In addition, we have found a significant elevation of diastolic [Na+]d in Chagas’ cardiomyocytes (FCII>FCI) that was greater than control. Exposure of cardiomyocytes to agents that enhance inositol 1,4,5 trisphosphate (IP3) generation or concentration like endothelin (ET-1) or bradykinin (BK), or membrane-permeant myoinositol 1,4,5-trisphosphate hexakis(butyryloxy-methyl) esters (IP3BM) caused an elevation in diastolic [Ca2+] ([Ca2+]d) that was always greater in cardiomyocytes from Chagas’ than non- Chagas’ subjects, and the magnitude of the [Ca2+]d elevation in Chagas’ cardiomyocytes was related to the degree of cardiac dysfunction. Incubation with xestospongin-C (Xest-C), a membrane-permeable selective blocker of the IP3 receptors (IP3Rs), significantly reduced [Ca2+]d in Chagas’ cardiomyocytes but did not have a significant effect on non-Chagas’ cells. The effects of ET-1, BK, and IP3BM on [Ca2+]d were not modified by the removal of extracellular [Ca2+]e. Furthermore, cardiomyocytes from Chagas’ patients had a significant decrease in the sarcoplasmic reticulum (SR) Ca2+content compared to control (Control>FCI>FCII), a higher intracellular IP3 concentration ([IP3]i) and markedly depressed contractile properties compared to control cardiomyocytes. These results provide additional and convincing support about the implications of IP3 in the pathogenesis of Chagas cardiomyopathy in patients at different stages of chronic infection. Additionally, these findings open the door for novel therapeutic strategies oriented to improve cardiac function and quality of life of individuals suffering from chronic Chagas cardiomyopathy (CC).Chagas cardiomyopathy is the most severe manifestation of human Chagas disease and represents the major cause of morbidity and mortality in Latin America. We previously demonstrated diastolic Ca Trypanosoma cruzi, is an endemic disease of Latin-American countries, affecting 10 million people are estimated to be infected with T. cruzi, and more than 120 million inhabitants are at risk of infection. The parasite is transmitted to humans in a vectorial way by infected triatomines and through other non-vector mechanisms such as the oral route, congenital transmission, organ transplants or blood transfusions. Due to immigration towards non-endemic regions, the disease can spread and affect people around the world via blood transfusions. The pathogenesis of this disease is still unwell understood; we previously demonstrated that cardiomyocytes isolated from Chagas patients have an intracellular Ca2+ overload, which appears to be associated with changes in the inositol 1,4,5 trisphosphate (IP3) signaling pathway. This study corroborates that human cardiomyocytes isolated from Chagas’ patients have an increase in [Ca2+]d and a partial membrane potential depolarization, which corresponds with the degree of cardiac dysfunction determined by the NYHA classification (23). In this report, we showed, for the first time, that IP3R activators, e.g., IP3BM, ET-1, and BK-induced a more significant elevation of [Ca2+]d in Chagas’ compared to non-Chagas’ human cardiomyocytes, which was not modified by the removal of [Ca2+]e. Additionally, these findings open the door for novel therapeutic strategies oriented to improve cardiac function and quality of life of individuals suffering from chronic Chagas cardiomyopathy (CC).Chagas disease, caused by the parasite  Trypanosoma cruzi (T. cruzi), which is transmitted to humans by blood-sucking triatomine bugs and by non-vectorial mechanisms, such as contaminated blood transfusion, organ transplantation, and congenital infection . In some experiments, cell integrity was further determined by the ability of the cardiomyocyte to exclude the dye trypan blue.Cardiomyocytes were studied if they had sharp outlines and rod-shaped, clearly visible striations, without developing subsarcolemmal blebs, and showing spontaneous contractile activity in the presence of 1.8 mM extracellular [Ca2+ and Na+ selective microelectrodes were prepared as described previously [2+ concentration ([Ca2+]d) and diastolic Na+ concentration ([Na+]d) as described before [2+ selective microelectrodes with a linear relationship between pCa 3 and 7  were used experimentally. The Na+ selective microelectrodes gave virtually Nernstian responses at free [Na+]e between 100 and 10 mM. However, although at concentrations between 10 and 1 mM [Na+]e, the microelectrodes had a sub-Nernstian response (40–45 mV), their response was of sufficient amplitude to be able to measure [Na+]d. The response of the Ca2+ and Na+-selective microelectrodes were not directly affected by any of the drugs used in the present study.Double-barreled Caeviously . Each iod before . Only th2+ tolerant cardiomyocytes were transferred to poly-L-lysine-coated coverslips for 45 minutes in a small Plexiglas chamber filled with normal Tyrode solution containing 20 mM BDM at 37°C. Only rod-shaped cardiomyocytes without any signs of deterioration and spontaneous activity at rest were used for experiments [2+ selective microelectrodes with the aid of an inverted microscope fitted with an x10 eyepiece and an x40 oil objective. The potentials from the 3 M KCl barrel -resting membrane potential (Vm)- and the Ca2+ barrel (VCaE) were recorded via a high-impedance amplifier . The potential of the voltage microelectrode (Vm) was subtracted electronically from the potential of the Ca2+ electrode (VCaE) to obtain the differential signal (VCa) representing the resting [Ca2+]d. Vm and VCa potentials were acquired at a frequency of 1,000 Hz with AxoGraph software , and stored in a computer for further analysis. Two criteria were used as key elements to accept or to reject individual [Ca2+]d measurements performed in cardiomyocytes from control and Chagas’ patients: i) polarize resting membrane potential -more negative than -80 mV in control and more than -75 in Chagas cardiomyocytes- and ii) stable recording potentials for no less than 40 seconds .Within 1–2 h after isolation, human Caeriments , 34. Car2+ stored in the sarcoplasmic reticulum (SR), control and Chagas’ cardiomyocytes were loaded with 5 μm Fluo-4-AM for 30 min at 37°C. Fluo-4 loaded cardiomyocytes were transferred to a small Plexiglas chamber filled normal Tyrode solution containing 20 mM BDM and placed on Plexiglass chamber on the stage of an inverted microscope equipped with epifluorescence illumination (XCite® Series 120 or Lambda DG4) equipped with a CCD cooled camera . The excitation wavelength of the argon-ion laser was set to 488 nm, and fluorescence emission was measured at wavelengths >515 nm. The experiments were conducted in a Ca2+-free solution to prevent the Ca2+ uptake by the SR. The Ca2+ transient elicited by 10 mM caffeine (2 min stimulus) was used as an index of the Ca2+ content of the SR, which was estimated by taking the area under the curve of the signal induced by caffeine [To estimate the total amount of Cacaffeine . The exp3] was determined in cardiomyocytes biopsies from control subjects and Chagas’ patients using a competitive radioligand binding assay, as previously described d was carried out. The area-under-the-curve for the caffeine-induced release of Ca2+ from the SR was calculated by the trapezoid rule (GraphPad Prism software 7.0). Statistical analysis was performed using a two-tailed paired and unpaired t-test or one-way analysis of variance coupled with either Tukey’s or Dunnett’s t-test for multiple measurements to determine significance. Significance was accepted at p<0.05 level. Statistical analysis was done using GraphPad Prism 7.03 .All values are expressed as mean±SD; 2+]d in CC patients, which correlate directly with the extent of their cardiac dysfunction (NYHA class) regardless of gender [2+]d in single cardiomyocyte isolated from control (A), FCI (B), and FCII (C) Chagas’ cardiomyocytes. An elevation of [Ca2+]d and a partial depolarization were observed in cardiomyocytes isolated from FCI and FCII Chagas patients. In control cardiomyocytes [Ca2+]d was 123±3 nM (n = 40), while that in CC patients from FCI patients [Ca2+]d was 262±25 nM (n = 35) (p≤0.001 compared to control), and in cardiomyocytes from FCII patients [Ca2+]d was 378±34 nM (n = 32) (p≤0.001 compared to control and FCI)  . No gend+]d was observed in cardiomyocytes isolated from FCI and FCII Chagas patients compared to control. In control [Na+]d was 8±0.1 mM (n = 13) compared to 12±1 mM (n = 16) and 17±1.2 mM (n = 17) in FCI and FCII cardiomyocytes respectively (p≤0.001 compared to control) (2+ and Na+ overload in chagasic cardiomyocytes compared to control cells.A significant difference for d in both control and Chagas’ cardiomyocytes, but the elevation was greater in the cardiomyocytes isolated from Chagas’ patients than control (FCII>FCI>control) (3BM elevated [Ca2+]d from 122±3 nM (n = 30) to 202±22 nM (n = 36) (p≤0.001), while in FCI-cardiomyocytes [Ca2+]d rose from 255±40 nM (n = 33) to 462±44 nM (n = 31) (p≤0.001). In FCII-cardiomyocytes, [Ca2+]d increased from 374±43 nM (n = 30) to 759±43 nM (n = 30) (p≤0.001) (3BM] (up to 30 μM) still evoked a differential pharmacological effect on [Ca2+]d between Chagas’ and control cardiomyocytes. The incubation in L-myoinositol 1,4,5-trisphosphate hexakis(propionyloxy-methyl) ester (L-IP3PM) did not induce changes in [Ca2+]d either in control or CC indicating that the action of the ester was highly specific The role of IPtivation . 