Activation of the A2A receptors can promote the occurrence of delayed depolarization, while activation of the A1 receptors can shorten the action potential’s duration and induce the resting membrane’s potential hyperpolarization, which promote pulmonary vein firing, stabilize the AF rotors and allow for functional reentry. Moreover, the A2B receptors have been associated with atrial fibrosis homeostasis. Finally, the adenosinergic system can modulate the autonomous nervous system and is associated with AF risk factors. A question remains regarding adenosine release and the adenosine receptors’ activation and whether this would be a cause or consequence of AF.Atrial fibrillation (AF) is the most common arrhythmia in the world. Because the key to developing innovative therapies that limit the onset and the progression of AF is to fully understand the underlying molecular mechanisms of AF, the aim of the present narrative review is to report the most recent advances in the potential role of the adenosinergic system in the pathophysiology of AF. After a comprehensive approach describing adenosinergic system signaling and the mechanisms of the initiation and maintenance of AF, we address the interactions of the adenosinergic system’s signaling with AF. Indeed, adenosine release can activate four G-coupled membrane receptors, named A Atrial fibrillation (AF) is a multifactorial sustained cardiac arrhythmia, and it is now considered a real worldwide public health issue, affecting more than 50 million individuals ,2. BecauAmong these molecular mechanisms, the implication of the adenosinergic system in AF has increased, since the accumulation of experimental data suggests that the increase in the adenosine blood level and the remodeling expression of the adenosine receptors might be part of the AF pathophysiology. Unfortunately, the adenosinergic system still has a Janus face in cardiac arrythmias, since adenosine can have both antiarrhythmic or proarrhythmic actions, along with adenosine receptors, which can lead to either profibrotic or antifibrotic effects ,4. FurthAs the key to developing innovative therapies that limit the onset and progression of AF is to fully understand the underlying molecular mechanisms of AF, the aim of the present narrative review was to report the most recent advances on the potential role of the adenosinergic system in the pathophysiology of AF. After describing the adenosinergic system’s signaling and the pathophysiology of AF, this review focuses on the arrhythmogenic effects of adenosine and concludes with the association between the AF risk factors and adenosinergic system disturbance.Since Drury and Szent-Györgyi first observed the cardiac effect of adenine compounds in 1929  and BurnBlood concentrations of adenosine are limited in time and space by its catabolism and reabsorption by balancing nucleoside transporters (ENTs), particularly in red blood cells ,15,16. ADuring hypoxia, ischemia or inflammation, ATP metabolism is modified leading to increased production of adenosine, which is associated with a decrease in its rephosphorylation. Inflammation or hypoxia can modulate the adenosinergic system thanks to the transcription factor NF-κB, which increases the transcription of adenosine receptors and hypoxia-inducible factor-1α (HIF-1α) genes ,21,22,231 (A1R), A2A (A2AR), A2B (A2BR) and A3 (A3R) receptors, which are classified according to their primary sequence, their associated G-protein and their pharmacological profile (binding affinity of agonists and antagonists) [1R and A3R are coupled to the Gi/o proteins leading to the inhibition of adenylyl cyclase (AC), whereas A2AR and A2BR are associated to Gs proteins, which stimulate the adenylyl cyclase and increase the intracellular production of cyclic-AMP (cAMP). A1R and A2AR have a high affinity for adenosine with a higher binding affinity of A1R. A2BR and A3R have a lower affinity for adenosine [Adenosine receptors are subdivided in four G protein-coupled receptors, named Agonists) ,31. A1R denosine .1R is highly expressed in the atrial myocardium, the sinoatrial node (SAN), the atrioventricular node (AVN) and the His–Purkinje system and presynaptically on adrenergic nerve varicosities but have a lower expression in ventricular myocytes [1R by adenosine induces direct negative chronotropic and dromotropic effects [2ARs are particularly expressed in vessels and coronary arteries with a vasodilator effect [1R, A2AR directly enhanced the inotropic effect of cardiomyocyte by increasing the cytosolic Ca2+ level and the Ca2+ sensitivity of the myofilaments [2A or A2B receptors nor the brief activation of these receptors impacts ischemia-reperfusion injury [2AR or A2BR knockout mice, the beneficial effects of A1R stimulation on post-ischemic function and infarct size were reduced [2BRs are expressed in coronary arteries and mediates coronary vasodilation, with a minor and supportive role compared to A2AR [2BRs has also been reported in atrial and ventricular cardiomyocytes [2BRs’ activation on cardiac fibroblasts reduces collagen and total protein synthesis suggesting that adenosine may protect against cardiac fibrosis [3Rs’ activation mediated coronary vasodilation [Adenosine receptors are widely distributed in various cells and tissues, including the cardiovascular system ,34. All myocytes . The act effects  and an i effects ,39,40  [1R is direct and cAMP-independent by modulating an inwardly rectifier potassium current, which is activated by both adenosine and acetylcholine , herein further referred to as IK,Ado [Adenosine is believed to induce a cAMP-dependent (indirect effects) anti-β-adrenergic action by a dual mechanism: the activation of the presynaptic Anephrine  and the    and the   . Neverths IK,Ado ,64.1Rs enhances the IK,Ado thanks to G-protein-coupled, inwardly rectifying K+ (GIRK) channels [iα subunits enhance the gating of the GIRK channels, in the same manner as acetylcholine on the muscarinic-2 receptor, which causes a vagally mediated negative chronotropy upon on atrial pacemaker activity [1R in the heart and, more particularly, in the SAN and atria [1Rs regulated the cAMP level by the inhibition of the adenylate cyclase activity through the Giα-subunit. Therefore, the cAMP-dependent protein kinase A (PKA) activity and the PKA-dependent phosphorylation of Ca2+-handling proteins are reduced, which modulates Ca2+ homeostasis and reduces isoproterenol-stimulated ICa,L [The activation of Achannels . After tactivity ,66. The activity ,68,69. Tnd atria ,71,72. Tnd atria . Complemed ICa,L .S-protein coupled receptors, A2AR stimulation can activate a cAMP-dependent pathway modulating Ca2+ handling. However, the precise mechanism of the A2AR-induced transduction signal remains controversial. Indeed, some authors report that the stimulation of A2ARs by a specific agonist  did not alter the cAMP level in rat ventricular cardiomyocytes [2AR [On the contrary, like other Gmyocytes , whereastes [2AR .2AR-induced inotropic effect was associated with a Ca2+-dependent pathway, but it was also considered as predominantly mediated by a Ca2+-independent