FLT3 gene mutations are among the most common genetic aberrations detected in AML and occur with a frequency of approximately 30%, mainly as internal tandem duplications (FLT3-ITD). As a novel finding, it has been reported that the specific insertion sites (IS) of FLT3-ITD exhibit marked heterogeneity in both biological and clinical features. Thus, the so-called non-juxtamembrane domain (non-JMD) FLT3-ITD insertions have been shown to be associated with worse clinical outcomes and resistance to both chemotherapy and tyrosine kinase inhibition. This present review summarizes our current knowledge of the biological and clinical impact of FLT3-ITD inserting at the non-JMD level. Recent evidence suggests that conformational changes depending on FLT3-ITD localization affect downstream signaling networks and the oncogenic potential. We propose that refined risk stratification guidelines integrating the negative prognostic impact of non-JMD FLT3-ITD are warranted. Overcoming therapy resistance in non-JMD-inserting FLT3-ITD-mutated AML may lead to promising treatment approaches.FLT3 gene are among the most common genetic aberrations detected in AML and occur mainly as internal tandem duplications (FLT3-ITD). However, the specific sites of FLT3-ITD insertion within FLT3 show marked heterogeneity regarding both biological and clinical features. In contrast to the common assumption that ITD insertion sites (IS) are restricted to the juxtamembrane domain (JMD) of FLT3, 30% of FLT3-ITD mutations insert at the non-JMD level, thereby integrating into various segments of the tyrosine kinase subdomain 1 (TKD1). ITDs inserted within TKD1 have been shown to be associated with inferior complete remission rates as well as shorter relapse-free and overall survival. Furthermore, resistance to chemotherapy and tyrosine kinase inhibition (TKI) is linked to non-JMD IS. Although FLT3-ITD mutations in general are already recognized as a negative prognostic marker in currently used risk stratification guidelines, the even worse prognostic impact of non-JMD-inserting FLT3-ITD has not yet been particularly considered. Recently, the molecular and biological assessment of TKI resistance highlighted the pivotal role of activated WEE1 kinase in non-JMD-inserting ITDs. Overcoming therapy resistance in non-JMD FLT3-ITD-mutated AML may lead to more effective genotype- and patient-specific treatment approaches.Mutations of the  FLT3). The receptor tyrosine kinase FLT3 is composed of an extracellular module with five immunoglobulin-like domains, a transmembrane domain, and an intracellular module consisting of a juxtamembrane domain (JMD) and two tyrosine kinase subdomains (TKD1 and TKD2) connected by a kinase insert domain  has increasingly focused on genetic aberrations to identify and monitor molecular markers and their prognostic significance . These ft domain  2]. In . In FLT3patients . Furtherpatients ,6,7. Difpatients ,9. FLT3 % in AML ,11, the % in AML . Thus, mFLT3, Breitenbuecher et al. detected ITDs inserted at the non-JMD level in approximately 30% of FLT3-ITD-mutated AML patients [Re-examining the common assumption that ITDs exclusively occur in the JMD of patients . Non-JMDpatients ,14,15. MFLT3 is a membrane-bound, ligand-activated class III receptor tyrosine kinase that is physiologically expressed on hematopoietic stem and progenitor cells of both the lymphoid and myeloid lineage . ConformFLT3-mutated AML [FLT3 gene mutations [FLT3-mutated AML patients. Midostaurin and gilteritinib bind to the adenosine triphosphate (ATP)-binding site of FLT3 in an activated conformational state and are designated as type I inhibitors along with others, including sunitinib and crenolanib. Type II inhibitors such as sorafenib, ponatinib, and quizartinib bind to the ATP-binding site and to a region adjacent to it and only in an inactivated conformational state of FLT3 [FLT3-mutated cells after first-line treatment may be of particular importance. In vitro, Ba/F3 cells retrovirally transduced with different FLT3 mutations showed differential sensitivity to the TKI tandutinib [FLT3 gene, as well as increased stimulation from overexpression of FLT3L and FLT3 itself contribute to resistance and subsequently decreased TKI impact or chemotherapy susceptibility [Following the detection of FLT3 and especially FLT3-ITD as a molecular marker in AML, larger cohorts of AML patients were studied to determine its prognostic value, and first efforts were made to establish a specific and targeted therapy