC by using a Superdex 200 column 26. The purity of the finally purified protein preparations was >95%, as judged by SDS-PAGE. Proteins were concentrated, flash frozen in liquid nitrogen, and stored at −80 °C until their usage for SAXS or HDX-MS experiments.The DNA constructs of rat NCX2-CBD12, NCX3-CBD12-AC, and NCX3-CBD12-B were cloned into the pET28a vector, confirmed by sequencing and expressed in 19. MODELLER was used to generate the models45.The models of NCX2-CBD12, NCX3-CBD12-AC, and NCX3-CBD12-B were generated based on the crystal structure of NCX1-CBD12-E454K (PDB 3US9)−1. Ten frames of two-dimensional images were recorded for each buffer or sample, with an exposure time of 2 sec per frame. The 2D images were reduced to one-dimensional scattering profiles and the scattering of the buffer was subtracted from the sample profile using the software at the site. The buffers contained 100 mM KCl, 20 mM β-mercaptoethanol, 20 mM Tris-HCl at pH 7.2, and either 10 mM CaCl2 or 10 mM EDTA. To account for possible inter-particle effects, each sample was measured at four concentrations: ~10, ~5, ~2.5, and ~1.5 mg/mL. In the presence of such effects, the lowest concentration curve was merged with a higher concentration curve at q ~ 0.2 Å−1 to prevent distortion of the low-angle data while preserving a high signal-to-noise ratio at the higher angles, which are far less sensitive to interparticle effects33. The experimental radius of gyration (Rg) was calculated from data at low q values in the range of qRg < 1.3, according to the Guinier approximation: lnI(q) ≈ ln(I(0)) − Rg2q2/3 using PRIMUS46. The Dmax values and the Porod volume were derived from the paired-distance distribution function (PDDF or P(r)) calculated using GNOM47. Ab initio shape reconstructions were performed with the ATSAS software suite, using scattering data of the q range between 0.02 and 0.30 Å−1 47.SAXS data were measured at the beamline BM29 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Data were collected at 4 °C with the X-ray beam at wavelength λ = 1.0 Å, and the distance from the sample to detector  was 2.867 meters, covering a scattering vector range (q = 4πsinθ/λ) from 0.0025 to 0.5 Å45, based on the crystal structure of NCX1-CBD12-E454K (PDB 3US9)19. Unstructured residues at the N- and C-termini of CBD1 and CBD2 and the large flexible FG-loop of CBD2 were truncated. The software RANCH48 was used to generate a pool of 10,000 stereochemically feasible structures with a random CBD1-CBD2 linker, N- and C-termini, and a CBD2 FG-loop. These pools were used as input for GAJOE48, which selects an ensemble with the best fit to the experimental data using a genetic algorithm: 50 ensembles of 20 orientations each were “crossed” and “mutated” for 1,000 generations and the process was repeated 50 times.NCX2-CBD12, NCX3-CBD12-AC, and NCX3-CBD12-B were modelled with MODELLER2. The HDX reaction was initiated by mixing 2ul of protein samples with 28 µL of D2O buffer  containing 1.42 mM EDTA, or 10 mM CaCl2 for 10 sec, 100 sec, 1,000 sec, or 10,000 sec at 4 °C. The reaction was stopped by adding 30 µl of ice-cold quench buffer . For non-deuterated (ND) samples, 2 µl of protein samples were mixed with 28 µl of H2O buffer , and 30 µl of ice-cold quench buffer was added.Purified proteins were prepared at a concentration of 100 µM in a buffer composed of 20 mM Tris-HCl, pH 7.2, 100 mM KCl, 20 mM β-mercaptoethanol either with 1.42 mM EDTA or 10 mM CaCl29. Mass-spectral analyses were performed with a Xevo G2 Quadruple-time of fly (Q-TOF) equipped with a standard electrospray ionization (ESI) source in MSE mode in positive ion mode. All setting conditions for the system were reported previously49. Identification and HDX-MS data processing of peptic peptides were performed by ProteinLynx Global Server 2.4 (Waters) and DynamX 2.0 (Waters) as previously described31. All of the data were derived from at least three independent experiments, and the statistical significance was analyzed by t-test.The quenched samples were digested and analyzed by the HDX-UPLC-ESI-MS system (Waters) as previously describedSupplementary Information