10 μM Icontrol) . IP3BM ep≤0.001) . Incubatspecific . The Ca23] and causes IP3-dependent Ca2+ release [3 in the pathogenesis of Chagas’ heart disease cardiomyocytes from control and Chagas’ patients were exposed to ET-1 and [Ca2+]d determined. Incubation in 100 nM ET-1 for 15 min induced an increase in [Ca2+]d that was significantly higher in Chagas’ than in control cells (FCII>FCI>control) (2+]d from 123±3 nM (n = 30) to 187±14 nM (n = 31) (p≤0.001 compared to untreated control). In Chagas’ cardiomyocytes from FCI hearts [Ca2+]d rose from 258±34 nM (n = 24) to 443±42 nM (n = 28) (p≤0.001 compared to untreated cardiomyocytes). In cardiomyocytes isolated from FCII patients, it increased from 378±35 nM (n = 36) to 746±42 nM (n = 30) (p≤0.001 compared to untreated cardiomyocytes) .Endothelin (ET-1) is a peptide that increases endogenous d in all cells analyzed. However, the increase in [Ca2+]d was greater in Chagas’ than in control cardiomyocytes (FCII>FCI>control) (2+]d from 123±4 nM (n = 24) to 210±33 nM (n = 22) in control cardiomyocytes (p≤0.001 compared to untreated cardiomyocytes). In FCI cardiomyocytes, [Ca2+]d was increased from 253±24 nM (n = 25) to 477±45 nM (n = 22) (p≤0.001 compared to untreated cardiomyocytes). In FCII cardiomyocytes, [Ca2+]d rose from 385±38 nM (n = 24) to 799±54 nM (n = 25) (p≤0.001 compared to untreated and FCI cardiomyocytes) (2+ did not modify the BK effect on [Ca2+]d in control or Chagas’ cardiomyocytes (see Extracellular Ca2+contribution).To further test the role of IP C (PLC)  which ha C (PLC) . Incubatcontrol) . BK raisyocytes) . The omi3R [2+ in Chagas’ cardiomyocytes. [Ca2+]d was measured before and after incubation for 15 minutes with 5 μM Xest-C. Treatment with Xest-C caused a significant reduction in [Ca2+]d in Chagas’ cardiomyocytes but not in control cardiomyocytes (123±3 nM (n = 25) versus 120±2 nM (n = 23) (p>0.05 compared to untreated cells)  to 160±23 nM (n = 27) (p≤0.001 compared to untreated cardiomyocytes), and in FCII cardiomyocytes [Ca2+]d decreased from 368±37 nM (n = 28) to 190±29 nM (n = 27) (p≤0.001 compared to untreated and FCI cardiomyocytes) (2+]d in Chagas’ cardiomyocytes was reversed by continuous washout from the bath (at least 15 minutes). Furthermore, Xest-C prevented the elevation of [Ca2+]d in control and Chagas’ cardiomyocytes elicited by IP3BM (The effects of xestospongin C (Xest-C), a membrane-permeable selective blocker of the IP3R , were ind cells) . In cardyocytes) . The effby IP3BM  and ET-1by IP3BM   than control cardiomyocytes (n = 10) (p≤0.001), and in the FCII was reduced by 61% (n = 9) (p≤0.001) (2+ content in FCI and FCII Chagas’ cardiomyocytes (2+ content was increased by 25% in FCI (n = 11) (p≤0.001 compared to untreated cells) and by 71% in FCII (n = 9) (p≤0.001 compared to untreated cells) in Chagas’ cardiomyocytes. No significant difference was observed in control cardiomyocytes after Xest-C treatment (n = 11) (p>0.05). These results suggest that the reduction in the SR Ca2+ levels appears to be mediated by an IP3Rs-Ca2+ leak from the SR.The level of the SR Cacaffeine . Under tp≤0.001) . Moreovemyocytes . SR Ca2+3, as determined by the competitive radioligand-binding assay were significantly higher in ventricular cells in patients with Chagas’ disease than in control. The basal level of [IP3]i was 5.4±0.6 pmol/mg protein (n = 11) in control cardiomyocytes (3]i was 8.1±0.8 pmol/mg protein (n = 14)  and in those classified as FCII [IP3]i was 14±2 pmol/mg protein (n = 10)   .2+ in the elevated [Ca2+]d observed in Chagas’ cardiomyocytes, we conducted experiments in Ca2+-free medium  to 96±6 nM (n = 13) (p≤0.001 compared to untreated cells), In FCI from 261±39 nM (n = 24) to 172±31 nM (n = 20) (p≤0.001 compared to untreated cells) and in FCII from 377±44 nM (n = 15) to 207±33 nM (n = 18) (p≤0.001 compared to untreated cells)  . Removalmyocytes  (p>0.05)vs. 1.89±0.02 μm, n = 12 and 1.85±0.02 μm, n = 13 for FCI and FCII respectively (p≤0.001 compared to control and p≤0.01 compared FCI versus FCII) (Heart failure is the most significant and severe manifestation of human CC . We founus FCII) . The peaus FCII) . PS was us FCII) . +dL/dt us FCII) . Furtherus FCII) . Xest-C us FCII) . It must2+ regulation. CC is the most severe and life-threatening manifestation of human Chagas disease and is one of the most common causes of heart failure and sudden death in Latin America. This disease has become a public health concern that is not limited to populations in Latin America but also poses a global problem because of migration of infected individuals for economic and/or political reasons to developed countries, mainly Europe and the United States.The current study reinforces our previous finding that a progressive deterioration of cardiac function in CC is associated at the cellular level with a defective intracellular Ca2+]d and a partial membrane potential depolarization, which corresponds with the degree of cardiac dysfunction determined by the NYHA classification d in Chagas’ compared to non-Chagas’ human cardiomyocytes, which was not modified by the removal of [Ca2+]e. Furthermore, Chagas’ cardiomyocytes had a reduced SR Ca2+ loading and a higher level of intracellular IP3 with compromised contractile properties compared to control. Treatment with Xest-C, an IP3R blocker, improves [Ca2+]d, increased SR-Ca2+ loading and ameliorates contractile dysfunction in Chagas’ cardiomyocytes.The present study confirms that human cardiomyocytes isolated from Chagas’ patients have an increase in [Cafication . In this2+ handling. Normal functioning of multiple mechanisms like plasma-membrane exchanger (Na+/Ca2+ exchanger) and pumps (PMCa2+ and SERCA-ATPase pumps) which control Ca2+ influx-efflux and reuptake allow for maintaining proper [Ca2+]d during the rest period of the cardiac cycle (diastole) within a physiological range (~100 nM) [2+]d values obtained from the control cardiomyocytes concur with previous estimations of the diastolic Ca2+ level in human ventricular myocytes using Ca2+-selective microelectrodes d as observed in Chagas’ cardiomyocytes is deleterious to muscle cell function because increase calpain activation and impairment of autophagy and mitochondrial function d found in Chagas’ cells are qualitatively similar to those reported in human epithelial cells infected with T. cruzi [2+]d observed in Chagas’ patients is related to the CC and not a resultant side effect from the patient’s pharmacological treatment because all medications were suspended 48 h before the endomyocardial biopsy. The observed partial depolarization in Chagas’ cardiomyocytes from FCI and FCII patients may relate to a diastolic Na+ overload found in human Chagas’ cardiomyocytes (1.4-fold in FCI and 2.1-fold in FCII compared to control). A membrane depolarization associated with intracellular Na+ overload has been described in skeletal muscle cells [+]d can contribute to a further intracellular Ca2+ overload through the reverse mode of sarcolemmal Na+/Ca2+ exchanger [The changes in the [CaT. cruzi . We consle cells . Besidesxchanger .2+ homeostasis and the intracellular messenger IP3 [2+]d in the Chagas’ cardiomyocytes d d . In this [Ca2+]d  have sugmyocytes . Additio failure .3 involvement in CC was obtained by showing that exposure of cardiomyocytes to agents that enhance endogenous generation or concentration of IP3 like IP3BM, ET-1 or BK [2+]d which was always greater in cardiomyocytes from Chagas’ patients than non-Chagas’ subjects and related to the degree of cardiac dysfunction (FCII>FCI). The differential pharmacological effect of IP3BM on [Ca2+]d in Chagas’ cardiomyocytes persists up to a concentration of 30 μM, where the [IP3]i levels would be equivalent between control and Chagas’ cardiomyocytes, suggesting a greater IP3Rs expression in Chagas cardiomyocytes compared to control. The IP3BM, ET-1, or BK effects on [Ca2+]d were not modified by the removal of extracellular Ca2+, but it was inhibited by Xest-C, suggesting that their pharmacological action is mediated through IP3-dependent Ca2+ release. These results reinforce the notion that increased [Ca2+]d observed in Chagas’ cardiomyocytes is mediated in part by activation of IP3Rs.Further support for the IP-1 or BK , 40 caus3PM did not induce any change in [Ca2+]d either in control or Chagas’ cardiomyocytes indicates that the action of IP3BM was highly specific. Individuals with CC had increased levels of ET-1 in plasma d may enhance the IP3 sensitivity of IP3Rs [2+]d. An increase in IP3Rs expression has been reported in atrial myocytes of humans and dogs during atrial fibrillation and in human heart failure [3R expression is significantly elevated in rat cardiac tissue from aorta-banded hypertrophic mice and human ischemic heart with dilated cardiomyopathy [3R expression may represent a plausible explanation for the increased [Ca2+]d observed in Chagas’ cardiomyocytes.In Chagas’ cardiomyocytes, chronic elevated i was higher compared to those from control subjects. It has been previously shown in various types of cells that elevation of IP3 production, which release Ca2+ from intracellular stores [2+]d  can have two possible sources i) the plasma membrane of parasites in intracellular forms, such as amastigotes [3 derived from the plasma membrane of the host changes due to changes in IP3 synthesis and/or degradation [3]i may provoke an increase in Ca efflux from the SR, which could end in a depletion of intraluminal sarcoplasmic reticulum Ca2+ content d, also elicits a Ca2+ depletion of the SR, depressing the amount of Ca2+ for release upon electrical stimulation [2+]d. Pretreatment with Xest-C partially reverse the contractile dysfunction in CC by significantly increasing PS, +dL/dt, and -dL/dt. The enhancement of contractile function induced by Xest-C may be related to the inhibition of IP3Rs and the prevention of SR Ca2+ depletion.Chagas’ cardiomyocytes exhibit markedly depressed contractile properties versus control across all parameters studied, such as peak shortening, maximal velocity of shortening (Control>FCI>FCII), which may be related to a reduced SR Ca failure , 75. Chamulation , 73. Fur2+ provoked a more significant reduction of [Ca2+]d in Chagas than control cardiomyocytes. Several mechanisms of Ca2+ entry non-voltage dependent have been described in cardiac cells; among them, the TRPC, a diversely regulated family of plasma membrane permeable cation channels, which are activated by diacylglycerol, by depletion of intracellular Ca2+ stores or by stretch [3R [An interesting observation was that depletion of extracellular Ca stretch . Biochemetch [3R . Further2+]d that appears to be mediated by IP3Rs and is associated with the deterioration of cardiac function (FCII>FCI). Consistent with these results, agents that enhance intracellular IP3 generation like ET-1, BK, or membrane-permeant IP3 esters caused a further elevation in [Ca2+]d more significant in cardiomyocytes from Chagas’ than non-Chagas’ subjects- and Xest-C an IP3Rs blocker decreased [Ca2+]d, and improved cardiomyocytes contractile response from Chagas’ patients. Furthermore, Chagas’ cardiomyocytes had a higher level of intracellular [IP3] with compromised SR-Ca2+ loading compared to control.In conclusion, patients suffering from CC have a chronic elevation of  and intracellular [IP3] in cardiomyocytes isolated from chagasic patients should be interpreted with caution. Both changes may occur as an epiphenomenon in a heart as a consequence of multiple pathological alterations observed in CC. However, despite the above limitations, we have confirmed the involvement of intracellular Ca2+ dysregulation, and we unmask a thus far unrecognized involvement of IP3 in the pathophysiology of CC.The major limitation of this study is that downstream IPantation , 29 or dantation , where mS1 Fig2+]d was measured using Ca2+-selective microelectrodes before and after treatments with L-myoinositol 1,4,5-trisphosphate hexakis(propionyloxy-methyl) ester (L-IP3PM). The incubation in L-IP3PM did not induce significant changes in [Ca2+]d either in control (CTR) or Chagas’ cardiomyocytes. Cardiomyocytes were obtained from 8–10 control individuals, 7–9 Chagas’ FCI, and 6–8 Chagas’ FCII patients, respectively; n represents the number of cardiomyocytes in which a successful measurement of [Ca2+]d was carried out. Data are expressed as means ± S.D. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparison tests, *** p≤0.001.[Ca(TIF)Click here for additional data file.