inotropic pathway, which may rely on an increased myofibrillar Ca2+ sensitivity [2ARs’ stimulation by CGS 21680 induces a PKA-mediated increase in the frequency of spontaneous calcium release from the sarcoplasmic reticulum (SR) without a significant increase in the ICa,L amplitude [Moreover, in rat ventricular cardiomyocytes, the Asitivity . In humamplitude .2+-dependent pathway, the PKA-dependent phosphorylation might increase the activation of L-type Ca2+ channels (LTCC) initiated by membrane depolarization. Following LTCC opening, the inward Ca2+ current , in turn, triggers a Ca2+-induced Ca2+ release (CIRC) from the sarcoplasmic reticulum via the phosphorylated cardiac ryanodine receptors (RyR2) and which is responsible for numerous spontaneous Ca2+ release events (Ca2+ spark and Ca2+ waves) during systole [2AR stimulation in atria and ventricles through the Ca2+-induced myofibrilla contraction responsible for the cardiac electromechanical coupling [2+ handling proteins [In the canonical Ca systole ,76. Thiscoupling . The strcoupling ,78. Thiscoupling  and the proteins ,80.2+ content at the end of the systole to hasten the relaxation (positive lusitropic effect), it is interesting to note that in mice overexpressing human A2AR, the stimulation of A2AR increased the phosphorylation states of phospholamban (PLN) and the inhibitory subunit of troponin (cTnI) [2+ from troponin C, allowing for a faster relaxation rate [2+ ATPase (SERCA) pump which, in turn, reuptakes cytosolic Ca2+ and leads to a lower Ca2+ content during the diastolic phase [2+ extrusion is also mediated by the Na+-Ca2+ exchanger (NCX: stoichiometry of 1Ca2+:3Na+) allowing for cell relaxation. Indeed, the beat-to-beat electromechanical coupling stability is determined by the ability of the myocyte to maintain equilibrium between the SR calcium’s uptake and release [Because an appropriate cardiac function requires a tight regulation of the Can (cTnI) ,81. The ion rate  The phosic phase . During  release .2+ handling protein phosphorylation, and to a GIRK-induced potassium outward current  , the conductive tissues and in the working myocardium are largely based on the modulation of adenylate cyclase activity, which induces cAMP-dependent PKA signaling, leading to Ca .2+ channels, which induce a low slope of depolarization (phase 0). Lastly, while the Ca2+ channels close, the rapid and slow delayed rectifier current  induces outward K+ currents, which are responsible for the repolarization  and β-adrenergic stimulated conditions  [1R activation limits the cAMP-dependent currents , which further decreases the slope of the diastolic depolarization [The action potential of the sinoatrial node cells is characterized by a more depolarized −60 mV) and more labile resting membrane potential than contractile cardiomyocytes due to the almost lack of Kir2-encoded inwardly rectifier potassium currents (IK1) ,86. Afterization A. Even id ICa,L) . The dirrization ,60,63  through the voltage-gated sodium channels Nav1.5 [+ current (IK1) in atrial cells, the human atrial action potential typically demonstrates a triangular morphology and a more depolarized resting membrane potential (~−70 mV) compared to the spike-and-plateau shape of ventricular cardiomyocytes (−90 mV) [+ current (INa), followed by an inward Ca2+ current due to the opening of L-type Ca2+ channels , which finally close during repolarization, while inward rectifier K+ channels open  . The iniels open B. Even i mV . The reduced ,60,91 Mo reduced .On ventricular cardiomyocytes, adenosine action does not cause any significant effect on the resting membrane potential, action potential duration or amplitude .Using these characteristics, the major diagnosis interest and clinical use of adenosine is due to the fact of its action on an atrioventricular node. It confers to adenosine its diagnosis properties used in supra ventricular tachycardia ,61. WhilAtrial fibrillation (AF) is the most common pathologic tachyarrhythmia, defined by the absence of distinct repeating P-waves, irregular atrial activations on surface ECGs and irregular R-R intervals. At the beginning, episodes of sinus rhythm are punctuated by periods of arrhythmia . AF progression leads to more frequent and longer episodes until they last more than 7 days (persistent AF). After one year, in the absence of spontaneous or medical cardioversion, which allow a return to sinus rhythm, AF is then considered as a “long-standing persistent” AF. Finally, AF can be defined as a “permanent” AF when the therapeutic attitude is to consider that no further attempts to control sinus rhythm will be undertaken . AF can The pathophysiology of AF is supported by anatomical and electrophysiological changes in atria for which all of the exact molecular mechanisms are still unknown. Understanding of this complex AF pathophysiology is essential to identify the underlying molecular mechanisms. According to the Coumel triangle, AF pathophysiology first depends on trigger factors, which are rapid focus firing activity, initiating the arrhythmia. Then, the arrhythmogenic substrate are electrophysiological, mechanical and anatomical characteristics of the atria that sustain AF. Further, multiple neuro-humoral dynamic modulators also interfere with the initiation, progression and termination of AF. Therapeutic targets for a rhythm control strategy should consider all aspects of this classification . UnfortuRepeated atrial ectopias are known to initiate AF and are considered as triggers of AF . They caPV ectopias originate all along the myocardium sleeve of PV and present unpredictable firing with various, intermittent and delayed conduction to the left atrium. They are defined as focal discrete sites of early and centrifugal activation . Compare2+ handling abnormalities leading to cytosolic Ca2+ overload. Indeed, the Ca2+ overload leads to the diastolic leak of Ca2+ from sarcoplasmic reticulum (SR), called Ca2+ sparks and Ca2+ waves. The SR Ca2+ released is supported by the ryanodine receptors type-2 (RyR2), whereas Ca²+ storage is endorsed by the sarco-endoplasmic reticulum Ca²+ ATPase (SERCA2). Dysfunction of one or the other can induce diastolic Ca²+ leaks [2+ overload activates the Na+-Ca2+ antiporter (NCX) to remove Ca2+ from cells with a stoichiometry of 3Na+:1Ca2+ [NCX) can induce delayed afterdepolarizations (DADs) [PV ectopia beats are mainly related to Ca²+ leaks ,107. Cona+:1Ca2+ . The asss (DADs) , which cs (DADs) .Secondary to PV ectopy, firing can be related to DAD due to the fact of abnormal diastolic spontaneous calcium release . InducedTwo major mechanisms of perpetuation supported by anatomical and/or electrophysiological changes are described by the multiple wavelets hypothesis ,118 and The multiple-wavelet phenomenon hypothesizes that multiple random wavefronts propagate through the atria until depolarizable tissue is available. Secondary to AF, atrial enlargement increases the critical atrial mass that is required to self-perpetuate this mechanism . Within AF drivers are defined as a localized source of fast and repetitive activity during AF episodes