The current analysis thus represents a new sub-analysis of the AML-001 trial, which is directly compared with a new sub-analysis of the AAR. Baseline characteristics, azacitidine application, response rates and OS were comparable between all patient cohorts within the trial or registry setting. Median OS was 9.9 versus 10.8 months (p = 0.616) for “AML-001” versus “AAR (001-like)” cohorts, respectively. Secondly, we pooled data from both cohorts (n = 309) and assessed the outcome. Median OS of the pooled cohorts was 10.3  months, and the one-year survival rate was 45.8%. Thirdly, we compared data from AAR patients meeting AZA-AML-001 trial inclusion criteria (n = 95) versus all AAR patients with World Health Organization (WHO)-defined AML (“AAR (WHO-AML)” cohort; n = 193). Within the registry population, median OS for AAR patients meeting trial inclusion criteria versus all WHO-AML patients was 10.8 versus 11.8 months (p = 0.599), respectively. We thus tested and confirmed the efficacy of azacitidine as a front-line agent in patients with AML, >30% BM blasts and WBC ≤ 15 G/L in a routine clinical practice setting. We further show that the efficacy of azacitidine does not appear to be limited to AML patients who meet stringent clinical trial inclusion criteria, but instead appears efficacious as front-line treatment in all patients with WHO-AML.We recently published a clinically-meaningful improvement in median overall survival (OS) for patients with acute myeloid leukaemia (AML), >30% bone marrow (BM) blasts and white blood cell (WBC) count ≤15 G/L, treated with front-line azacitidine versus conventional care regimens within a phase 3 clinical trial . As results obtained in clinical trials are facing increased pressure to be confirmed by real-world data, we aimed to test whether data obtained in the AZA-AML-001 trial accurately represent observations made in routine clinical practice by analysing additional AML patients treated with azacitidine front-line within the Austrian Azacitidine Registry  and directly comparing patient-level data of both cohorts. We assessed the efficacy of front-line azacitidine in a total of 407 patients with newly-diagnosed AML. Firstly, we compared data from AML patients with WBC ≤ 15 G/L and >30% BM blasts included within the AZA-AML-001 trial treated with azacitidine (“AML-001” cohort;  Acute myeloid leukaemia (AML) is predominantly a disease of the elderly, with a median age at diagnosis of roughly 70 years . Older pp = 0.1009) was reported by part of our group on 16 July 2015 [In 2010, the international phase 3 randomised AZA-AML-001 clinical trial testing azacitidine versus conventional care regimens  in AML patients older than 65 years with newly-diagnosed AML, >30% BM blasts and ≤15 G/L white blood cell (WBC) count was initiated. In this trial, a clinically-meaningful improvement in OS for azacitidine versus conventional care regimens  patients, they are considered to include extremely selected, skewed and/or not fully-representative patient populations . ResultsTherefore, a cooperative data analysis between the sponsors and data owners of the AZA-AML-001 trial and the AGMT-AAR-registry was agreed upon, with the aim to test whether the use of azacitidine within the routine clinical practice setting can recapitulate the median OS time of 10.4 months observed in AML patients with >30% BM blasts and ≤15 G/L treated with azacitidine front line within the AZA-AML-001 trial. Of note, 27 of the 241 patients (11.2%) treated with azacitidine within the AZA-AML-001 trial were excluded from the current analysis, as they had ≤30% BM blasts after central pathology review and, therefore, did not fulfil the inclusion criteria of this study . The curOur predefined criteria (>30% BM blasts and ≤15 G/L WBC) for the direct comparative analysis of the clinical trial with registry data were met by 214 patients of the AZA-AML-001 trial (“AML-001” subset) and 95 patients from the AAR (“AAR (001-like)” subset) . This reBaseline characteristics were similar between the “AML-001” subset and the “AAR (001-like)” subset, including gender, median age, median BM blast count, WBC count, haemoglobin, absolute neutrophil count (ANC), platelets (PLT), prior myelodysplastic syndromes (MDS), transfusion dependence (TD) and National Comprehensive Cancer Network (NCCN) cytogenetic risk . The onlTreatment-related characteristics, such as median number of azacitidine cycles (six vs. five), percentage of patients receiving ≥6 cycles (50% vs. 46%) or ≥12 cycles of azacitidine (29% vs. 24%), median total days of azacitidine application (42 vs. 34 days), daily dose (130 vs. 132 mg), patient status at data cut-off and reasons for azacitidine discontinuation were similar between the clinical trial and registry cohorts, respectively .p = 0.9553), median relapse-free survival , median event-free survival , median complete response (CR)/CR with incomplete blood count recovery (CRi) duration , one-year survival rates  and 30-day mortality rates  were comparable between the clinical