Urticaria is common in pediatric population and is caused by various etiologies which usually differ among different age groups. The different etiologies require different management strategies. Thus, understanding detailed of the etiologies of urticaria in children would help pediatricians to perform appropriate initial treatment.A cross-sectional epidemiological study of all patients aged under 18-year-old with the diagnosis of urticaria from any causes entered in the emergency department during January 1st, 2016 to December 31st, 2019 by collecting the data from the Health Object Program®, an authorized electronic medical records program, at the Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand.There were total of 515 urticaria patients aged under 18 years old at the emergency department. The ages of patients ranged from 8 months to 18 years with a median age of 7 years (IQR 3.17–12.08). The majority of the patients were in the preschool-aged group (40.97%), followed by the school-aged (28.16%), adolescent (22.14%), and infant (8.74%). Six major etiologic categories were identified in the present study. The most common cause of urticaria was infection (51.26%), followed by idiopathic urticaria (34.37%), inhalants (6.99%), drugs (4.08%), foods (2.52%), and insect stings (0.78%).Having underlying allergic diseases had a strong association with all identified causes of urticaria in the study population, of which, food and inhalation etiologies had a significant difference when compared to the other identified causes. The present study has found that infection was the most common cause of acute urticaria in children. This etiology (infection-induced urticaria) usually presents concurrent with fever, however, non-febrile symptoms were also presented. Therefore, in the pediatric population, pediatricians should always look for infection as the cause of urticaria even in patients without pyrexia. Urticaria is common in pediatric population and is one of the most common dermatologic conditions presenting at the emergency departments . The preThe authors conducted a cross-sectional epidemiological study from January 1st, 2016 to December 31st, 2019 by collecting the data from the Health Object Program®, an authorized electronic medical records program, at the Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand. All patients aged under 18-year-old with the diagnosis of first episode urticaria from various causes, entered at the emergency department, Faculty of Medicine, Khon Kaen University were included in to the study.The study was approved by the institutional review board of the Khon Kaen University, Human Research Ethics Committee (#HE631510). The waiver for the informed consent was approved and all methods were performed in accordance with the relevant guidelines and regulations.P < .05 were considered to indicate statistical significance. Multinomial logistic regression and post hoc analysis were used to test for the association between etiologic factors of urticaria.At the end of the study, the collected data were analyzed using STATA software version 10 (StataCorp LP). Descriptive statistical methods, means, standard deviations (SDs), medians and frequencies were used to analyze the demographic data. Values of There were total of 515 urticaria patients aged under 18 years old at the emergency department during the study period. There were 283 (54.95%) male and 232 (45.05%) female patients; thus, the ratio of males to females was 1.2. The ages of patients ranged from 8 months to 18 years with a median age of 7 years (IQR 3.17–12.08). The age range were classified in to four groups; infant (< 1-year-old), preschool-aged (1 to 6-year-old), school-aged (7 to 12-year-old), and adolescent (13 to 18-year-old). The majority of the patients were in the preschool-aged group (40.97%), followed by the school-aged (28.16%), adolescent (22.14%), and infant (8.74%). There were 68 patients (13.2%) with fever and 27 patients (5.24%) with angioedema. The patients’ underlying allergic diseases  were documented in 59 patients (11.45%). Majority of patients (79.42%) had their onset of urticaria less than 24 h prior to visiting the emergency room. Table Six major etiologic categories were identified in the present study. The most common cause of urticaria was infection 51.26%), followed by idiopathic cause (34.37%), inhalants (6.99%), drugs (4.08%), foods (2.52%), and insect stings (0.78%). We did not find acute urticaria caused by physical stimuli from the present study. Figure .26%, folThere were various sources of infection in infection-induced urticaria in the study population. Respiratory tract infection was the most common cause , followed by gastrointestinal infection , unidentified source of infection , and otitis media . Figure P < 0.001). Two etiologies of urticaria  showed a significant difference from other causes , Table There were 59 patients (11.45%) with at least one underlying allergic disease . Multinomial logistic regression and post hoc analysis revealed an association between urticaria  and patients with underlying allergic diseases  had fever, of which, infection-induced urticaria was diagnosed in 62 patients. However, infection-induced urticaria can be found in nonfebrile patients. This may due to the fact that, patients can develop urticaria at a time of infection or days to weeks after. Thus, normal temperature in infection-induced urticaria can be found. Therefore, in the pediatric population, pediatricians should always look for infection as the cause of urticaria even in patients without pyrexia. The present study performed a correlation test between fever and the cause of urticaria by using multinomial logistic regression analysis, nevertheless, no associations were found.Angioedema was found in 27 patients 5.24%). This symptom can indicate an early sign of anaphylaxis. Angioedema can be found together with urticarial rashes or else as an isolated finding %. This s–26. TherOnset of the rashes in urticaria patients usually present as acute onset in nature. The authors tried to find the association between the onset of rashes prior to the emergency room of less and more than 24 h and the etiology of urticaria. However, this factor has no correlation to any urticaria causes, Table P < 0.001  were documented in 59 patients (11.46%). A recent publication among a large cohort study in adolescent has found a strong association between chronic spontaneous urticaria and atopic diseases . These a01 Table . Among tFrom January 2016 to December 2019, etiologies of urticaria in children did not change over time  were fully excluded. Moreover, some essential information in some patients, such as history of allergy was also missing. In patients who had inhalants allergens  listed as the cause of their urticaria, it was not clear whether this occurred through contact or other routes.The present study revealed several identified causes of urticaria . All etiologies had a strong association with the underlying allergic diseases, of which food and inhalation causes had a significant difference when compared to the other identified etiologies. The present study has found that infection was the most common cause of acute urticaria in children. This etiology (infection-induced urticaria) usually presents concurrent with fever, however, probable non-febrile symptoms can also exist. Therefore, in pediatric population, pediatricians should always look for the underlying causes of urticaria from infections even in patients without pyrexia symptoms.


Using a combination of biochemical, Ca2+ imaging, and electrophysiological techniques, we determined the effect of 24-h aldosterone treatment on the TRPCs/Orai-dependent SOCE in adult rat ventricular cardiomyocytes (ARVMs). The 24-h aldosterone treatment (from 100 nM to 1 µM) enhanced depletion-induced Ca2+ entry in ARVMs, as assessed by a faster reduction of Fura-2 fluorescence decay upon the addition of Mn2+ and increased Fluo-4/AM fluorescence following Ca2+ store depletion. These effects were prevented by co-treatment with a specific mineralocorticoid receptor (MR) antagonist, RU-28318, and they are associated with the enhanced depletion-induced N-phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2)-sensitive macroscopic current recorded by patch-clamp experiments. Molecular screening by qRT-PCR and Western blot showed a specific upregulation of TRPC1, TRPC5, and STIM1 expression at the messenger RNA (mRNA) and protein levels upon 24-h aldosterone treatment of ARVMs, corroborated by immunostaining. Our study provides evidence that the mineralocorticoid pathway specifically promotes TRPC1/TRPC5-mediated SOCE in adult rat cardiomyocytes.Whereas cardiac TRPC  channels and the associated store-operated Ca The Furmin and Rmax, Fura-2-loaded ARVMs were firstly exposed to Ca2+-free solution with 10 mM caf + 5 µM Tg for 5 min, to empty Sarcoplasmic Reticulum (SR) Ca2+ stores. The bath solution was then switched to K+ buffer  containing 10 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). To obtain Rmin, the cells were incubated for 30 to 45 min with 20 µM non-fluorescent Ca2+ ionophore, ionomycin, and 10 µM carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone in K+-buffer Ca2+-free solution, and measurements were taken at both wavelengths after the fluorescence reached stable values. Then, Rmax was obtained by saturating the indicator with 10 mM or 20 mM CaCl2 in the presence of 10 mM EGTA.For in situ measurements of RD, a dose response ) was performed in K+ buffer. As a double-log plot, the Ca2+ response of the indicator was linear with the x-intercept equal to the log of the apparent KD of the indicator. We calculated Rmin = 0.66, Rmax = 1.84, and β = 1.35 with KD = 377 nM. Of note, aldosterone treatment did not change any parameters.To calculate the K2+ influx was measured on Fura-2/AM loaded ARVMs as described previously [2 solution was perfused in the presence of NIF and KB-R7943, and the initial linear slope of the Fura-2 fluorescence’s decrease was measured, reflecting the SOC channel activity. The quenching rate of fluorescence intensity (F360) was estimated using linear regression of the initial decaying phase  just after Mn2+ addition, expressed as a percentage decrease in F360 per min . Photobleaching was <0.5%/min during measurements. All experiments were done at controlled 37 °C.The Mneviously . Fura-2 ISOC current was recorded as described previously [2+ stores were depleted by successive perfusion of 5 μM Tg and 10 mM caf, and then the currents (ISOC) were elicited by a 1.2-s voltage ramp from −100 mV to +60 mV, preceded by 0 to +40 mV prepulse (400 ms) to inactivate the voltage-dependent Na+ channels. The SOCI were recorded before and after 5 µM BTP2 cell perfusion, and then normalized to membrane capacitance to account for cell size variations.The eviously . The SR Western blot was performed as described previously  using thARVMs were fixed and permeabilized with 2% paraformaldehyde (PFA)/0.5% Triton X-100 as previously described . Images The RNA extraction and RT-qPCR quantification were performed as previously described , using tn) or animals (N) studied per experiment is indicated. Statistical analyses, described in the figure legends, were performed using GraphPad Prism 6.0 software . The variance assumption was automatically tested. A value of p < 0.05 was considered significant.The number of cells , caffeine , and nifedipine , the subsequent addition of 1.8 mM Ca2+ in the presence of NIF and KB-R7943  induced a moderate increase of fluorescence in 69% of ctrl ARVMs (gray trace), while a stronger response was observed in a dose-dependent manner in all aldosterone-treated cells . On average  antagonist RU-28318 (RU at 10 µM). Similar results were obtained using the ratiometric Fura-2/AM fluorescent probe (2+-quenching microfluorimetry in the presence of major cardiac Ca2+ entry pathway inhibitors (NIF and KB). 