from which activation propagates and breaks down into fibrillation of the rest of the atria . The driAnatomical reentries are defined by the presence of an unidirectional slow conduction area or block resulting in a fixed cycle length and localization circuit. Atrial fibrosis favors these slow conduction areas . The nonBoth mechanisms of reentry can be present within the same atrium. There is evidence of intermittent and spatially unstable drivers anchored to the structural heterogeneities of the atrial fibrosis area, which is considered to be the major mechanism of human AF perpetuation .The AF perpetuation is caught in a vicious circle. The triggers and perpetuation mechanisms are dependent on multiple underlying electrical remodeling and atrial fibrosis which are also induced by AF. This vicious circle is supported by the fact that a complete electrical reverse remodeling occurred within weeks after medical or electrical cardioversion ,133. TheAtrial cardiomyopathy has been defined as “any complex of structural, architectural, contractile or electrophysiological changes affecting the atria with the potential to produce clinically relevant manifestations” . Thus, aThe onset and the termination of AF episodes are likely related to dynamic modulators, which interact with both the trigger and substrate of AF. They exhibit multiple timescales and sites of action, including immediate and long-term electrophysiological atrial changes, ion channel conductance and homeostasis, and local and general inflammation status as well as fibrosis promotion . NonexhaHigh adenosine plasma levels have been found in the left atria of patients during episodes of paroxysmal AF and in persistent AF . The adeA high adenosine plasma concentration could also be a consequence of energy use in specific underlying cardiovascular conditions, including hypertension ,139, chr2AR and A2BR expressions in the left and right atrium in postoperative AF [1R in AF, especially in the right atrium [2A and A2B receptors, as was also described in peripheral blood mononuclear cells (PBMCs), was correlated with the atrial expression of adenosine receptors, suggesting that the peripheric status of the adenosinergic system could be a mirror of the underling atrium disease [The expression of adenosine receptors is modified in AF. Our team recently described the upregulation of Aative AF . This ovative AF . Li et at atrium . In pati disease ,137.1R agonist (CCPA) produced a profound negative chronotropic effect, whereas the selective A1R antagonist (PSB36) produced a nonsignificant positive chronotropic effect [1R activation [1R through IK,Ado modulation induces a resting membrane potential hyperpolarization, a reduction of the action potential duration and a shortened effective refractory period (ERP) on atrial cardiomyocytes [In Langendorff preparation of rat hearts, the injection of a specific Ac effect . The inctivation . Indeed,myocytes  , which is usually specifically located within the sinoatrial node cells. However, their presence has been demonstrated within PV cardiomyocytes [In a canine model, Datino et al. showed that adenosine enhanced a larger IK,Ado current in PV cardiomyocytes compared to left atrium cells, which resulted in the heterogeneity of the hyperpolarization. Conversely, the ERP of PV cardiomyocytes and left atrial cells both similarly decreased . Hyperpomyocytes .Following PV ectopic activity, a shortened effective refractory period allows for PV firing. Furthermore, a similar decrease in the ERP between PV and LA cells does not modify the underlying heterogeneity of the ERP between both structures . HyperpoObservations by Li et al. support this concept. They showed that adenosine-induced APD shortening was more significant in the right atria than in the left atria. They also showed that adenosine-induced atrial fibrillation was mainly associated with reentrant drivers localized in the lateral right atria, which is associated with a higher expression of A1R and GIRK4 . In thisInterestingly, in AF, the reduced supply of oxygen in localized atrial regions of a canine model showed a profound conduction slowing within the ischemic zone, which promoted reentry mechanisms . Further1R overexpression [1R expression in a sinoatrial node and atrial cardiomyocytes was associated with adenosine-induced sino-atrial node conduction abnormalities and shorter atrial repolarization, which increase the risk of AF [1R inhibition limits its practical use [The brady-tachycardia syndrome associates a sinus bradyarrhythmia with tachyarrhythmia, such as AF, which is a hallmark of heart failure . Chronicpression . In a cask of AF . These esk of AF . In clinical use .+ current  density [2+ release from the sarcoplasmic reticulum through the ryanodine receptor [2+ overload responsible for delayed afterdepolarization (DAD), which promotes AF [1R activation inducing a c-AMP-dependent decrease in ICa,L, it has limited or no effect on the action potential duration [2AR in Ca2+ handling remodeling in AF.In atrial cardiomyocytes, a reduced L-type Ca2 density  and incrreceptor  are implmotes AF ,117. Theduration . Indeed,2AR was reported in transgenic mice with cardiac overexpression of human A2AR [2AR can increase the frequency of a PKA-dependent spontaneous Ca2+ release from the sarcoplasmic reticulum without modulating L-type Ca2+ current [2AR overexpression is associated with an increased phosphorylation state of the ryanodine receptor, an increase in Ca2+ spark and wave frequency and a subsequent increase in the spontaneous INCX frequency [2AR activation in these right atrial samples did not modulate the Ca2+ content of sarcoplasmic reticulum, the amplitude of ICa,L and the INCX. However, the remodeling of Ca2+ handling associated with A2AR activation by adenosine has been reported to alter the ability of human atrial myocytes to maintain a uniform beat-to-beat response [2AR activation was linked to an increased spontaneous Ca2+ release, which can elicited DAD by the arrhythmogenic transient inward current (Iti)  .2BR has an important role in cardiac fibroblast homeostasis, but the role of A2B in fibrosis is controversial, since both profibrotic [2BR activation plays an essential role in the inhibition of angiotensin-II-induced collagen production [2BR increases the production of collagen and increases the release of IL-6 in human cardiac fibroblasts, resulting in a profibrosis state [2BR that is time dependent on the A2BR, with early protective effects in acute post-myocardial ischemia and a later inappropriate healing runaway [The activation of Afibrotic ,159,160 fibrotic ,164,165.oduction . Howeveris state . These o runaway .2BR in atrial fibrillation and fibrosis proliferation. Recently, our group demonstrated an overexpression of A2BR in AF, especially within the left atrium [2BR antagonist reduces ventricular remodeling and tachycardia vulnerability after myocardial infarction [2BR may be implicated in fibrosis modulation in atrial fibrillation.Until there, scarce data are published on implication of At atrium . In the farction , A2BR ma2AR could also reduce