trial and the registry cohorts , which was due to a lower rate of CR/CRi in the registry population.Median OS , age < versus ≥75 years , PLT-TD , ECOG-PS  and NCCN cytogenetic risk  had a significant impact on survival in univariate analysis (p = 0.8998).Univariate and multivariate Cox regression analyses of OS were performed to evaluate the similarity of the two groups adjusted for baseline covariates . The basanalysis . Of notep = 0.0057), ECOG-PS  and NCCN cytogenetic status  remained significant. As the baseline covariate “AML-001” versus “AAR (001-like)” was the variable most critical to this publication, this variable was kept in the final multivariate model, and the two groups were comparable even after adjustment for significant baseline covariates , indicating no relevant difference between the outcomes of patients from either cohort  for outcome analyses: median OS was 10.3  months, and the Kaplan–Meier (KM) estimate of one-year survival rate was 45.8%.Having determined no relevant differences in baseline variables, treatment-related characteristics or various measures of outcome between the AZA-AML-001 trial and the “real-world” cohorts of the AAR, both cohorts were pooled ”) and all WHO-AML patients included within the registry (“AAR (WHO-AML)”) treated with azacitidine front-line, with the exception of BM blast count, which was expectedly lower for the “WHO-AML” patient subgroup as these also included patients with 20%–30% BM blasts . TreatmeResponse, including ORR, TI and median CR/CRi duration, was similar between the “001-like” and “WHO-AML” subsets . Other oCurrently, both physicians and regulatory agencies are placing a stronger focus of attention on the performance of new drugs in the routine clinical practice setting, rather than merely relying on results obtained in clinical trials, and pressure is rising to recapitulate such results in unselected patients in clinical practice ,32,33,34The EMA has reacted to this pressure by publishing its intention to “expand the use of patient registries” .In anticipation of a need to test and potentially confirm the efficacy of azacitidine in a real-world population, the AAR established a platform to document the off-label use of azacitidine even before azacitidine was approved for the treatment of AML in the European Union and before the AZA-AML-001 trial was initiated. This indicates the lack of alternative treatments, an unmatched medical need and the high expectation of physicians that azacitidine would provide clinical benefit for their patients. Physicians’ anticipation and conviction that they were acting in the best interest of their patients, independent of the current EMA label, were based on previously published results in patients with higher-risk MDS and AML ,8,9,10. In a collaboration effort with the sponsors, data owners and authors of the AZA-AML-001 trial, this study compared data of AML patients treated with azacitidine front-line within the setting of the AZA-AML-001 clinical trial   [p = 0.250) [p = 0.104) [We further aimed to evaluate whether the efficacy of azacitidine might be limited to patient subsets fulfilling the AZA-AML-001 clinical trial inclusion criteria . In this regard, no relevant difference in baseline parameters, treatment-related parameters, TEAEs, response and survival outcomes existed between patients of the AAR that met clinical trial inclusion criteria versus all WHO-AML patients A. These . 14.0%) , and we = 0.104) .It is clear that observational studies cannot replace clinical trials, but they do play a well-accepted complementary role. One might argue that the retrospective, uncontrolled nature of a registry setting is a drawback of the current analyses. However, precisely this fact represents one of the acknowledged advantages of registry settings. The necessity to assess the effect of new drugs in patients that often escape inclusion in clinical trials, in order to gain additional insights into a drug’s ability to achieve its intended use, safety and outcome in the routine clinical practice setting, in contrast to the confined and controlled environment of a clinical trial, is supported by several groups, including the EMA ,49,50,51n = 8580) from the Medicare database (cut-off date: 31 December 2012) published similar findings, namely that only 14.7% of MDS patients aged ≥66 years had received hypomethylating agents [Another recurrent concern about registries is the potential presence of a bias regarding the inclusion of patients. In the AAR, 20% of all patients (and 40% of patients aged ≥70 years) diagnosed with AML across