against FLT3. In accordance with these studies, the occurrence of FLT3-ITD is associated with leukocytosis, a high percentage of peripheral blood and bone marrow blast cells, an increased relapse risk (RR) along with reduced RFS, and worse OS ,40,41,42ated AML ,44,45, autations ,47. Althutations  and quizutations ,50, thes of FLT3 ,52,53. I of FLT3 ,55. For ndutinib . Furthertibility ,61,62,63tibility .NPM1 mutation [NPM1 mutation status [NPM1 mutation was associated with an intermediate risk, and the co-occurrence with wild-type NPM1 was related to unfavorable risk. Currently, the updated 2022 ELN risk classification no longer considers the FLT3-ITD allelic ratio [NPM1 mutation. Mutated NPM1 is considered a favorable risk only in the absence of an FLT3-ITD mutation. The differences in the 2017 and 2022 ELN risk classifications are depicted in FLT3 gene mutations. According to the meanwhile revised 2017 National Comprehensive Cancer Network (NCCN) guidelines, FLT3-ITD-mutated AML, even with normal cytogenetics, is linked with a poor risk [FLT3 mutations within AML diagnostics. A Japanese multicentric prospective observational cohort study (Hokkaido Leukemia Net) recently demonstrated the inferior OS of FLT3-ITD-mutated AML patients compared to FLT3-mutation negative patients and highlighted FLT3-ITD as an independent poor prognostic factor [FLT3 mutation status and the assessment of the genetic landscape beyond are crucial for subsequent therapeutic decisions [ITDs were shown to vary in length from 3 bp to more than 400 bp . The promutation . In the n status ,73,74. Aic ratio ,76. In toor risk ,77. Thusc factor . In addiecisions ,79.Unexpectedly, the investigation of ITD IS in 753 patients with unselected FLT3-ITD-positive AML showed that about 30% of ITDs display IS at the non-JMD level of FLT3, particularly within the β1-sheet of TKD1 ,13. ITDsNPM1 mutation on achieving CR were related only to JMD-inserting ITDs [Later on, additional independent studies confirmed that about 30% of FLT3-ITD mutations insert at non-JMD sites ,7,14,80.In summary, a large body of evidence highlighted the negative prognostic impact of FLT3-ITD mutations inserted at non-JMD sites in various AML patient cohorts and at different times of treatment. Thus, in addition to the negative prognostic impact of an FLT3-ITD mutation on CR rates, OS, and RFS already acknowledged within current risk stratifications, ITDs inserted at the non-JMD level are associated with an even worse high-risk prognostic phenotype in FLT3-ITD-mutated AML.Interestingly, in vitro and in vivo preclinical models showed heterogeneous effects on TKI treatment depending on the particular localization of FLT3-ITD IS . Arreba-FLT3-mutated AML [FLT3-mutated AML. A study by the German–Austrian AML Study Group showed improved event-free survival with midostaurin treatment in FLT3-ITD-positive AML patients aged 18–70 years [p = 0.032). The 4-year OS rates were 29%, 44%, and 50%, respectively. In addition, there was a higher 4-year CIR in patients with ITD insertions in TKD1-sole compared to JMD-sole (60% vs. 45%). The rate of CR achievement was independent of FLT3-ITD IS [p = 0.047) for JMD-sole. The negative prognostic impact of FLT3-ITD inserting at the TKD1 level was not reversible via midostaurin treatment [The prospective randomized, placebo-controlled phase III CALGB 10603/RATIFY trial NCT00651261) investigated the therapeutic benefit of midostaurin as a multi-targeted kinase inhibitor in combination with standard chemotherapy with cytarabine and daunorubicin in patients with newly diagnosed ated AML . A previated AML . A totalated AML . Thus, i70 years . In 202170 years . Dependi1261 inve70 years ,14,80. B3-ITD IS . Multiva3-ITD IS . The secreatment . This fireatment ,26. Nextreatment ,86,87,88reatment . Anotherreatment . Regarding FLT3-ITD-mediated oncogenic signaling pathways, Fleischmann et al. investigated the susceptibility of various ITD IS at either the JMD or TKD1 level to inhibition of N-glycosylation using tunicamycin, heat shock protein 90 (HSP90) using 17-AAG, or histone deacetylation using valproic acid. After treatment with tunicamycin and incubation with 17-AAG, Annexin V-negative cells were significantly decreased regardless of ITD IS. However, Ba/F3 cells that harbor the FLT3-ITD variant G613E inserted at the TKD1 level showed higher sensitivity to 17-AAG. Within the Ba/F3 G613E variant, expression of ERK and STAT5 was decreased upon valproic acid treatment . SimilarMCL-1-specific