the angiotensin-II-induced collagen production, especially in the case of hypertension [2BR, explaining perhaps a lower interaction with left atrium fibrosis modulation [2BR activation or blockade in AF and on atrial fibrosis are needed.Other ARs have also been implicated in profibrotic action or reduction of myocardial remodeling. Similarly, activation of Artension . Neverthrtension . Howeverdulation . FurtherDysregulation of the autonomous nervous system are characterized by an excessive sympathetic activation, and a diminished parasympathetic influence is central to the pathogenesis of cardiovascular diseases, including heart failure, hypertension and AF . Combine2+ channels (LTCC) and is responsible for large increases in the Ca2+ flux [2AR agonist, the increase in the heart rate was attenuated by a β-blocker. Thus, Wragg et al. demonstrated a direct activation of the sympathetic nervous system by A2AR stimulation [Atrial sympathetic innervation is controlled by adrenergic receptors, which are divided into three major subfamilies: α1-, α2-, and β-adrenergic receptors. They are all coupled to different classes of heteromeric G proteins. The α1 and α2 receptors are coupled to Gq/11 and Gi/o, respectively. β-Adrenergic receptors are coupled to Gs. The α1 and α2 receptors induce a strong extracellular ATP release, while the β-adrenergic receptors activate cAMP production . The acta2+ flux . Interesmulation .2Rs). As with A1R stimulation, M2R activation leads to the opposite effect on β-adrenergic stimulation. The M2R stimulation activates inhibitory G proteins, subsequently reduces the activity of the HCN and LTCC and leads to decreased automaticity and conduction velocity in the nodal cell. M2R also activates the GIRK channel responsible for hyperpolarization [In parallel, atrial parasympathetic innervation is controlled by muscarinic receptors .The remodeling of the adenosinergic system has been described in many cardiovascular diseases; however, it is of note that the repetition of pathophysiological characteristics by exogenous adenosine injection have been described only in syncope  and atriThe question remains whether adenosine release would be a cause or a consequence of AF. On the one hand, the release of adenosine could be a consequence of increased energy requirements related to AF risk factors, which then secondarily enhance AF. On the other hand, adenosine release could be just a consequence of the cardiac hypoxia associated with AF.As illustrated in this review, multiple electrophysiological modulations secondary to adenosine receptor activation and overexpression may coexist as part of the initiation, recurrence or maintenance of AF. To go further, continuous monitoring of adenosine plasma levels could help to understand whether AF or adenosine is the chicken or the egg.It is also hypothesized that the vicious circle of AF initiation and maintenance may also be driven by the modulation of adenosine receptors’ expression. Until now, the antiarrhythmic action of adenosine is still debated, and the results of studies evaluating the antiarrhythmic actions of adenosine agonists or antagonists are controversial. To overcome this barrier, research efforts in AF could explore other pharmacological approaches, such as biased agonist at adenosine receptors. The specific modulation of adenosine receptor signaling and activity may then play a protective antiarrhythmic role in AF.


To determine the impact of glucose levels at admission and during first week (early phase) on clinical outcomes in patients with acute pancreatitis (AP) and to investigate the relationship between stress hyperglycaemia (SHG) and hypertriglyceridaemia (HTG).Two independent and prospective databases were retrospectively analysed (n = 1792). Patients admitted with pain of less than 48 hours and confirmed AP were included. SHG was defined as admission blood glucose ≥ 10.00 mmol/L (non-diabetic) or ≥ 16.67 mmol/L (diabetic). Blood glucose records for the first week were inspected to determine whether SHG lasted ≥ 48 hours (persistent) or < 48 hours (transient). Clinical outcomes were compared between designated patient groups using multivariate and trend analyses. The correlation between SHG and HTG (serum triglyceride ≥ 5.65 mmol/L) was also analysed.P < 0.05). Duration of SHG was also associated with worsened clinical outcomes . In HTG-AP patients, more severe clinical outcomes were observed in those who concomitantly had SHG (P < 0.05). On admission, SHG was present in 27.8% (499/1792) patients; during the first 48 hours of admission, transient and persistent SHG was found in 31% (556/1792) and 8.0% (144/1792) patients, respectively. Admission SHG was associated with higher incidence of persistent organ failure, acute necrotic collection, major infection, and mortality as well as prolonged length of hospital stay (all Admission and persistent SHG during the first week of admission worsens clinical outcomes of AP patients. These effects are more pronounced when admission HTG co-existed. Acute pancreatitis (AP) is one of the leading acute gastrointestinal diseases which has no effective and targeted drug treatment  and causIt is now well known that acute illness or injury can result in hyperglycaemia, insulin resistance and glucose intolerance, collectively termed stress hyperglycaemia (SHG) . SHG is We have recently investigated the specific BG levels that define SHG in AP patients with or without pre-existing DM . It was ).The present study was based on the retrospective analysis using the STROBE guidelines  of two lAll patients followed uniform diagnostic criteria for AP according to Revised Atlanta Criteria (RAC) . ComprehThe inclusion criteria were adult patients (18-80 years) who had pain for 48 hours or less prior to admission, including those referred from other hospitals. The BG levels were determined at the time of admission and during the first week of admission at a frequency of at least every 48 hours.The exclusion criteria were admission hypoglycaemia (BG level < 3.9 mmol/L) ; use of Blood glucose: admission BG data used in this study were derived from the first blood biochemistry analysis of patients who presented at emergence department and very few were obtained at general ward. Subsequent daily BG levels during the first week were from the biochemistry analysis of blood drawn prior to any potential food intake in the morning (fasted at least for 8 hours overnight).Stress hyperglycaemia: defined as BG ≥ 10.00 mmol/L or ≥ 16.67 mmol/L for non-diabetic and diabetic patients, respectively , regardlPersistent SHG: defined as SHG not resolved after 48 hours of treatment. Patients with early fulminant pancreatitis who died within 48 hours after admission and hence were not able to develop SHG lasting > 48 hours, were included in the group of patients with persistent SHG , 29.Transient SHG: defined as SHG of less than 48 hours duration regardless of insulin treatment.Pre-existing DM: diagnosed based on disease and medicine history, or serum glycated haemoglobin  as per American Diabetes Association (ADA) criteria .HTG-AP: defined as AP