2+ entry, were increased up to 1.5-fold after aldosterone incubation; the effect was blunted in ARVMs co-treated with 10 μM RU-28318  for 24 h. Of interest, 24-h aldosterone treatment did not induce cellular hypertrophy as assessed by the similar membrane capacitance of isolated ARVMs treated 24 h with or without 1 μM aldosterone . As exemplified in  average B, we dennt probe C,D. To cRU-28318 F. These 2+ and K+ currents by including inhibitors (NIF and KB) and Cs+ to block K+ channels. After SR Ca2+ store depletion (see above), the SOC currents (ISOC) were elicited by a standard ramp protocol before and after the perfusion of the non-selective SOC channel blocker BTP2 (5 µM). As shown in ISOC was increased in aldosterone-treated ARVMs , a moderated SOCE activity might be observed [As previously reported , ARVMs s in 2004  remains ar cells ,17,35,45ar cells ,46, evenobserved ,47,48,492+ release-activated Ca2+ channel supported by the Orai1 channel to relatively Ca2+ non-selective SOC channels supported by TRPC channels [Reflecting previous reports in adrenal chromaffin cells  or coronchannels . Unlike channels , we did channels ,10 mightchannels , in corochannels ,56, and channels . These achannels ,58. A nuchannels ,60,61,62channels , in the channels , in humachannels , in NRVMchannels , and in channels ,46,67. Schannels , contribchannels ,69. In achannels ,74,75,763) sensitive Ca2+ store depletion remain to be performed.Our findings provide direct support for the presence of STIM1, TRPC1, and TRPC5 in ARVMs and for their participation in aldosterone/MR-enhanced SOCE. Taking into account that G-protein-coupled receptor (GPCR) activation promotes TRPC1 and TRPC5 translocation to the plasma membrane in a STIM1-dependent manner  and the


IL13rs1295686A/A genotype had an increased risk of AML (PCorr = 0.0144) whereas carriers of the VEGFArs25648T allele had a decreased risk of developing the disease (PCorr = 0.00086). In addition, we found an association of the IL8rs2227307 SNP with a decreased risk of developing AML that remained marginally significant after multiple testing (PCorr = 0.072). Functional experiments suggested that the effect of the IL13rs1295686 SNP on AML risk might be explained by its role in regulating IL1Ra secretion that modulates AML blast proliferation. Likewise, the protective effect of the IL8rs2227307 SNP might be mediated by TLR2-mediated immune responses that affect AML blast viability, proliferation and chemorresistance. Despite the potential interest of these results, additional functional studies are still warranted to unravel the mechanisms by which these variants modulate the risk of AML. These findings suggested that IL13, VEGFA and IL8 SNPs play a role in modulating AML risk.The purpose of this study was to conduct a two-stage case control association study including 654 acute myeloid leukaemia (AML) patients and 3477 controls ascertained through the NuCLEAR consortium to evaluate the effect of 27 immune-related single nucleotide polymorphisms (SNPs) on AML risk. In a pooled analysis of cohort studies, we found that carriers of the  Epidemiological studies suggested that AML onset can be triggered by multiple factors including age, sex, lifestyle, exposure to chemicals and a number of blood and congenital disorders2. However, the biological mechanisms underlying AML aetiology remain largely elusive. Even though cytogenetic analysis have allowed the stratification of AML patients into favourable, intermediate and unfavourable classes and has improved our ability to predict clonal evolution and disease progression3, many AML patients (~ 45%) have a normal karyotype, which suggests that additional genetic alterations are needed to develop the disease. Sequencing studies identified genes frequently mutated in AML, some of which predict poor prognosis 4. Furthermore, it is increasingly evident that host immunity might also be implicated in AML risk and survival5. AML blasts activate immunosuppressive mechanisms to evade the immune system whereas immune response changes induced by the gut microbiota can also influence the anti-leukaemic effects of immune cells6. In addition, the efficacy of allogeneic stem cell transplantation (SCT) in eradicating AML is linked to the appearance of the graft-versus-leukaemia effect, mediated by the recognition of major histocompatibility antigens present in malignant blasts by T cells7. Likewise, the disappearance of these circulating T cells recognising AML or the loss of costimulatory  or inhibitory interactions (PD-1/PDL-1) eventually leads to relapse8 and the infusion of donor-derived CD8+ memory T cells induces remission in patients who relapsed following allogeneic SCT9. Considering that around two-thirds of AML patients relapse within the first 18 months after first-line therapy, clinical trials are trying to assess the efficacy of immunotherapies in AML and to unravel the interplay between the immune system and AML blasts. Considering the aspects detailed above, the purpose of this study was to conduct a two-stage case control association study including 654 AML patients and 3477 controls ascertained through the NuCLEAR consortium to evaluate whether 27 single nucleotide polymorphisms (SNPs) within the IL4, IL8, IL8RB (CXCR2), IL12A, IL12B, IL13, IFNG, IFNGR2, CCR5, MIF and VEGFA loci influence the risk of developing AML. We also decided to investigate the correlation of selected SNPs with serum steroid hormone levels and their role in modulating immune responses after stimulation of whole blood, peripheral mononuclear cells (PBMCs) and macrophages with lipopolysaccharide (LPS), phytohemagglutinin (PHA), Pam3Cys and CpG.Acute Myeloid Leukaemia (AML) is a common haematological malignancy characterised by the clonal transformation of haematopoietic precursors that alter normal hematopoietic cell growth and differentiation10. Healthy controls included 667 Spanish blood donors from the REPAIR consortium11, 1000 German controls came from the Heinz-Nixdorf Recall (HNR) study12 and 22 donors of allogeneic stem cell transplantation from the Medical University of Innsbruck . In accordance with the Declaration of Helsinki, all study participants provided their written informed consent to participate in the study and the ethical committees of all participating centres and hospitals approved the study.We conducted a two-stage genetic association study to assess whether 27 functional single nucleotide polymorphisms (SNPs) within host immunity-related genes could influence AML risk. The discovery population consisted of 2027 European subjects . AML patients were diagnosed by experienced clinicians and ascertained through the iNternational Consortium for LEukaemiA Research , Regulome (https://www.regulomedb.org/regulome-search/), Blood eQTL browsers , and linkage disequilibrium values  or the Maxwell® 16 Blood DNA Purification kit (Promega) according to manufacturer’s instructions. SNP selection criteria were based on previous associations with haematological malignancies  or solid tumours and clinical related parameters  but also according to their functionality in Haploreg (χ2). Logistic regression adjusted for sex and country of origin was used to assess the associations of the SNPs with AML risk assuming log-additive, dominant and recessive models. According the Meff method14, 24 of 27 SNPs were independent and, consequently, the study-wide significant threshold was set to 0.0007 (0.05/24SNPs/3models). Statistical power was calculated using Quanto (v.12.4) assuming a log-additive model of inheritance.Deviation from Hardy-Weinberg Equilibrium (HWE) was tested in the controls by chi-square (P < 0.05) were replicated in a cohort of 2104 subjects . AML cases were recruited from an independent Spanish medical institution , from the University Hospital of Würzburg (Germany) and from two Italian medical institutions  between 2015 and 2017. Five hundred and seven Spanish controls were blood donors recruited from the Blood Transfusion Centre , 194 Italian controls from the REPAIR consortium, 1068 German controls from a second and independent set of the Heinz-Nixdorf Recall (HNR) study  and 19 donors of allogeneic stem cell transplantation from the University of Würzburg (Germany). The ethical committees of these centres approved the study.For replication purposes, the most relevant findings . The Arnhem-Nijmegen Ethical Committee approved the study (42561.091.12) and biological specimens were collected after informed consent was obtained. We investigated whether any SNP was correlated with cytokine levels  after stimulation of peripheral blood mononuclear cells (PBMCs), whole blood or monocyte-derived macrophages from 408 healthy subjects with LPS (1 or 100 ng/ml), PHA (10 μg/ml), Pam3Cys (10 μg/ml), and CpG (100 ng/ml). After log transformation, linear regression analyses adjusted for age and sex were used to determine the correlation of selected SNPs with cytokine expression quantitative trait loci (cQTLs). All analyses were performed using R software (www.r-project.org/). In order to account for multiple comparisons, we used a significant threshold of 0.00006, i.e., the quotient of 0.05/(24 independent SNPs × 9 cytokines × 4 cell stimulants).In order to determine the biological function of the most relevant SNPs, cytokine production in response to stimulation was measured in the 500 Functional Genomics cohort from the Human Functional Genomics Project  supplemented with gentamicin (10 mg/ml), l-glutamine (10 mM) and pyruvate (10 mM). PBMC stimulations were performed with 5×105 cells/well in round-bottom 96-wells plates (Greiner) for 24 h in the presence of 10% human pool serum at 37 °C and 5% CO2. Supernatants were collected and stored in −20 °C until used for ELISA. LPS (100 ng/ml), PHA (10μg/ml) and Pam3Cys (10 μg/ml) and CpG (100 ng/ml) were used as stimulators for 24 or 48 h. Whole blood stimulation experiments were conducted using 100 μl of heparin blood that was added to a 48 well plate and subsequently stimulated with 400 μl of LPS and PHA  for 48 h at 37 °C and 5% CO2. Supernatants were collected and stored in −20 °C until used for ELISA. Concentrations of human TNFα, IFNγ, IL1β, IL1RA, IL6, IL8, IL10, IL17, and IL22 were determined using specific commercial ELISA kits , in accordance with the manufacturer’s instructions.Detailed protocols for PBMCs isolation, macrophage differentiation and stimulation assays have been reported elsewheren = 280). Complete protocol details have been reported elsewhere17. Steroid hormones were analysed by liquid chromatography tandem–mass spectrometry (LC–MS) after protein precipitation and solid-phase extraction as described in Ter Horst et al.17 . Significance thresholds were set to 0.0003 (0.05/24 independent SNPs/7 hormones).Given the impact of steroid hormones in modulating immune responses, we also evaluated the correlation of SNPs with serum levels of 7 steroid hormones  in a subset of subjects without hormonal replacement therapy or oral contraceptives  and showed a slightly increased male/female