small interfering ribonucleic acid (siRNA) restored susceptibility to midostaurin [Upregulation of MCL-1 has been shown in different cell lines with varying FLT3-ITD IS at the JMD  or TKD1 level  compared to FLT3 wildtype (RS4-11) . Upregulostaurin . Suppresostaurin . Thus, Mostaurin . Currentostaurin ,96. Massacci et al. examined mechanisms of TKI resistance in FLT3-ITD-mutated AML by combining high-sensitive transcriptome, phosphoproteome, and proteome analysis with literature-derived signaling networks using the SignalingProfiler computational module . For thiIn conclusion, the current body of evidence supports the view that distinct conformational changes in the FLT3 receptor mediated by JMD- versus TKD1-inserting ITDs lead to differences in phosphorylation patterns of FLT3 downstream signaling molecules, which in turn alter the response to therapeutic agents ,97,98. IIn first-line therapy, cytarabine is the backbone in the induction and consolidation therapy of AML patients, except in acute promyelocytic leukemia (APL) ,102,103.Pugliese et al. comprehensively examined the response to cytarabine depending on the ITD IS in vitro. The Ba/F3 and 32D cell lines, harboring FLT3-ITD either at the JMD or TKD1 level, were treated with cytarabine . Cell liFollowing the discovery of FLT3-ITD mutations as molecular markers in AML patients, their prognostic value was recognized and integrated into risk stratification guidelines for AML used to date. Non-JMD-inserting FLT3-ITD are associated with inferior CR rates, PFS, and OS. In vitro and in vivo analyses revealed inferior susceptibility to TKI treatment and chemotherapy in AML expressing FLT3-ITD with IS at the TKD1 level. However, despite marked differences in survival and therapy resistance depending on ITD IS, previous risk stratifications do not consider the crucial role of non-JMD ITDs. Furthermore, current studies often do not distinguish between JMD- and non-JMD-inserting FLT3-ITD, limiting data density. To our knowledge, the expression of FLT3-ITD in relation to different ITD IS has also not been examined in pediatric cohorts. We propose recognizing non-JMD ITDs as an independent adverse prognostic factor. Thus, refined risk stratification guidelines highlighting the prognostic differences between JMD and non-JMD IS in FLT3-ITD-mutated AML are needed. Due to its considerable prognostic and therapeutic importance, the determination of FLT3-ITD IS should be a mandatory part of the daily diagnostic routine in AML. Overcoming TKI and chemotherapy resistance in FLT3-ITD-mutated AML, especially associated with ITDs inserted at the non-JMD level, could lead to promising genotype- and patient-specific treatment approaches.


A fixed 20:1 combination of cafedrine and theodrenaline has been widely used to treat perioperative hypotension and emergency hypotensive states since the 1960s; however, its effect on the intracellular Ca2+ concentration ([Ca2+]i) of respiratory epithelium remains unknown. Therefore, human tracheal epithelial cells were exposed to the clinically applied 20:1 mixture of cafedrine/theodrenaline and the individual substances separately. [Ca2+]i was assessed by FURA-2 340/380 fluorescence ratio. Pharmacological inhibitors were applied to elucidate relevant signal transduction cascades, and reverse transcription polymerase chain reaction (RT-PCR) was performed on murine tracheal epithelium to analyze ryanodine receptor (RyR) subtype expression. All three pharmacological preparations instantaneously induced a steep increase in [Ca2+]i that quickly returned to its baseline value despite the persistence of each substance. Peak [Ca2+]i following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone increased in a dose-dependent manner, with median effective concentrations of 0.35 mM , 3.14 mM, and 3.45 mM, respectively. When extracellular Ca2+ influx was inhibited using a Ca2+-free buffer solution, the peak [Ca2+]i following the administration of cafedrine alone and theodrenaline alone were reduced but not abolished. No alteration in [Ca2+]i compared with baseline [Ca2+]i was observed during β-adrenergic receptor inhibition. Depletion of caffeine-sensitive stores and inhibition of RyR, but not IP3 receptors, completely abolished any increase in [Ca2+]i. However, [Ca2+]i still increased following the depletion of mitochondrial Ca2+ stores using 2,4-dinitrophenol. RT-PCR revealed RyR-2 and RyR-3 expression on murine tracheal epithelium. Although our experiments showed