with serum triglyceride (TG) levels ≥ 5.65 mmol/L on admission after ruling out common aetiologies . DefinitThe primary clinical outcome was persistent organ failure, defined as at least one of the systems  having modified Marshall organ failure score ≥ 2 and lasting ≥ 48 hours . Secondath-75th percentile and compared using Mann–Whitney U test, Kruskal-Wallis H test, or Cuzick’s trend test analyses. Categorical data are expressed as number with percentages and compared using Chi-square test (or Fisher’s test), linear trend test or proportional trend test analyses.Continuous data are displayed as medians with 25P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS® 26.0 . The E-value and 95% CI were calculated using an online calculator: https://www.evalue-calculator.com/evalue/  with 95% confidence intervals (CIs). Cox proportional hazards analysis was used to report LOHS and expressed as hazard ratios (HRs) with 95% CI (days for deceased patients were considered as truncated data). In both multivariate logistic regression and Cox proportional hazards analyses, baseline factors including age, or those of important clinical significance were adjusted. To quantify the effect of unmeasured potential confounding factors, we report the E-value, which represents the minimum strength of association on the risk ratio scale that an unmeasured confounder would need to have with both the exposure (with SHG) and the outcomes to fully explain away an association between the two . Surviva/evalue/ , 32.P > 0.05) of the present study design, albeit with the demographic, aetiologies, and admission clinical severity scoring systems varied.The demographics and clinical outcomes are shown in P < 0.05) between patients with and without SHG.SHG on admission was present in 499 (27.8%) patients Table 1.P < 0.05), corresponded to E-values of 2.18, 2.00, 3.87 and 3.64, respectively; the respective E-values for the low 95% CIs were 1.76, 1.64, 2.41 and 1.24. Admission SHG was also significant associated with increased LOHS  when compared with those without SHG, E-value with the high 95% CI were 1.92 and 1.63.Results for comparing clinical outcomes between patients with and without admission SHG are shown in P > 0.05). In patients with increased duration of SHG there was a significant increase in BMI, HTG aetiology, admission BG and TG levels, and clinical severity scores .Transient SHG was found in 556 (31%) and persistent SHG in 144 (8.0%) patients (139 were non-diabetic) and comparison between these two groups is demonstrated in P < 0.001). There were no significant differences between transient SHG and no SHG groups . Across these 3 groups there was a step-wise increase in the incidence of persistent organ failure, MODS, acute necrotic collection, major infection, and mortality with corresponding prolonged LOHS . The same was true for HTG-AP patients (lower panel). These results were confirmed after adjusting for age, gender, BMI, Charlson comorbidity index, time to admission, and referral status  into no, transient, and persistent SHG which were associated with escalating disease severity and adverse clinical outcomes; clinical outcomes were worse in patients with persistent SHG compared with those with admission SHG. Furthermore, the association of SHG with adverse clinical outcomes remained robust in HTG-AP patients.A large number of studies have focused on the probability of DM after pancreatitis and its possible mechanism , 33–35. While the definition of SHG varied among studies , 38, thePersistent hyperglycaemia is a common parameter used to evaluate blood glucose fluctuations, and our finding of their association with adverse clinical outcomes in AP patients is similar to that in acute ischaemic stroke, acute myocardial infarction, intracerebral haemorrhage, and other critical illness , 40, 41.HTG has been reported as the aetiology of AP in more than a third of cases in some large Chinese AP cohorts –8. This post hoc analysis, the BG data were not available for every day (data were mainly missing on day 4 and day 6), which may cause the lower proportion of transient SHG than it actually was. Secondly, only more than half of the patients had HbA1c measured on admission and thus we most likely underestimate the prevalence of pre-existing DM in out cohorts. Thirdly, we could not determine from both databases what the timing of and which treatment strategies affected the glucose levels. Therefore, we did not try to perform an analysis of glycaemic variability. Further research with individual-level data on treatment type and timing may help clarify these questions. Finally, we did not investigate the impact of admission and duration of SHG on the probability of developing post-pancreatitis DM, which will comprise a separate study.Our study has several limitations. Firstly, as the nature of In conclusion, we demonstrate that the admission and duration of SHG had important impact on development adverse clinical outcomes in AP patients. Screening, monitoring, and targeting AP patients with high-risk of developing SHG may have beneficial clinical implication.The original contributions presented in the study are included in the article/XY and NS contributed equally to this study. XY, NS, WHe, LL, YL, LY, LD, TJ, NL, and YZ: acquisition of data. XY, NS, and WHu: drafting of manuscript. XY, NS, WHe, PZ, TL, LD, TJ, and WHu: analysis and interpretation of data. SheL and PZ: statistical analysis supervision. ShiL, RS, and JW: important intelligence input. RS, JW, YZ, and QX: critical revision of the manuscript. WHu, QX, and YZ: study concept and design, obtained funding, study supervision. All authors contributed to the article and approved the submitted version.This work was supported by National Natural Science Foundation of China ; Key Research and Development Programme of Science and Technology Department of Sichuan Province ; China Postdoctoral Science Foundation ; NIHR Senior Investigator Award (RS).These authors thank all the staff from the pancreas multidisciplinary teams at West China Hospital of Sichuan University and First Affiliated Hospital of Nanchang University for their continuous support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically (p = 0.02) and molecular  response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model.Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O NPM1-mutated (NPM1c) AML is the most frequent [The outcome of patients diagnosed with acute myeloid leukemia (AML) remains unsatisfactory and new therapeutic approaches are required . Drug sefrequent .FLT3 mutations), and some active drugs may lack strong genetic biomarkers  [After the failure of conventional therapies, there is no standard salvage regimen and patients are increasingly proposing personalized therapies based on their physical status and the genetic make-up of their disease. However, genetics may not suffice to choose the optimal therapy, because some targets may have multiple available inhibitors  –6.DSS datasets can be leveraged at the population level, e.g., to identify drugs active in pre-defined AML subsets, or at the individual level, to tailor therapy to each patient. So far, most AML DSS platforms have tested drugs across broad concentration ranges in standard culture conditions, using the global viability of minimally fractionated primary samples to estimate drug sensitivity –6. HowevNPM1-mutated AML, we perform clinical and genetic validation of the platform and nominate novel sensitizers to “7 + 3” daunorubicin (DNR) and cytarabine (AraC) combination chemotherapy in AML.Here we report the development and validation of an ex vivo drug screening platform for primary human AML cells that combines a niche-like culture system with a multiparametric flow cytometry readout. Focusing on Supplementary Appendix (online only).Detailed Methods are provided in the Fresh or viably frozen Bone Marrow (BM) or peripheral blood (PB) mononuclear cells (MNCs) from AML patients were collected after informed consent at the time of diagnosis or relapse by the INCa-labeled Hôpital Saint-Louis Tumor Bank. The project was approved by INSERM IRB CEEI-15-220). Clinical and genetic annotations are provided in Supplementary Tables 5-220. Cl2 and 5% CO2 . Details are provided in the Supplementary Appendix.All experiments were conducted in 96-well plate format. hTERT-MSC-GFP immortalized human mesenchymal stromal cells (MSCs) were provided by JP Bourquin) . AML MNCSupplementary Appendix.Cells were washed and stained with Fixable Viability Stain eFluor 780 (Thermo Fisher Scientific), anti-CD45 PerCPCY5.5, anti-GPR56 PE, anti-CD11b APC, anti-CD14 APC, anti-CD15 APC, anti-CD3 BV421 and anti-CD19 BV421  and processed on an Attune Next (Thermo Fischer Scientific) flow cytometer. Cell counts were obtained after manual gating on FlowJo V10.6.2 . Details are provided in the PhamarcoGx R package [Supplementary Appendix.Cell counts in each gate were normalized to negative controls (DMSO 0.1% vehicle wells). Drug activity was determined as the actual  area over the curve (AOC) of cell counts (ie without fitting a dose-response curve) without truncation, using the  package . With AOhttps://www.R-project.org/). Details are provided in the Supplementary Appendix.Statistical analyses were conducted in Prism 8.0.1  or R version 3.6.0 (Bulk and single-cell RNA-Seq will be available at European Genome-phenome Archive (EGA) under accession code EGAS00001006265. Other data will be available upon reasonable request to the principal investigator.2  and is stable upon short-term ex vivo culture [2) and MSC co-culture enhanced the number of viable GPR56+ leukemic cells after 72-hour culture ex vivo over each feature alone or standard  culture  growth of CD34+ hematopoietic stem/progenitor cells from healthy donors in niche-like  co-culture dose-response assays, whichever was lowest , unsupervised clustering revealed different patterns of drug activity, including one with predominant activity on the leukemic bulk with lower efficacy on LSCs and differentiation (“cytotoxic” pattern A), one with predominant activity on the proportion of LSCs (“stemness-specific” pattern B) and one with prominent differentiating activity  without impact on the total number of leukemic cells upon single-agent exposure to the BCL-2 inhibitor venetoclax, the relative sparing of LSCs with the cytotoxic DNR-AraC combination, and the differentiating activity of ATO-ATRA, also resulting in a seemingly unchanged count of total leukemic bulk. Altogether, these results are in keeping with known pre-clinical or clinical data regarding these agents in NPM1-mutated AML [We next interrogated the patterns of activity of 25 drugs (or combinations) at fixed concentrations in the niche-like culture system. Clinically relevant maximal concentrations for ex vivo drug testing were defined as either the peak plasma drug concentration in available pharmacokinetics (PK) studies, or as the concentration inhibiting 40%  and EPO  to the output of short-term ex vivo cultures of primary AML cells using multiparametric flow cytometry. Cells were treated with vehicle (DMSO) or with a mini-panel of six drugs/combinations with reported activity in NPM1-mutated AML in 4-point 10-fold dilution dose-response assays  to those in full “pseudo-niche” conditions . Overall, there was no systematic bias between culture conditions  after standard culture versus pre-culture, compared to only 244  after niche-like culture  or 20% of Cmax from PK data, whichever was lower . In 21 NPM1-mutated AML patients with available post-induction NPM1c transcript MRD data, MRD levels did not differ between patients with chemosensitive versus chemoresistant blasts (p = 0.35). Conversely patients with chemosensitive GPR56 + LSCs (n = 11) had lower post-induction MRD than those with chemoresistant LSCs , independently of adverse European LeukemiaNet risk . The niche-like platform also validated known genetically targeted therapies , and to a lesser extent on LSCs (p = 0.05) of FLT3-ITD mutated samples (n = 16), while addition of the more potent FLT3-ITD inhibitor crenolanib had superior activity on both blasts (p = 0.006) and LSCs (p = 0.009). Finally, the IDH1 inhibitor ivosidenib led to enhanced killing of blasts (p = 0.04), but not LSCs (p = 0.35) in IDH1-mutated samples (n = 9). Of note, no difference was noted for the activity of the combination with the IDH2 inhibitor enasidenib between IDH2 mutated (n = 7) and wildtype (n = 38) samples on bulk (p = 0.20) or LSC (p = 0.47) populations (not shown). Systematic inspection of chemogenomic relations further revealed that FLT3 mutations significantly sensitized blasts and LSCs to the addition of venetoclax to DNR-AraC  in 45 patient samples, including 37 NPM1c AMLs , whereas the addition of ruxolitinib to doxorubicin-cytarabine further reduced leukemic infiltration over chemotherapy alone  and the addition of ruxolitinib to chemotherapy also significantly improved survival compared to chemotherapy alone  of 45 tested patients, “private” optimal combinations with nine different third agents were nominated for 17 (38%) patients, while only 1 (2%) had no benefit of any of the 23 third agents tested  still being detectable in all tested patients, although the full resolution of clonal architecture would require whole-exome sequencing. Our bulk RNA-Seq results suggest a benefit of niche-like culture over standard culture in preserving leukemic transcriptional states, though a significant depletion of the promonocyte-like state was also noticeable upon niche-like culture, while single-cell RNA-sequencing revealed a skewed cell cycle distribution in residual cells after standard, but not niche-like culture.NPM1-mutated AMLs, most of whom achieve remission after 7 + 3 chemotherapy [NPM1-transcript MRD, but also event-free survival. Of note, CD34 expression was not accounted for in this study, given its variable expression in NPM1-mutated LSCs [FLT3 or IDH1 mutations predicted the response to combinations including FLT3 or IDH1 inhibitors, respectively. Of note, no such correlation was noted with the IDH2 inhibitor enasidenib. Though this could simply reflect the limited size of the studied cohort , this could reflect a potential