ratio compared to healthy controls . In addition, we observed that each copy of the IL4rs2243268C allele was associated with a 1.31-fold increased risk of AML . On the other hand, we found that carriers of the IL8rs2227307G and VEGFArs25648T alleles had a significantly decreased risk of AML  whereas each copy of the IL8rs4073A, CCR5rs1799987G, CCR5rs2734648T alleles was associated with ~ 20–25% decreased risk of AML . Even though only the association of the VEGFArs25648 SNP with a decreased risk of developing AML remained significant after correction for multiple testing in the discovery cohort (PCorr = 0.0014), we found that the association of IL8rs2227307 and IL13rs1295686 with AML risk was confirmed in the replication population  whereas carriers of the IL8rs2227307G allele had a decreased risk of developing the disease that remained marginally significant after correction for multiple testing . Interestingly, although it was not statistically significant in the replication population likely due to the relatively limited power, the pooled analysis also revealed a strong association of the VEGFArs25648T allele with a decreased risk of AML that largely surpassed the stringent study-wide significant threshold , a locus involved in chemokine-mediated immune responses. Interestingly, although it neither reached statistical significance after multiple testing correction, we also found a negative correlation between the IL13rs1295686A allele and levels of IL1Ra after stimulation of PBMCs with LPS  within this gene have been consistently associated, at GWAS level, with immune-related diseases19 and haematological malignancies20. In this two-stage case control association study we found a consistent and statistically significant association of the IL13rs1295686A/A genotype with an increased risk of developing AML that suggested a role of this locus in the pathogenesis of the disease. Mechanistically, we observed a negative correlation between the IL13rs1295686A allele and IL1Ra levels after stimulation of PBMCs with LPS  in the bone marrow that accounts for macrophage polarisation (toward the M2 phenotype) and B cell differentiation and survival23. Although at this point it is tempting to speculate that the IL13rs1295686A allele, which correlates with lower levels of IL1Ra secretion, might represent a biomarker with a potential benefit in AML by antagonising IL1 effects on blast proliferation and blocking inflammation, we believe that additional functional experiments are still warranted to explain the exact mechanism by which the IL13rs1295686 variant influence the risk of AML.The IL8rs2227307T allele with a decreased risk of developing AML. Although the association of the IL8rs2227307 SNP with AML risk remained only marginally significant after multiple testing correction, this finding suggested that the IL8 locus might play a role in the pathogenesis of AML. The IL8 gene is located on chromosome 4q12-q21 and encodes for IL8, a chemokine mainly produced by macrophages and epithelial cells. Previous studies have suggested that the blocking IL8-CXCR2 pathway might have a therapeutic potential in a variety of tumours27 including AML and myelodysplastic syndromes (MDS)28. However, the role of IL8 in AML is still scarce. A recent study has demonstrated that IL8 and its receptor are significantly overexpressed in AML and MDS patients28 and that the expression of these molecules also correlates with poor outcomes. In addition, it has been reported that the IL8-CXCR2 axis is highly expressed in hematopoietic stem cells and progenitor compartments in comparison with healthy controls28 and that this pathway plays a key role in the regulation of cancer stem cell function31 and mesenchymal stem cell-induced T cell proliferation. In addition, Schinke et al. (2015) have experimentally demonstrated that the inhibition of CXCR2 leads to decreased viability and clonogenic capacity of primary cells from AML patients, which pointed towards the use of IL8-CXCR2 pathway as novel therapeutic target28. In line with our genetic data and the notion of a role of the IL8 locus in the pathogenesis of AML, we found that carriers of the IL8rs2227307T allele had increased levels of IL1β after the stimulation of PBMCs with Pam3Cys , a locus involved in chemokine-mediated immune responses. These results suggest that the IL8rs2227307 polymorphism might also influence the risk of AML through chemotaxis stimulation in the microenvironment of the bone marrow (BM). In line with this notion, it has been demonstrated that IL8 is a hypoxia-regulated cytokine that promotes migration in mesenchymal stromal cells in the BM38 and that both endogenous and hypoxia-induced production of IL8 was higher in AML cases compared to controls and was prognostically unfavourable38. A more recent study has also suggested that IL8 blockade might be used as new therapeutic strategy for AML, as it prevents activated endothelial cell mediated proliferation and chemoresistance39.Another interesting finding of this study was the consistent association of the F4V Fig. , a locusVEGFArs25648 SNP to modulate immune responses, our genetic findings are in line with previous studies reporting an increased vascularity and VEGFA levels in AML patients, and a specific VEGFA-dependent vascular morphology in the leukemic BM40. In addition, it has been reported that VEGFA levels are an independent prognostic factor41 and that they modulate the appearance of graft versus host disease after SCT42. Based on the current evidence, we hypothesize that the VEGFArs25648 SNP might influence the risk of developing AML through changes in BM vascularity and morphology and migration of human leukemia cells.Finally, even though we did not find any functional effect of the α = 0.0007) for a SNP with a frequency of 0.25, which underlined the feasibility of the study design. Another important strength of this study is the development of cytokine stimulation experiments and the measurement of seven serum steroid hormones in a large cohort of healthy subjects, which allowed us to investigate the functional role of the most relevant markers in modulating immune responses but also in determining serological steroid hormone levels. A drawback is the multicentric nature of this study that placed inevitable limitations such as the impossibility of uniformly collect cytogenetic and mutation profiles for a significant set of patients. Another limitation was that age was unknown for a subset of German controls. However, given that selected SNPs have not been linked to survival in AML, we think that age is not a modifying factor that could significantly influence the results.One of the major strengths of our study is the inclusion of two large populations. In the combined analysis, we had 80% power to detect an odds ratio of 1.33  and using FAIR principles to optimise Findability, Accessibility, Interoperability and Reusability44.Functional data used in this project have been meticulously catalogued and archived in the BBMRI-NL data infrastructure (https://hfgp. bbmri.nl/) using the MOLGENIS open source platform for scientific dataSupplementary MaterialSupplementary Figure 1


In addition to the control of bioenergetics and ROS production, mitochondria are fine regulators of the cytosolic Ca2+ homeostasis that induce vital signalling mechanisms in excitable cells such as neurons. Impairment in the mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU) or release through the Na+/Ca2+ exchanger may lead to mitochondrial Ca2+ overload and opening of the permeability transition pore inducing neuronal death. Recent evidence suggests an important role for these mechanisms as the underlying causes for neuronal death in β-amyloid and tau pathology. The present review will focus on the mechanisms that lead to cytosolic and especially mitochondrial Ca2+ disturbances occurring in AD and tau-induced FTD, and propose possible therapeutic interventions for these disorders.Aggregation and deposition of β-amyloid and/or tau protein are the key neuropathological features in neurodegenerative disorders such as Alzheimer’s disease (AD) and other tauopathies including frontotemporal dementia (FTD). The interaction between oxidative stress, mitochondrial dysfunction and the impairment of calcium ions (Ca The present review will focus on the Ca2+ signalling impairment, with a special emphasis on the mitochondrial Ca2+ dysbalance occurring in AD and tau-induced FTD.Neurodegenerative disorders, characterised by progressive neuronal loss in specific areas of the brain, nowadays represent one of the biggest medical and social challenges: very few therapeutic strategies are available to slow down the course of these diseases. Aggregation and deposition of misfolded proteins are histopathological hallmarks in these conditions. Among them, β-amyloid plaques found in Alzheimer’s disease (AD) and tau aggregates present in AD, frontotemporal dementia (FTD) and up to other 20 diseases collectively termed tauopathies are one of the most studied . Many ac2+ signalling is a key mechanism in critical events for cell life, from gene transcription or cell growth, to cell-specific mechanisms, such as muscle contraction or egg fertilisation. In neurons, Ca2+ is involved in most aspects of neuronal function: differentiation and migration, synaptic transmission and plasticity, vesicle release, cell death and survival or neuronal–glial communication . The different kinetics, frequency, amplitudes or spatial locations of these transients entails a signalling mechanism able to exert specific impacts in their downstream effectors , accumulate close to the ER-plasma membrane junctions, and interact with the Store-Operated Calcium Channels (SOCCs) in the plasma membrane, allowing the entrance of Ca2+. Orai channels were identified as components of the SOCCs  and restore it to its basal levels to maintain Ca2+ homeostasis. These mechanisms include the efflux of Ca2+ by transporters through the plasma membrane, uptake by organelles such as the ER and the mitochondria and binding to Ca2+-buffering proteins.Either way, the duration and spread of the Ca2+ out of the neurons are the high-affinity, low capacity plasma membrane Ca2+-ATPase (PMCA), which hydrolyses ATP to pump Ca2+ against gradient, and the low affinity, high capacity Na+/Ca2+ exchanger (NCX), which is abundant in neurons and uses the electrochemical gradient of Na+ to extrude Ca2+ , such as the synapses, acting as highly localised Ca2+ buffers able to shape the local Ca2+ signals and regulate neuronal activity , via direct and indirect mechanisms like calpain-induced degradation, pH, PKC or PKA, as recently reviewed in , mitochondrial Ca2+ content must be tightly regulated. Mitochondrial Ca2+ overload, especially under conditions of oxidative stress, triggers the opening of the mitochondrial permeability transition pore (mPTP), a high-conductance mitochondrial channel whose composition and structure are still under debate. While firmly closed under physiological conditions, after mPTP opening, the mitochondrial inner membrane becomes unselectively permeable to small solutes, leading to ΔΨm collapse and eventually mitochondrial swelling and necrotic and apoptotic cell death. mPTP opening has been implicated as the mechanism of cell death in many human diseases, thus representing a major therapeutic target  is able to activate NADPH oxidase, enhancing ROS production in neurons and leading to cell death. Ca2+ signals and increased ROS