that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone release Ca2+ from intracellular stores through mechanisms that are exclusively triggered by β-adrenergic receptor stimulation, which most probably lead to RyR activation, clinical plasma concentrations are considerably lower than those used in our experiments to elicit an increase in [Ca2+]i; therefore, further studies are needed to evaluate the ability of cafedrine/theodrenaline to alter mucociliary clearance in clinical practice.Mucociliary clearance is a pivotal physiological mechanism that protects the lung by cleaning the airways from pollution and colonization, thereby preventing infection. Ciliary function is influenced by various signal transduction cascades, and Ca Ciliary cells prevent the accumulation of debris and colonization by microbial pathogens via an outward-directed transportation processes2. Cilia are hair-like protrusions located on the apical side of the respiratory epithelium along the airways; they are composed of unique structural proteins, including motor proteins, like the ATP-dependent dynein3. Due to their continuous synchronized and orally directed motion, inhaled particles and complex compositions of mucus, electrolytes, and endogenous defensive substances are removed from the lower airways and are subsequently coughed up under normal physiological conditions4. While the ciliary apparatus continuously beats without external stimulation, various events can provoke an increase in ciliary beating frequency, when necessary4. Exogenous, endogenous, and paracrine effects moderate ciliary activity, and Ca2+ functions as a central second messenger. Therefore, intracellular Ca2+ concentration ([Ca2+]i) represents a keystone regulating ciliary activity because most signal transduction cascades, which contribute to the ciliary beating frequency, induce at least a temporary increase in [Ca2+]i5. Intracellular Ca2+ is foremost stored in the endoplasmic reticulum (ER) and is released following inositol trisphosphate (IP3) or ryanodine receptor (RyR) activation3. Furthermore, the ER regulates [Ca2+]i via the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), which pumps cytosolic Ca2+ ions to its internal stores, maintaining the balance of [Ca2+]i7. Mitochondria also contribute to intracellular Ca2+ homeostasis by buffering Ca2+ ions when [Ca2+]i exceeds a threshold of 500 nM9. In addition, other cell organelles contribute to intracellular Ca2+ homeostasis, such as lysosomes, which contain Ca2+ but can also regulate mitochondrial Ca2+ dynamics, and peroxisomes; however, their individual influence on [Ca2+]i in ciliary cells and mucociliary clearance remains unknown11. [Ca2+]i is not only increased by Ca2+ release from intracellular stores; extracellular Ca2+ can enter the cell via plasma membrane-bound Ca2+ channels. Although voltage-gated calcium channels and transient receptor potential channels are located at the plasma membrane together, the predominant method of extracellular Ca2+ entry into non-excitable cells occurs via store-operated Ca2+ entry (SOCE)13. SOCE is triggered by an increase in [Ca2+]i that is predominantly caused by the liberation of Ca2+ from internal stores12. Stromal interaction molecule proteins are located at the ER membrane, and they activate SOCE when Ca2+ is released13. Therefore, changes in [Ca2+]i produce fundamental metabolic and functional alterations in ciliary cells and can be influenced by different drugs administered in various settings.Mucociliary clearance of the lower airways is a pivotal physiological mechanism that protects the lung by clearing the airways of pollution and colonization by pathogens, thereby preventing infection. Together with basal, suprabasal, and goblet cells, multiciliated epithelial cells constitute the complex mucociliary clearance function of the respiratory tract16. Covalently linked theophylline and norephedrine compose cafedrine, and theodrenaline is formed from theophylline and noradrenaline in the same manner17. Both compounds are administered in a fixed 20:1 combination of cafedrine and theodrenaline as an intravenous bolus in adults, and they restore mean arterial blood pressure by increasing preload, cardiac stroke volume, and cardiac output18. The clinical effects of cafedrine/theodrenaline are mediated through β1-adrenoreceptor and α-adrenoreceptor stimulation, and nonspecific inhibition of phosphodiesterases (PDEs) is believed to enhance their response17. In contrast with synthetic vasopressors , systemic vascular