IDH2-independent activity of enasidenib [Akin to several DSS studies , 48–51, otherapy , and usiotherapy , we coulted LSCs . This clasidenib .NPM1 mutation) confirmed the benefit of adding venetoclax or selinexor to intensive chemotherapy [FLT3 or signaling mutations and is reminiscent of the sensitization to BCL-2 inhibition by this kinase inhibitor recently reported by an ex vivo DSS study [NPM1-mutated PDX model, where engrafted mice received a ruxolitinib regimen in the lower range of reported in vivo experiments [NPM1-mutated AML cells [The standard randomized clinical trial approach, even when selecting patients based on a genetic biomarker, has so far yielded limited benefit, owing to the variable benefit of adding a third agent to the standard 7 + 3 backbone. Indeed, by screening the addition of 23 drugs at concentrations deemed clinically relevant based on available PK data, or on drug testing of healthy CD34+ cells, we could show that in 39% of patients, the optimal triplet therapy is a private one. This result praises the development of robust DSS assays and innovative clinical trial design to foster functional precision medicine in AML . Analyziotherapy . More suSS study . A combiSS study , but to eriments –58, and/ML cells .Prospective analytical and clinical validation of our proposed niche-like ex vivo drug testing assay and benchmarking to existing platforms are important future steps , 59. FurSupplementary Methods and Figures (Clean)Supplementary Tables 1–3 and 5-12Supplementary Table 4


Cytosolic calcium ([Ca2+]c) was measured using the high affinity sensor Fluo-2. We recorded two types of calcium events: local and global ones. Local events were limited to a small, 2–5 µm section of the dendrite, presumably caused by local synaptic activity, while global events were associated with network bursts and extended throughout the imaged dendrite. In both cases, cytosolic surges were followed by a delayed rise in [Ca2+]m. In global events, the rise lasted longer and was observed in all mitochondrial clusters. At the end of the descending part of the global event, [Ca2+]m was still high. Global events were accompanied by short and rather high [Ca2+]m surges which we called spikelets, and were present until the complete decay of the cytosolic event. In the case of local events, selective short-term responses were limited to the part of the mitochondrial cluster that was located directly in the center of [Ca2+]c activity, and faded quickly, while responses in the neighboring regions were rarely observed. Caffeine , and carbonyl cyanide m-chlorophenyl hydrazine  could affect [Ca2+]m in both global and local events. We constructed a computational model to simulate the fundamental role of mitochondria in restricting calcium signals within a narrow range under synapses, preventing diffusion into adjacent regions of the dendrite. Our results indicate that local cytoplasmic and mitochondrial calcium concentrations are highly correlated. This reflects a key role of signaling pathways that connect the postsynaptic membrane to local mitochondrial clusters.While neuronal mitochondria have been studied extensively in their role in health and disease, the rules that govern calcium regulation in mitochondria remain somewhat vague. In the present study using cultured rat hippocampal neurons transfected with the mtRCaMP mitochondrial calcium sensor, we investigated the effects of cytosolic calcium surges on the dynamics of mitochondrial calcium ([Ca For example, MT is connected to the endoplasmic reticulum via MAM , which is crucial for the regulation of [Ca2+] in the two compartments [Mitochondria (MT) are the main energy provider of living organisms . While iartments . The rul2+]c) [2+]m in a limited area near a synapse, with very little lateral diffusion, is of particular importance. Both local calcium stores [2+]c.In particular, one of the poorly studied issues is the function of MT localized in synaptic zones of dendrites, especially under dendritic spines and their immediate vicinity. MTs in these key regions are organized in relatively extended clusters, up to ≈5 µm in length. Their function may be to provide energy support to synapses and to ensure local protein synthesis in the area of clusters of dendritic spines, especially during synaptic plasticity and metabolic adaptation to stress . However2+]c) , which mm stores  and mito2+]m and [Ca2+]c and to propose that the latter regulates local variations in [Ca2+]m. In order to examine kinetics of postsynaptic mitochondrial calcium transients in response to cytosolic calcium bursts, we used two pharmacological agents, caffeine and CCCP, which are known to alter mitochondrial calcium transients. These results clarify MT functions in normal and functionally impaired neurons.In the present study, we employed cultured rat hippocampal neurons transfected with a molecular MT calcium sensor mtRCaMP  as well 2+]c events. The second type of activity is ‘local’, as it is restricted to a small (2–5 μm) section of the dendrite.Two types of spontaneous events can be detected in cultured networks of neurons. The first type, the synchronized activity, which is expressed as a network burst, involves neurons generating action potentials simultaneously. During a network burst, calcium surges involve all dendrites of the neuron. These are called ‘global’ [Ca2+]c events , while nearby ‘side’ and ‘far’ MT were observed in only 18% and 5% cases with the source in the spine (2+]c (top panel) and [Ca2+]m (bottom panel) are indeed restricted to a small region of the dendrite. Pearson’s correlation coefficient between [Ca2+]c and [Ca2+]m peaks amounted to 0.851. Furthermore, a region of the dendrite to the right of the central cluster in 2+]m levels detected in ROIs adjacent to the spines and away from it (‘side’) during the [Ca2+]c events is summarized on 2+]m in ROI were significantly larger than those detected in ‘side’ regions , but also contributed to the overall rise in network burst (red bars). 2+]c (top) and [Ca2+]m (bottom) in the same area as in 2+]m  produced a complex action; in the first five minutes after infusion, it caused an increase in number and amplitude of global c and [Ca2+]m is shown in 2+]c amplitude, CCCP did not affect the kinetics of global events in cytosol (top). At the same time both drugs had an explicit effect on the [Ca2+]m timing (bottom). Thus, in CCCP (gray curve), the initial part of [Ca2+]m rise was faster and higher than in control , while at the later part, associated with [Ca2+]c decay, the caffeine-related curve (purple) was dramatically higher than both of other traces, indicating the slower decline .The effect of both drugs on the time course of global [Ca2+]m rise. However, we do not have enough experimental evidence of our own to support this hypothesis. As for the effect of caffeine, the release of calcium from the ER stores is a relatively slower process, due to which the accumulation of calcium in mitochondria reaches significantly higher levels, and occurred a few seconds later.We hypothesize that the effect of CCCP may be associated with a