Austria were included in the registry between 2008 and 2012 . In compg agents . The Medg agents ). In lign = 214; “AML-001” subset) versus patients included within the AAR ” subset), using patient-level data from both cohorts , for outcome analyses ” subset ” subset) versus all comers (“AAR (WHO-AML)” subset) .The multicentre, randomised, open-label, parallel-group AZA-AML-001 trial  evaluated the efficacy and safety of azacitidine versus conventional care regimens in 488 patients aged ≥65 years with newly diagnosed AML with >30% BM blasts and ≤15 G/L WBC and was conducted in 18 countries. Details regarding the study design and the randomization process have recently been published  (study sn = 407), AML was defined according to WHO 2008 criteria [For all patients included in these analyses  to death from any cause. Event-free survival  and relapse-free survival  were assessed from Day 1 to the occurrence of an event . Participants who were still alive without any of these events were censored at the date of their last assessment.Response was determined based on International Working Group (IWG) Response Criteria for AML . MorpholAEs were assessed according to the Common Terminology Criteria for Adverse Events . The saInclusion criteria of the AZA-AML-001 trial comprised newly-diagnosed patients with AML, ineligibility for HSCT, age ≥65 years, BM blasts >30%, WBC ≤15 G/L, ECOG-PS ≤2 and intermediate- or poor-risk cytogenetics (NCCN 2009 criteria); exclusion criteria included antecedent chronic CMML or other myeloproliferative neoplasm (MPN) and concurrent malignancies . The onlThis is a non-interventional retrospective comparative analysis of patient-level data of the above-defined patient populations with the above-defined aims and using the below-defined methods.p-value < 0.10 were included in the multivariate model. A backward selection method with a stay-level of 0.1 was used as the criterion to select covariates for the final model. The study covariate  was kept in the final multivariate model.Patient-level data for both studies were analysed by the sponsor of the AZA-AML-001 trial , as well as in a wider AAR patient population that included all patients with WHO-AML. We thus not only confirm the value of azacitidine as reported by the clinical trial in a real-world scenario, but also expand on these data by showing that the safety and efficacy of azacitidine as a front-line agent is not limited to patients meeting stringent clinical trial inclusion criteria. Instead, azacitidine appears efficacious as a front-line treatment in a wider patient population that includes all patients with WHO-AML.


Here, we demonstrate that a direct interaction exists between RyR2 and CSQ2. Topologically, CSQ2 binding occurs at the first luminal loop of RyR2. Co-expression of RyR2 and CSQ2 in a human cell line devoid of the other quaternary complex proteins results in altered Ca2+-release dynamics compared to cells expressing RyR2 only. These findings provide a new perspective for understanding the SR luminal Ca2+ sensor and its involvement in cardiac physiology and disease.Cardiac muscle contraction requires sarcoplasmic reticulum (SR) Ca Summary: Direct physical and functional calsequestrin 2 association with the ryanodine receptor luminal domain is the minimal requirement to form the sarcoplasmic reticulum Ca2+ sensor, which governs cardiac Ca2+ homeostasis and reliable heartbeat. Upon plasma membrane depolarisation, Ca2+ influx through voltage-gated Ca2+ channels triggers Ca2+ release through the sarcoplasmic reticulum (SR)-located RyR2 to increase cytoplasmic Ca2+ concentration and transduce sarcomere contraction .Cardiac excitation–contraction coupling, where an action potential elicits cardiomyocyte contraction, is mediated by ‘Catraction . Terminaactivity . Luminal on RyR2  and throein CSQ2 . Abnorma failure . Catechohandling . CPVT tyASPH), which together form the luminal Ca2+ sensor . Using GST pull-down assays, we found strong GST–CSQ2 binding to recombinant human RyR2  resulted in co-precipitation of HA–CSQ2 , providing further evidence for direct complex formation between RyR2 and CSQ2. Our findings are consistent with previous work that indicates a physical association between the skeletal muscle isoforms, RyR1 and CSQ1, and that made use of purified native RyR1 with no detectable levels of endogenous triadin or junctin  was used as a positive control. Purified (1 µM) GST-fusion protein was incubated with CHAPS-solubilised RyR2 from pig cardiac SR and isolated with glutathione Sepharose A, bottom1093 A, top. Rman RyR2 B, indicaman