resistance and heart rate remain mostly unaffected, which makes cafedrine/theodrenaline especially appealing in obstetric surgery19. Although cafedrine/theodrenaline has been widely used for decades, little is known about its pharmacodynamics or pharmacokinetics in specific end organs16. This is surprising as the unique combination of three single drugs  in a 20:1 mixture might produce different effects in vivo as recently shown in human atrial myocardium and internal mammary arteries17. However, the influence of cafedrine/theodrenaline on the [Ca2+]i of human respiratory cilia cells has not yet been investigated. The aim of this study was to evaluate the influence of cafedrine/theodrenaline on the [Ca2+]i of human tracheal epithelial cells. Cells were exposed to the clinically used 20:1 mixture of cafedrine/theodrenaline and the individual substances alone. Our experiments elucidated the effects of the individual components and identified the relevant pharmacological signaling cascades leading to alterations in [Ca2+]i. Therefore, we applied specific inhibitory substances inhibiting distinct signal transduction cascades, and we evaluated the origin of the released Ca2+. Furthermore, we used reverse transcription polymerase chain reaction (RT-PCR) to detect the messenger ribonucleic acid (mRNA) expression of key receptor subtypes influencing [Ca2+]i following the administration of cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone.In Germany, intraoperative hypotension and emergency hypotensive states have been treated with a combination of cafedrine and theodrenaline  since the 1960s2, 10 mM glucose, 125 mM NaCl, 1.2 mM KH2PO4, and 1.2 mM MgSO4. NaOH was used to adjust the pH to 7.4 at 30 °C. To realize experiments in Ca2+-free solutions, CaCl2 was substituted with 1 mM ethylene glycol tetraacetic acid. The following drugs were applied during the experiments: 2-aminoethoxydiphenylborane , 2,4-dinitrophenol , cafedrine , cafedrine/theodrenaline 20:1 , caffeine , FURA-2 AM , ICI-118,551 , KCl (200 mM diluted in 66.6 µl H2O), ryanodine , and theodrenaline . The stated drug concentrations were achieved during the experiments after applying the stock solution to the buffer solution in the recording chamber. In control experiments, the buffer solution or the solvent alone was applied to rule out any contribution of the buffer, the solvent, or the mechanical application process to the Ca2+ signals.The historically established 20:1 ratio of cafedrine/theodrenaline refers to mass, not molarity. Therefore, due to the different molar masses of cafedrine (357.41 g/mol) and theodrenaline (375.38 g/mol), a 1 M solution of cafedrine/theodrenaline 20:1 with a molar mass of 7,882.98 g/mol consists of 1 M theodrenaline and approximately 21 M of cafedrine. Experiments were performed in HEPES solution consisting of 10 mM HEPES, 2.6 mM KCl, 2.5 mM CaCl2. Cells from passage one to three were seeded onto laminin-coated coverslips for [Ca2+]i measurements in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Dye loading was performed in the dark with 2.5 µM FURA-2 AM for 45 min at 37 °C. The cell-containing coverslip was then rinsed in fresh HEPES buffer and subsequently transferred to the recording chamber of an upright fluorescence microscope equipped with a 20 × immersion lens , where the coverslip was placed into a Delta T culture dish  containing 2 ml of fresh HEPES buffer. Excitation light was provided by a 50 W xenon lamp, and the microscope was equipped with a dichroic excitation longpass mirror at 400 nm. FURA-2 AM was excited at 340 nm and 380 nm while equipped with bandpass excitation filters. The emitted fluorescence was directed through a dichroic shortpass filter of 560 nm to a bandpass filter of 510 nm and was recorded with a scientific camera . Measurements of the FURA-2 AM 340/380 ratio were performed every second for 1,000 s using an automated protocol of the TiLLvisION Imaging software program . Cafedrine, theodrenaline, or 20:1 cafedrine/theodrenaline were added following a 100-s resting period to ensure adequate baseline calibration. After 800 s, 200 mM potassium chloride (KCl) was added to the buffer solution to completely invert the membrane potential, leading to massive influx of Ca2+ indicating the integrity of the analyzed cells up to the end of the respective experiment.Human tracheal epithelial cells  were cultured with the Airway Epithelial Growth Medium Kit (C-21160) containing the Airway Epithelial Growth Medium Supplement Pack  