production were observed after the acute application of tau aggregates, suggesting a mechanism by which extracellular tau fibrils can incorporate into the membranes and lead to neuronal dysfunction in the neighbouring neurons . On the other hand, mitochondrial depolarisation and bioenergetics dysfunction can be triggered by Ca2+ and prevented by the inhibition of mitochondrial Ca2+ uptake as previously shown in works by Abramov and Duchen [Tau and beta-amyloid dysfunction have been widely linked to altered cytosolic Cad Duchen ,118,119.2+ uptake in neuronal death induced by glutamate excitotoxicity [2+ uptake protecting neurons from excitotoxic cell death [2+ uptake without affecting ΔΨm or bioenergetics, and protects neurons against glutamate excitotoxicity [Several reports highlight the role of the mitochondrial Catoxicity ,121. Qiull death . Our grotoxicity . These r2+ uptake and prevent overload induced by β-amyloid, protecting neurons against cell death in AD [Other authors have proposed that the induction of a mild mitochondrial uncoupling with different agents such as non-steroidal anti-inflammatory drugs (NSAIDs) could also reduce mitochondrial Cath in AD . Resultsth in AD .2+ overload in a subset of neurons in the brain of these mice, which preceded neuronal death and could be prevented by MCU inhibition [2+ levels highlighting one more time the deleterious effect of mitochondrial Ca2+ overload. This work also evaluated available microarray and RNA-Sequencing datasheets to analyse the expression of mitochondrial Ca2+-related genes in patients with AD and found that all the genes involved in mitochondrial uptake were downregulated, while Slc8b1 gene encoding NCLX was significantly upregulated, suggesting a possible compensatory response to prevent mitochondrial Ca2+ overload [Recent in vivo imaging by Calvo-Rodriguez et al. in the APP/PS1 transgenic mouse model of AD has shown β-amyloid dependent mitochondrial Cahibition . Interesoverload . Howeveroverload .2+ from ER to mitochondria [2+ transfer, which might contribute to mitochondrial Ca2+ overload [2+ leak from the ER [The mitochondria-associated ER membranes are subcompartments of the ER connected physically and biochemically to the mitochondria allowing the communication between both organelles and the transfer of Cachondria . Many rechondria  and a hichondria ,131. As overload ,133. Somm the ER , thus com the ER .2+ overload is one of the mechanisms of β-amyloid- and tau-induced mitochondrial dysfunction and cell death [mPTP opening induced by mitochondrial Call death ,137,138.ll death . Reductill death . In addill death ,137.2+ efflux has not been explored in the pathogenesis of AD until very recently. Jadiya et al. have shown in different mouse and animal models of the disease that AD progression is associated with the loss of NCLX expression and functionality [Impairment of mitochondrial Caionality . Importa2+ efflux through NCLX in neurons [2+ oscillations in primary neurons after 24 h incubation. These oscillations were followed by mitochondrial Ca2+ uptake, and induced a gradual increase in basal cytosolic and mitochondrial Ca2+, suggesting an impaired Ca2+ handling induced by tau [2+ signal with glutamate (in neurons) or ATP (in astrocytes) incubated with tau further evidenced a slower cytosolic and mitochondrial Ca2+ efflux in both cell types. Experiments in permeabilised cells confirmed that the impairment was mediated by NCLX, as showed by the altered Na+ and Ca2+ currents. More importantly, tau-induced NCLX impairment led to a faster mitochondrial depolarisation when exposing the neurons to  repetitive Ca2+ stimulations, suggesting an increased vulnerability to Ca2+-induced cell death [MAPT were also more vulnerable to physiological and pathological Ca2+ stimulation, and presented an increased susceptibility to mPTP opening [Recent work from our group has shown for the first time the tau-induced altered mitochondrial Ca neurons . In thisd by tau . Stimulall death . iPSC-de opening . MAPT mutation also impairs neuronal excitability [MAPT neurons display an increased mitochondrial membrane potential [2+ overload, are the triggers for mPTP opening. Preliminary data shows that mitochondrial antioxidants are able to protect the 10+16 neurons and reduce their susceptibility to mPTP opening  and efflux (β-amyloid and tau). Specific targeting of the mechanisms leading to Ca2+ impairment, and especially mitochondria-targeted therapies emerge as potential treatments for these disorders.Ca


Previous evolutionary analyses of its pore-forming subunits MCU and EMRE, and gatekeeper MICU1, pinpointed an evolutionary paradox: the presence of MCU homologs in fungal species devoid of any other uniporter components and of mt-Ca2+ uptake. Here, we trace the mt-Ca2+ uniporter evolution across 1,156 fully-sequenced eukaryotes and show that animal and fungal MCUs represent two distinct paralogous subfamilies originating from an ancestral duplication. Accordingly, we find EMRE orthologs outside Holoza and uncover the existence of an animal-like uniporter within chytrid fungi, which enables mt-Ca2+ uptake when reconstituted in vivo in the yeast Saccharomyces cerevisiae. Our study represents the most comprehensive phylogenomic analysis of the mt-Ca2+ uptake system and demonstrates that MCU, EMRE, and MICU formed the core of the ancestral opisthokont uniporter, with major implications for comparative structural and functional studies.Calcium (Ca The mitochondrial calcium uptake system, crucial for cellular processes, evolved in ancient eukaryotes. Here, authors perform a phylogenomic analysis across 1,156 eukaryotes, and show that previously identified animal and fungal genes in this system originated from an ancestral duplication. Mitochondrial Ca2+ (mt-Ca2+) uptake is mediated by a highly selective channel, the mt-Ca2+ uniporter, that resides in the inner mitochondrial membrane and is powered by the negative transmembrane potential4. However, the functional role of mt-Ca2+ uptake and its pathophysiology has remained largely unknown, as the molecular identity of the mt-Ca2+ uniporter channel was only recently discovered7. To this end, comparative genomics analyses based on a few eukaryotic species, in combination with RNAi assays, were instrumental in identifying the first components of the mammalian uniporter, MCU and MICU1 (refs. 7). While MCU constitutes the pore-forming and Ca2+-conducting subunit of the uniporter8, the EF-hand-containing and Ca2+-sensitive protein MICU1 has been shown to function as a channel gatekeeper and cooperative activator9. Importantly, their discovery paved the way to the identification of additional structural and regulatory components of the mammalian uniporter, including a MCU-dominant-negative beta subunit (MCUb)10, three tissue-specific MICU1 paralogs or splice variants (MICU2 (refs. 12), MICU3 (ref. 13), and MICU1.1 (ref. 14)), and an essential MCU regulator (EMRE)15. When co-expressed with MCU, EMRE was shown to be necessary and sufficient to form a functional Ca2+ channel and to reconstitute mammalian-like uniporter activity even in yeast mitochondria16, which are otherwise incapable of Ca2+ uptake18.Mitochondria from several organisms play a key role in regulating intracellular Ca2+ uptake, the identification of several organisms with a different uniporter’s composition in different clades have raised interest in understanding the structural basis of uniporter activity24. For example, while MCU and MICU1 showed highly correlated evolutionary histories across 138 sequenced eukaryotic organisms, EMRE apparently lacked any homolog outside the metazoan lineage and was therefore suggested to be an animal-specific innovation26. Consistently, Dictyostelium discoideum, an amoebozoan that diverged earlier than the origin of opisthokonts, expresses both MCU and MICU1 orthologs, which form a functional uniporter in the absence of EMRE16. Despite the ancient origin of their interaction and the overall observed high co-evolution between MCU and MICU1, most fungi represented an exception to this rule, with most fungal species encoding homologs of MCU but not of MICU1 (refs. 26). The identification of MCU as the only uniporter component in Basidiomycota and filamentous Ascomycota  has been described as an evolutionary paradox (see ref. 27), but has also been interpreted as indicating that fungal MCUs could be sufficient for mt-Ca2+ uptake and regulated independently of MICU1 (refs. 26). Based on the assumption of an orthologous relationship between human and fungal MCUs28, several independent structural studies of Ascomycota MCUs have been performed to understand the basic principles of uniporter channel assembly and function24. However, those organisms had been shown to lack uniporter activity30 and their MCU homologs were unable to mediate mt-Ca2+ uptake when heterologously expressed in HeLa or yeast cells24. Not surprisingly, significant structural and sequence differences were found between fungal MCUs and their animal counterparts24, raising the question of whether fungal MCUs function as classical Ca2+ uniporters at all.Altogether, these findings have highlighted a complex multimeric nature for the mammalian uniporter, whose composition, stoichiometry, and regulation need to be fine-tuned to the physiological demands of each cell and tissue. However, the functional and mechanistic role of each uniporter component and the molecular basis of their interdependence are still unclear. Furthermore, although several observations have pointed to an ancient eukaryotic origin of mt-Ca2+ uniporter and show that a gene duplication at the opisthokont common ancestor of animals and fungi resulted in two distinct MCU paralogous clades, differentially retained in the two groups. When only the animal-like clade is considered, we observe fully consistent co-evolutionary patterns between MCU and MICU (MICU1’s family), across eukaryotes, and of these with EMRE, across opisthokonts. We find that only three early diverging fungi contain both the fungal paralog and the complete animal-like MCU complex . Consistently, we find that the heterologous expression of MCU and EMRE from fungal species with an animal-like MCU complex results in the functional reconstitution of Ca2+ uptake in mitochondria of HeLa and yeast cells. Conversely, representatives of the fungal paralogs do not show uniporter activity, suggestive of an alternative function or of the requirement for a yet unidentified regulator. Altogether, our phylogenomic and functional analyses of the mt-Ca2+ uptake system demonstrate that MCU, EMRE, and MICU represent the minimal core components of the ancestral opisthokont uniporter and pinpoint key targets for comparative structural and functional studies. Finally, this study also confirms the importance and power of thorough evolutionary analyses to understand the molecular basis of functional interactions within a protein complex.Here, we perform a comprehensive evolutionary analysis of the mt-Ca26. We confirmed the presence of MCU in at least some species of the major eukaryotic groups , and its absence in all sequenced Apicomplexans, Microsporidia, Trichomonas, and Giardia, and all yeasts in Saccharomycotina and most in Schizosaccharomyces,. Hence, mt-Ca2+ uptake appeared to have been lost many times independently during the evolution of eukaryotes. A significant number of these losses correlated with extreme streamlining of mitochondrial metabolism, as most MCU/MICU-lacking lineages