gradual disintegration of mitochondrial clusters and their transformation from long filamentous structures with a large surface area to dense, rounded formations as shown before ,20,21,222+]c and [Ca2+]m baselines for control and CCCP groups were the same but significantly elevated in caffeine  and the same applied to cytosolic local events , compared to this peak, while the presence of another obstacle in the side reduced the number of calcium ions to a negligible level (~0.88 ions maximum). Therefore, it is concluded that the presence of absorbing regions of calcium, similar to MT in the dendrite, could have high, non-linear reductions of calcium availability at the dendritic locations that are not directly below the spine.Then the simulation was repeated with a partially absorbing obstacle C showing2+]m following similar changes in [Ca2+]c, indicating that the control of variations in [Ca2+]m may lie in changes in concentration of free intracellular [Ca2+]c. The changes are found primarily but not exclusively in close proximity to local dendritic spines, and it is likely that they are caused by activation of AMPA and NMDA receptors in the postsynaptic membrane. The difference between global and local events is likely to reflect the striking activation of voltage gated NMDA receptors caused by the strong depolarization of the dendrites c as well as [Ca2+]m signal  have been reported before in cultured H9 cardiac cells , and sug2+]c rise. However, in control and in the presence of CCCP this increase rapidly degrades. Summarizing the caffeine-related data on duration and kinetics of both local events and spikelets, we find an increases in their amplitude, duration and a slower decay kinetics. In contrast, CCCP shortens the peak time and the frequency of [Ca2+]m transients. The kinetics of mitochondrial calcium decay is cell-specific. MT calcium uptake and release are important for IP3—associated cytosolic Ca2+ signaling [2+]m release in both global mitochondrial pool and in the local areas of individual mitochondrial clusters following caffeine, which targets ER-mitochondrial interaction and following CCCP, which disrupts their physiological functionality. Differential release of Ca2+ from mitochondria can contribute to the control of cytosolic signaling pathways and function along with calcium stores, other organelles and internal buffers to modulate and regulate global and compartmentalized cytosolic calcium levels.The frequency of spikelets increases along with [Caignaling . The pre2+]m employed Rhod-2 as the calcium sensor [2+]m. In any case, Rhod-2 is a diffusible agent, and is not likely to predict the precise localization of changes in [Ca2+]m. Moreover, the introduction of Rhod2 involves an injection of the agent into the cell. The genetically encoded mtRCaMP [2+]m.Early studies on the regulation of m and [Ca2+]c are different, and they do not share the same region of interest. Second, Fluo-2, being a diffusible sensor, may not obey the boundaries of the mitochondria, unlike mtRCaMP, and we have seen different regions of interest for the two agents   studied ing CCCP .Finally, a major field with special interest in MT research is Alzheimer’s disease (AD). While the main focus of recent AD studies is in the formation of amyloid plaques, there are compelling indications that malfunction of MT and intracellular calcium regulation might be the place where neurodegeneration is initiated. In line with this hypothesis, it has been found that one of the main mutated proteins associated with the disease is presenilin, localized in the mitochondria of affected individuals . The rolIn conclusion, our studies that link dendritic morphological compartments and local mitochondrial and cytosolic calcium interrelations with dendritic spines and synaptic or network activity gives new insights into dynamic mitochondrial functions within small dendritic compartments of central neurons.5 cells in 1 mL medium were plated in each well of a 24 well plate. Cells were left to grow in the incubator at 37 °C, 5% CO2 and the medium was replaced by FUDR (5-fluoro-2′-deoxyuridine)-containing medium to block proliferating glia.Animal handling was performed in accordance with the guidelines published by the Weizmann Institute’s Institutional Animal Care and Use Committee (IACUC), Rehovot, Israel, cultures were prepared as detailed elsewhere . BrieflyNeurons were transfected with eGFP or eBFP, for imaging cell morphology, and MT calcium sensor (mtRCaMP), using lipofectamine 2000 , at 6–7 days in vitro (DIV), and were imaged at 10–21 DIV. The transfection methodology was adopted from standard protocols .2+]c resulting from network activity. Cultures were then placed in the 3 mL perfusion chamber, on the stage of an upright Zeiss 880 confocal microscope using a 40× water immersion objective (1.0 NA), and imaged at a rate of 10–20 frames/s. No photobleaching was detected under these conditions. Standard medium for imaging contained (in mM) NaCl 129, KCl 4, MgCl2 1, CaCl2 2, glucose 10, and HEPES 10, pH was adjusted to 7.4 with NaOH and osmolality to 320 mOsm with sucrose. Imaging of cell morphology (405 nm), cytosolic (488 nm) and MT calcium (543 nm) were made simultaneously. All measurements were conducted with identical laser parameters for all groups  at room temperature. Pharmacological agents were applied into the chamber during recording. The substances were dissolved at working concentration and the initial volume was gradually replaced with this solution during 30–60 s. The substances were considered to be completely washed in or out the chamber after replacing the entire volume of the chamber two-three times.Cultures were initially incubated with Fluo-2AM  for 1 h at room temperature to image variations in [Cat-tests, as the case may be. Statistically significant differences were considered at p < 0.05. For data processing, we used MATLAB , Visual Studio Code , RStudio , KaleidaGraph , and Origin  software.High-resolution fluorescent images were analyzed using ImageJ  and MATLAB -based programs. Statistical comparisons were made with post hoc tests using ANOVA and Ca = 600 μm2/s−1. Initially (at t = 0), 1000 ions are located 10 μm below the topmost point of the spine head (top arrow). The interior surfaces of the spine head, spine neck and the dendrite act as fully-reflecting boundaries, while there are 50 calcium pumps (each with a radius of 10 nm) that are fully absorbing and uniformly distributed in the upper hemisphere of the spine head [We simulate three-dimensional calcium diffusion in a dendritic spine and a segment of a dendrite  Figure A. All ioine head . Therefo2+ diffusion is simulated until all ions are absorbed, either by Ca2+ pumps or at the two sides of the dendrite. When obstacles are added to the simulation, they are modeled as fully reflecting or partially-absorbing spheres with a radius of 700 nm, located as shown in The two ends of the dendrite are absorbing boundaries, separated by 5 μm, which is the length of the dendritic segment we simulated. CaAll simulations were implemented with Python 3.8 and the NumPy library.