in a humidified chamber at 37 °C with air containing 5% COMale C57BL6J mice (n = 5) weighing 25–35 g (aged 12–15 weeks) were purchased from Charles River  to characterize the expression of RyR in the respiratory epithelium. All procedures involving animals were conducted in compliance with the European legislation for the protection of animals used for scientific purposes, the ARRIVE guidelines and the standards for animal experiments according to the German animal welfare law and were approved by the local committee for animal care of the regional council of Giessen, Germany . After deep narcosis using 5% isoflurane , animals were sacrificed by cervical dislocation. Tracheae, tracheal epithelium, diaphragm, cardiac muscle, and skeletal muscle were subsequently collected. To obtain isolated tracheal epithelium, the epithelial layer was gently scrubbed from the opened trachea using a hygienic swab. All tissues were stored in RNA stabilization lotion  at − 20 °C until further processing. Tissue probes were then lysed in 350 µl RNeasy Lysis Buffer  containing 3.5 µl ß-mercaptoethanol and they were subsequently homogenized in a tissue homogenizer . RNA was extracted using the RNeasy Micro Kit, and DNA removal was performed using the RNase-Free DNase set .-3 expression in whole trachea and in the respiratory epithelium alone (each n = 5) was analyzed using specific primers at temperatures shown in Table https://www.ncbi.nlm.nih.gov/tools/primer-blast, National Institutes of Health, Bethesda, MD, USA). Skeletal muscle (RyR-1), cardiac muscle (RyR-2), and diaphragm (RyR-3) served as positive controls, and H2O was used as negative control. Primer concentration was set at 0.2 µM and reactions were performed in a Mastercycler gradient . TAQ polymerase  was activated at 95° for 3 min, followed by 40 cycles consisting of a 1-min denaturation step at 95 °C, a 45-s annealing step at a primer-specific temperature  according to the manufacturer’s protocol and stored at − 20 °C until further use. RyR-1, RyR-2, and RyR50) were calculated using the Hill equation. The Mann–Whitney U test was used to compare the peak measurement points from different experiments, while the Wilcoxon rank sum test was used to compare paired variables. In general, two-tailed values of p < 0.05 were considered statistically significant, while multiple comparison problems were counteracted by adjusting the α-level according to the Bonferrroni correction. GraphPad PRISM  was used for statistical analysis and figure creation.FURA-2 340/380 ratio was recorded from at least 30 cells per experimental setup. These 30 cells were collected from at least three different coverslips each evaluating 10 cells taken from cell passages one to three. Measurements were only included in the statistical analyses when clear response of the FURA-2-fluorescence ratio (doubling of the ratio) was detected after the application of KCl at the end of the experiments. Median effective concentrations , 3.14 mM (1.12 mg/ml), and 3.45 mM (1.30 mg/ml), respectively. All three substances provoked a sharp increase in [Ca2+]i that was observed immediately, within 5 s after application . Application of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone provoked a significant increase in [Ca2+]i , was applied, baseline i peaks were significantly higher in the presence of DNP compared with the [Ca2+]i peaks without depletion of mitochondrial Ca2+ stores (both p < 0.001). [Ca2+]i did not reach baseline following the administration of cafedrine alone or theodrenaline alone. Furthermore, caffeine-sensitive Ca2+ stores, which are mainly represented by the ER, were depleted using caffeine . Subsequent application of 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone did not increase [Ca2+]i  were identified by RT-PCR. RyR-1 mRNA was not detected in the whole trachea or tracheal epithelium. An overview of the RT-PCR results, including the controls, is presented in Table 2+]i, when applied to human tracheal epithelial cells. We observed sharp, transient peaks in the FURA-2 340/380 ratio, which followed a dose–response relationship and were described by the Hill equation. Herein, it should be noted that the effect curves of 20:1 cafedrine/theodrenaline and cafedrine alone showed a profile where the increase from 0 to 100% is already achieved within one log unit, which would be classically attributed to ion channel activation. In contrast, only the dose–response curve of theodrenaline clearly indicated receptor-mediated