RyR2 C, bottomR2 , or in the absence of Ca2+ (1 mM EGTA), as well as under conditions of high (5 mM CaCl2) or low (100 µM CaCl2) SR Ca2+ load. Using co-immunoprecipitation assays, we found comparable amounts of BT1D2 binding to HA–CSQ2 in the presence of 1 mM EGTA, and 100 µM and 1 mM CaCl2; however, recovery of BT1D2 in the anti-HA immunoprecipitate was diminished with 5 mM CaCl2 (n=4) following densitometry analysis and normalisation for the amount of protein expressed in the cell lysate indicate equivalent amounts of HA–CSQ2 binding to BT1D2 for Ca2+ concentrations up to 1 mM ; however, 5 mM CaCl2 substantially reduced binding by >50% . These results suggest that at Ca2+ levels within the physiological SR range (≤1 mM), the RyR2–CSQ2 interaction remains intact but that it is diminished at supra-millimolar Ca2+ levels, in agreement with previous reports . Co-expression of RyR2 in the CSQ2-expressing stable cell line resulted in an increase in the duration between Ca2+-release events (inter-transient duration), decreasing their overall frequency  underlying arrhythmias and sudden cardiac death.In conclusion, our data indicate a direct functional RyR2 interaction with CSQ2, shedding more light on the quaternary structural assembly of the SR luminal Ca1093, epitope at residues 4454-4474; 1:500)  from Roche, CHAPS from Merck, nProtein-A Sepharose and glutathione Sepharose 4B from GE Healthcare, secondary antibodies from Santa Cruz Biotechnology, enhanced chemiluminescence reagents from Pierce, DNA restriction endonucleases from New England Biolabs, Pfu DNA polymerase from Promega, oligonucleotides and all other reagents were from Sigma. Antibodies used for immunoprecipitation: (1 μg) rabbit anti-HA antibody  and non-immune rabbit IgG were from Santa Cruz Biotechnology. Antibodies used in immunoblotting were mouse anti-Myc  from Santa Cruz Biotechnology, mouse anti-HA  from Covance; rabbit anti-GST (1:2000) , 2006, a; 1:500) , 2006 an(+)hygro (Life Technologies). Human RyR2 constructs were generated by performing PCR amplification [using human (h)RyR2 cDNA as template] and cloning into a modified pCR3 vector containing an N-terminal Myc peptide epitope tag. For bacterial expression of the LL1 construct, the cDNA was subcloned into the pGEX6P1 vector. Oligonucleotide primers and restriction enzyme cloning sites used are available upon request. All plasmids were verified by direct DNA sequencing .Human CSQ2 cDNA containing the entire open reading was generated by performing PCR amplification from a human heart cDNA library (Clontech) and was cloned into the bacterial expression vector pGEX6P1 . For transient mammalian expression, CSQ2 cDNA was subcloned into a modified pCR3 vector (Life Technologies) containing an N-terminal HA peptide epitope tag, whereas for stable expression, it was cloned into pcDNA3.1®) were cultured in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine and 10% fetal bovine serum under a humidified atmosphere of 5% CO2 at 37°C. On reaching ∼70% confluence, cells growing on a 100-mm Petri dish were transiently transfected with 24 μg of plasmid DNA using the calcium phosphate precipitation method, as described elsewhere (−1). For Ca2+ imaging, stable cells from this clonal population were transiently transfected with eGFP–hRyR2 using Effectene® (Qiagen) according to the manufacturer's instructions.HEK293 cells (ATCClsewhere . Cells w5) were seeded onto poly-lysine-coated glass-bottomed dishes (MatTek™) before transient transfection with eGFP–hRyR2. After 48 h, cells were loaded with Fluo-3 AM (10 µM) for 45 min at 30°C. Following de-esterification, cells were immersed in Krebs-Ringer-Hepes buffer for imaging . RyR2-mediated spontaneous Ca2+-release events were monitored using a laser scanning confocal microscope (Leica SP5) with an oil immersion 63× objective lens with excitation at 488 nm, and Ca2+-dependent fluorescence emission was detected over a 520±28 nm range. Using Leica Microsystems software (LAS-AF), Ca2+-release events were recorded for 2 min (five frames per second) at 512×512 pixel resolution, and acquired regions of interest representing global Ca2+ environments  were selected. Amplitude and temporal characteristics of spontaneous Ca2+ release were quantified and are displayed as means±s.e.m. Statistical analysis was performed using unpaired Student's t-test.Cells stably expressing CSQ2 (∼1×10t-test.Bacterial expression and purification of GST-fusion proteins, GST pull-down and co-immunoprecipitation assays, SR preparation and immunoblotting were performed as described previously , 2006. D