encompassed relict forms of anaerobic mitochondria, such as mitosomes  or hydrogenosomes (Trichomonas)31. Our homology-based results confirmed the above-mentioned anomaly that most fungal genomes, for which our dataset is particularly rich—776 species as compared to 50 in previous studies26—encode for MCU but not MICU or EMRE. Unexpectedly, our analysis uncovered the presence of EMRE outside Holozoa, identifying reliable orthologs in three chytrid fungi—an early diverging zoosporic fungal lineage: Allomyces macrogynus, Catenaria anguillulae, and Spizellomyces punctatus. Additional searches in public databases confirmed that EMRE was not present in other sequenced fungi.We assessed the evolution of each uniporter component across 1156 fully-sequenced eukaryotic genomes  to each non-vertebrate animal MCUs, and also to all MCUs from protists and plants, explaining why Trypanosoma’s MCUb does not functionally complement human MCUb34. It also implies that shared physical interactions between paralogous MCU proteins in hetero-oligomers in different organisms, as shown for human and Trypanosoma brucei MCUb proteins34, are the result of parallel evolution. Importantly, one duplication event in the MCU gene family occurred in the common ancestor of opisthokonts and was followed by differential losses that distinguished Holomycota  from the other opisthokonts, i.e. the Holozoa . Most fungal species kept only one of the two MCU paralogs that is referred here as the fungal-specific MCU paralog (MCUP). Holozoa, instead, retained the other MCU paralog, the bona-fide animal MCU. Only three chytrid fungi in our dataset, A. macrogynus, C. anguillulae, and S. punctatus retained both the MCUP and the animal MCU, and these are also the only fungi encoding MICU1 and EMRE homologs. This striking, previously undetected, co-evolution pattern between MCU, MICU, and EMRE in chytrids suggests a strong interdependence, and even stronger considering that Blastocladiales (Allomyces and Catenaria) and Spizellomyces do not form a monophyletic clade35. Based on these findings, we hypothesized that these animal-like MCUs present in chytrids should require EMRE to drive mt-Ca2+ uptake, similarly to their human ortholog.To clarify the underlying evolutionary history of the uniporter, we reconstructed and inspected the molecular phylogenies of MCU Fig.  and MICU2+ uptake in the opisthokont ancestor, preceding the diversification of animals and fungi. Consistently, the heterologous expression of MCU from Dictyostelium discoideum, representing an amoebozoan lineage that diverged earlier than the origin of opisthokonts, is alone sufficient to reconstitute mt-Ca2+ uptake in yeast mitochondria, while human (Hs-) MCU only does so in the presence of EMRE16. Similarly, we hypothesized that co-expression of animal MCU and EMRE proteins from chytrids would be necessary and sufficient to reconstitute uniporter activity. The phylogenetic distribution profile (presence/absence) across the MCU complex components revealed a strong co-evolution pattern, when only the true orthologous sequences were considered  or S. punctatus (Sp-MCU) with their respective EMREs  . In contrast, we did not detect any mt-Ca2+ uptake in yeast strains expressing MCUP proteins from A. macrogynus (Am-MCUP1) and S. punctatus (Sp-MCUP), despite proper expression and localization  with Hs-EMRE in yeast was able to reconstitute mt-Ca2+ uptake to a similar extent of a strain expressing Hs-MCU and Hs-EMRE  Fig. . Particu36. Notably, animal and chytrid EMREs appeared highly divergent  were fully conserved. Interestingly, the fungal EMRE sequences contained an extra C-terminal domain that was not found in Holozoa, suggesting some degree of specialization. Thus, we hypothesized that Am-MCUa and Sp-MCU would have evolved to interact with EMRE proteins from the same or related species. To this goal, we measured mt-Ca2+ uptake in HeLa cells infected with lentiviral particles expressing either Hs-MCU or Am-MCUa and Sp-MCU  . Instead, unlike Hs-MCU, all the fungal MCUs and MCUPs were unable to rescue mt-Ca2+ uptake despite showing proper expression and insertion into the inner mitochondrial membrane . These MCUPs, which are paralogous to Hs-MCU, have been so far wrongly considered functionally equivalent to Hs-MCU and deeply studied for this reason. This is the case, for instance, of the Ascomycota MCUP proteins  that have been recently used as models for understanding structure and regulation of the human uniporter channel24. Consistently, we and others find that fungal MCUPs are unable to reconstitute or rescue mt-Ca2+ uptake in yeast or HeLa cells lacking MCU, respectively29. Similarly, the same fungal MCUP sequences used for investigating structure and function of the mammalian uniporter24 failed to mediate mt-Ca2+ uptake when expressed in yeast mitochondria or in MCU knockout HeLa cells , whereas only 12 species have one of the two components. Most such incongruencies are likely to result from assembly, annotation, or identification errors. In fact, we expect MICU to be encoded in all species with a functional MCU complex, with the only likely exception being Onchocercidae, a family of parasitic nematodes. In our dataset all three species in the clade  appear to lack MICU1 homologs in the presence of MCU  and using the Gathering Cut-Off threshold (–cut_ga) when the raw HMM profile from Pfam was used, an e-value threshold of 10−2 otherwise. For all BLAST searches low-complexity regions in the query sequence (default parameter) were filtered out to minimize the number of false positives and an E-value threshold of 10−5 was used. Conserved domains in all retrieved sequences were annotated using the HMM profiles of Pfam release 30.0. For MCU, proteins in our database containing at least one MCU (Pfam: PF04678 [https://pfam.xfam.org/family/PF04678]) domain were detected using an HMMsearch. In total, 1076 protein sequences were selected for subsequent analysis. For MICU, 2105 protein sequences were retrieved from a BLAST search using Hs-MICU1 (Uniprot: Q9BPX6 [https://www.uniprot.org/uniprot/Q9BPX6]) as a query. HMMscan was used to search for additional domains in the retrieved sequences using all the Pfam domain profiles, and all the sequences with detected at least one Mito_carr domain (Pfam: PF00153 [https://pfam.xfam.org/family/PF00153]) were classified as the members of the mitochondrial carrier family . The Aralar-related sequences (Mito_carr domain containing) were clustered together clearly as a monophyletic clade in a phylogenetic tree, in the exclusion of known MICU sequences, and were excluded. The remaining 651 MICU sequences were re-aligned and a new phylogenetic tree with the same methods was reconstructed. EMRE sequences are characterized by a DDDD (Pfam: PF10161 [https://pfam.xfam.org/family/PF10161]) domain. Their short length and low sequence conservation makes detection strikingly difficult, which explains why in some cases EMRE appears to be missing even from some animal genomes. HMMER searches with the “gathering” (–cut_ga) threshold were performed, and all detected homologs were retrieved and re-aligned, for a new HMM profile to be built and used to search back the genome database in a second iteration. One EMRE sequence was detected in A.macrogynus in the first search, while one more sequence in the same species, as well as in S. punctatus and C. anguillulae, were detected in the second iteration. The detected EMRE sequences in this second iteration are those that were further considered for all analyses. For NCLX (NCKX6), the Na_Ca_ex Pfam domain was initially used to retrieve protein sequences. However, this domain characterizes a ubiquitous superfamily of sodium/calcium exchangers that regulate intracellular Ca2+ concentrations in many cell types. Therefore, selection of NCKX6 (NCLX) homologs based on the Na_Ca_ex Pfam domain using HMMER returned 4024 hits. To narrow down the number of hits for more accurate alignment and phylogenetic reconstruction, we used the human NCLX sequence as a query (uniprot: Q6J4K2 [https://www.uniprot.org/uniprot/Q6J4K2]) for a blast search, retrieving 2105 sequences for phylogenetic analysis. Using the human members as reference, 1391 sequences across eukaryotes were selected as related to the NCLX clade (NCLX orthologs).The protein sequences encoded in 1156 completely sequenced eukaryotic genomes were retrieved from Ensembl DB v91, and v37 of Ensembl Metazoa, Plants, Fungi, and Protists (see Supplementary Data 38)  and a soft trimming was applied, filtering out positions in the alignment with gaps in more than 99% of the sequences using trimAl39 (−gt 0.01). IQ-TREE v1.6.8 (ref. 40) was used to derive Maximum Likelihood (ML) trees. LG was selected as the base model to test all rate-heterogeneous models using the “-mset LG” parameter . The best-fit models, chosen according to the Bayesian information criterion (BIC), were LG + R10 for the MCU and MICU alignments and LG + F + R10 for NCLX alignment. Branch supports were obtained using the ultrafast bootstrap implemented in the IQ-TREE program with 1000 replicates. The ETE Toolkit41 was used for all taxonomic and phylogenetic tree operations and visualization. Sequence alignments were visualized using Jalview2 (ref. 42), and the Multi-Harmony43 method was used identify patterns of variation across the different protein clades, positions conserved across the animal MCUs and the animal-like fungal MCUs but not in the MCUPs. Raw phylogenetic trees in newick format can be found in Supplementary Data Sequences of all the protein families described were aligned, and the alignment was trimmed and used to compute a phylogenetic tree. The selected homologous proteins were aligned with MAFFT v7.394 . Control cells were generated using the empty vector pLKO (Addgene #8453). Mt-AEQ MCU knockout (MCU-KO) HeLa cells transiently expressing Hs-MCU, Ce-MCUP, Fg-MCUP, Ma-MCUP, and Nf-MCUP from the pLX304 lentiviral vector were generated by transfection using the X-tremeGene HP DNA transfection reagent (Roche), according to the manufacturer’s instructions. cDNAs for Sp-MCUP, Sp-MCU, Am-MCUP1, Am-MCUa, Am-MCUb, Ce-MCUP, Fg-MCUP, Ma-MCUP, and Nf-MCUP expression without a stop codon were codon optimized for human expression, synthesized de novo in the PuC57 vector, and amplified with flanked attB1 and attB2 sites by PCR  were generated as in ref. 17,18. To generate yeast strains co-expressing mt-AEQ together with different combinations of fungal and human MCU and EMRE orthologs, cDNAs were amplified from the pLX304 vector  by EcoRI digestion and cloned upstream of the ubiquitous CAG promoter. Two sets of single-guide RNA (sgRNA) were designed using an online tool CRISPOR . For each sgRNA, complementary oligonucleotides containing the target sgRNA sequences were annealed and cloned into the BbsI site of the pX330-Puro-ccdB vector45. After which, pX330-Puro-ccdB vector containing sgRNAs were transfected into mt-AEQ HeLa cells using the Lipofectamine 3000 Reagent (Life Technologies), according to the manufacturer′s instructions and selected with 2 μg/ml puromycin for 2 days. Cells were then seeded at a density of 1 cell per well and expanded. Gene knockout was screened by PCR  and confirmed by sequencing and western blot using the following antibodies: anti-MCU , anti-EMRE , anti-ACTIN .Mt-AEQ HeLa cells with knockout of MCU were generated by CRISPR targeting17. Briefly, HeLa cells were grown to confluency, rinsed with PBS, and resuspended in ice-cold isolation buffer . Cells were permeabilized by nitrogen cavitation at 600 psi for 10 min at 4 °C and then centrifuged at 600 × g for 10 min. The supernatant was transferred into new tubes and centrifuged at 8000 × g for 10 min at 4 °C. The resulting pellet containing crude mitochondria was resuspended in IB for protein topology analysis.Crude mitochondria were isolated from HeLa cells as described17. Roughly, 30 µg of freshly isolated mitochondria were gently resuspended in 30 µl of IB buffer with either increasing concentrations of digitonin or 1% Triton X-100 in the presence of 100 µg/ml PK and incubated at room temperature for 15 min. The reaction was stopped by the addition of 5 mM PMSF, followed by incubation on ice for 10 min. Samples were mixed with 10 µl of 4× Laemmli buffer containing 10% 2-mercaptoethanol and boiled for 5 min at 98 °C for immunoblot analysis. Immunoblotting was performed according to the standard procedures using the following antibodies: anti-V5 ; anti-TIM23 ; anti-TOM20 ; anti-Cyclophilin D (Cyp D) . TOM20, TIM23, and Cyp D were used as controls for integral mitochondrial outer membrane, inner membrane, and soluble matrix-targeted proteins, respectively.Proteinase K (PK) protection assay was performed on mitochondria isolated from HeLa cells17. Briefly, yeast cells were grown at 30 °C in a selective lactate medium supplemented with the respective selection markers till an OD ~0.8. The cell pellet was resuspended in a buffer containing buffer 0.6 M sorbitol, 20 mM HEPES/KOH pH 7.2, 80 mM KCl, and 1 mM PMSF, and vortexed five times for 30 s with glass beads (425–600 μm diameter), with a 30 s cooling interval in between to break cell wall and plasma membrane. After the first centrifugation step at 1000 × g for 5 min at 4 °C, the supernatant was further centrifuged at 20,000 × g for 10 min at 4 °C to obtain the mitochondrial fraction (pellet). The supernatant (cytosolic fraction) was precipitated with trichloroacetic acid at −20 °C for 1 h, washed once with cold acetone, and centrifuged at 20,000 × g for 10 min at 4 °C. Both cytosolic and mitochondrial fractions were analyzed with immunoblotting according to the standard procedures using the following antibodies: anti-MCU ; anti-EMRE ; anti-V5 ; anti-AEQ ; anti-YME1; anti-PGK1 .Expression and subcellular localization of heterologous expressed proteins in yeast was tested by immunoblot analysis of cytosolic and mitochondrial fractions isolated from recombinant yeast strains2+ uptake in intact yeast and HeLa cells were performed using an aequorin-based measurement46. Aequorin is widely used as a genetically encoded Ca2+ indicator (GECI) for measurements of mt-Ca2+ kinetics because it offers several advantages. Besides being targeted with precision to the organelle, it functions over a wide range of Ca2+ concentrations, and shows low buffering capacity, despite achieving a limited spatial resolution compared to fluorescent GECIs46. Briefly, yeast cells were collected at an OD ~0.8, washed three times with milliQ water, and starved for 1.5 h at room temperature in a nutrient-free buffer . Afterwards, cells were collected at 3500 r.p.m. for 5 min and resuspended in NFB to a higher density (25 × 108 cells per ml) in the presence of 50 μM native coelenterazine  to reconstitute the photoprotein aequorin. After 30 min in the dark at room temperature, 0.5 × 108 cells per well were plated into a white 96-well plate and Ca2+-dependent light kinetics was recorded upon stimulation with 1 mM CaCl2 and 100 mM glucose, at 469 nm every 0.5 s interval in a MicroBeta2 LumiJET Microplate Counter. At the end of each experiment, cells were lysed with 1 mM digitonin for 5 min at 37 °C and any residual aequorin counts were collected upon the addition of CaCl2 to a final concentration of 140 mM. For mt-Ca2+ uptake measurement in mt-AEQ HeLa cells, those were seeded at 25,000 cells/well of white 96-well plates in growth medium overnight. Afterwards, mt-AEQ was reconstituted with 2 μM native coelenterazine for 2 h at 37 °C and Ca2+-dependent light kinetics were recorded upon 100 μM histamine stimulation at 469 nm every 0.1 s interval. At the end of each experiment, cells were lysed with a solution containing 0.5% Triton X-100 and 10 mM CaCl2 to record the released residual aequorin counts.In vivo analyses of mitochondrial Ca2+ concentration was performed using a MATLAB software as in ref. 18. The dynamics of mt-Ca2+-dependent luminescence signal was smoothed by the cubic spline function:p is a smoothing parameter, controlling the tradeoff between fidelity to the data and roughness of the function estimate, f is the estimated cubic spline function to minimize the above function, and xi and yi are the dynamical data points. Here, p is set at 0.5. Parametrization of the Ca2+-dependent luminescence kinetics was performed in order to determine the maximal amplitude of the luminescence signal (peak) and the left slope of the bell-shaped kinetic trace. Aequorin-based luminescence signal calibration into mt-Ca2+ concentration was performed using the algorithm reported in ref. 46 for wild-type aequorin and native coelenterazine, with the following formula:λ = 1, KR = 7.23 × 106, KTR = 120, and n = 2.99 are the calibration values used for WT aequorin and native coelenterazine.Quantification of mt-CaData are represented as mean ± SEM and the statistical analysis of each experiment is described in the figure legends including the statistical tests used and the exact value of biological replicates. For each biological replicate experiment at least three technical replicates were used for quantification and data analysis. Normal distribution was tested by Shapiro–Wilk normality test. Statistical tests between multiple datasets and conditions were carried out using one-way analysis of variance followed by Dunnett’s multiple comparison tests. Statistical analyses were performed using GraphPad Prism .Further information on research design is available in the Supplementary InformationPeer ReviewReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


This enzyme, which belongs to the CaMKII family, is the predominant family member in the heart and functions as a messenger toward adaptive or detrimental signaling in cardiomyocytes. Classically, the nuclear CaMKIIδB and cytoplasmic CaMKIIδC splice variants are described as mediators of arrhythmias, contractile function, Ca2+ handling, and gene transcription. Recent findings also put CaMKIIδA and CaMKIIδ9 as cardinal players in the global CaMKII response in the heart. In this review, we discuss and summarize the new insights into CaMKIIδ splice variants and their (proposed) functions, as well as CaMKII-engineered mouse phenotypes and cardiac dysfunction related to CaMKIIδ missplicing. We also discuss RNA splicing factors affecting CaMKII splicing. Finally, we discuss the translational perspective derived from these insights and future directions on CaMKIIδ splicing research in the healthy and diseased heart.RNA splicing has been recognized in recent years as a pivotal player in heart development and disease. The Ca Heart disease is a major cause of death around the world, but the mechanisms underlying its development are not completely understood . In receIn the heart, alternative splicing participates in pre- and postnatal development, as well as in the development and progression of heart disease . The funTTN gene, is a sarcomeric protein that acts as a scaffolding filament, a signaling platform, and is a principal regulator of contraction in striated muscle. Mutations located in the RBM20 gene are linked to Titin missplicing toward a larger isoform termed N2BA-Giant, both in animal models and human dilated cardiomyopathy (2+ channel ryanodine receptor type 2 (RYR2), the L-type Ca2+ channel (LTCC) alpha 1C subunit (CACNA1C/Cav1.2), and the Ca2+/calmodulin-dependent protein kinase II delta (CAMK2δ). Missplicing of these genes may contribute to the severity of the cardiomyopathy  in the heart, from the CaMKIIδB and CaMKIIδC isoform to the CaMKIIδA and CaMKIIδ9 isoform  belongs to the multifunctional CaMK family. In mammals, CaMKs are divided into 3 classes: CaMKI, CaMKII, and CaMKIV; which in total comprise more than 27 proteins  maladaptive and detrimental effects on cardiac integrity , a regulatory segment (exons 11–12), a variable linker (exons 13–19), and a hub domain  gene reporter as well as Anf protein expression  and RyR2 (Ser-2814) phosphorylation  increases both SR Cad by TAC . Notablyd by TAC . These efunction . Howeverfunction . This su2+ handling defects seen in transgenic mice overexpressing CaMKIIδC, which is in line with the hypothesis that the decreased SR Ca2+ uptake through SERCA underlies the Ca2+ handling defects. However, while the SR Ca2+content and Ca2+ transient amplitude were normalized, left ventricular dilation, ventricular function, apoptosis, and mortality were exacerbated  attenuates the Cacerbated . This capoptosis . On the poptosis .2+ handling are distinct from the effects of CaMKIIδB overexpression, but overexpression of both splice variants similarly leads to HDAC4 phosphorylation (Ser-632), which inhibits nuclear import, and subsequently induces MEF2 transcriptional activity , which is the transmembrane protein that allows the passage of Ca2+ from cytoplasm into the inner mitochondria  opening, triggering programmed cell death . ASOs in their original form are short, single-stranded, synthetically produced oligodeoxynucleotides that bind to mRNA targets in a complementary manner and thus modulate mRNA and protein expression . The firin vivo, since ASOs circulate in the bloodstream after intravenous or subcutaneous injection and can accumulate in the liver, kidney or spleen  its levels are upregulated in both experimental models of heart failure and human samples which suggests a causal role for increased CaMKIIδ activation in disease, (2) the transgenic overexpression of CaMKIIδA, -δC and -δ9 leads to adverse cardiac remodeling accompanied by contractile dysfunction, and most importantly (3) CaMKIIδ inhibition is generally associated with protection against cardiac dysfunction and a reduction in cardiac damage in animal models subjected to detrimental stimuli. Nevertheless, the importance of CaMKIIδ in physiological processes, as well as the proposed protective effects of, e.g., CaMKIIδB suggest that CaMKIIδ-directed therapeutics require high specificity [For further details see ].2+ in the heart came from work of Ringer in the nineteenth century who accidentally used tap water instead of distilled water in his NaCl solution, and then found out that Ca2+ was necessary for contraction  studies for decades  could clarify why these isoforms exert different and/or redundant functions. Moreover, the 4 isoforms described in this review are not the only CaMKIIδ splice isoforms expressed in the heart. Even though less highly expressed, other splice isoforms such as CaMKIIδ4 are also found in the heart, and the function and relevance of these isoforms has not been explored. The fact that CaMKIIδ9, after being overlooked for a long time, turns out to be one of the highest expressed isoforms with a relevant function in the heart, illustrates that lesser known isoforms should not be neglected (The first report of the role of Catraction . Since t decades . These s decades . Based o decades  are only decades . It wouleglected . AnotherAll authors contributed to writing and revising the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.