effects because the increase from 0 to 100% required two log units. However, although the formal criteria for receptor-mediated Ca2+ release were only barely met in our dose–response curves following the application of 20:1 cafedrine/theodrenaline and cafedrine alone, our following experiments clearly proved adrenergic and ryanodine receptor-mediated Ca2+ release following the application of all three substances analyzed in our experiments. Interestingly, the EC50 of cafedrine alone and theodrenaline alone were within comparable ranges; however, when applied as the clinically used 20:1 mixture, much more cafedrine was required to achieve a significant effect on [Ca2+]i, and the applied concentration of theodrenaline seemed almost negligible in the light of the calculated EC50 of theodrenaline alone. Moreover, [Ca2+]i peaks observed after the application of 20:1 cafedrine/theodrenaline were consistently lower than those observed following applications of the individual substances alone. Interestingly, clinically irrelevant high concentrations of 20:1 cafedrine/theodrenaline ultimately led to cell lysis, which was also observed following the administration of high concentrations of cafedrine or theodrenaline alone; however we never observed a higher FURA-2 340/380 ratio than that shown in our dose–response curves. Although the pharmacokinetics of cafedrine and theodrenaline remain largely unknown, the immediate response in blood pressure was attributed to theodrenaline, while the effects of cafedrine were observed after a 20-min delay20. Therefore, it has been hypothesized that cafedrine may not provoke any sympathomimetic actions alone; it may be metabolized into active metabolites that exert its clinical effects17. In our experiments, the effect observed after the application of 20:1 cafedrine/theodrenaline could only be attributed to theodrenaline alone if strong synergistic effects would apply in the presence of cafedrine. Because the concentration of theodrenaline alone in the 20:1 mixture was insufficient to provoke any significant changes in [Ca2+]i, we conclude that the foremost effect on changing [Ca2+]i in our experiments was induced by cafedrine. This assumption is supported by the observation that very high concentrations of cafedrine were applied using the 20:1 combination compared with the dose–response relationship of cafedrine alone. However, our model cannot represent the effects of potential drug metabolites, as we used isolated cells, and the drugs were directly applied to the buffer solution. Furthermore, clinical data reported a plasma concentration of 6 µg/ml after the intravenous application of one ampoule of 2 ml cafedrine/theodrenaline; therefore, all concentrations used in our experiments were significantly higher than clinically used concentrations20. However, the transient intravascular and intraepithelial concentrations immediately after injection are unknown and might be much higher than those observed after distribution to the body compartments; therefore, our data should be interpreted with caution, and further studies are necessary to evaluate the transferability of the concentrations used in our experiments.Our experiments revealed that 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone increased i rapidly returned to its baseline3. The rapid restoration of baseline [Ca2+]i is primarily explained by SERCA activity, which pumps Ca2+ into the ER after cytosolic Ca2+ release, or by mitochondrial Ca2+ buffering22. However, almost every other small cell organelle contributes to the rapid restoration of baseline [Ca2+]i, and prolonged alteration of ciliary beat frequency is induced by transient changes in [Ca2+]i. Immediately after purinergic or cholinergic stimulation, tracheal epithelial cells exhibited a transient increase in [Ca2+]i; however, ciliary beat frequency remained consistently increased24. Therefore, we conclude that analogous mechanisms are mediated after the stimulation of β-adrenergic receptors, which were the pivotal receptors involved in [Ca2+]i alterations in our experiments. The increase in [Ca2+]i completely vanished after 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone were added during non-selective adrenergic receptor inhibition. These results are in line with the clinical effect of cafedrine/theodrenaline, which increases cardiac stroke volume via β1-adrenergic receptor activation14. In-vitro studies using human atrial myocardium and coronary arteries also elucidated β1-adrenergic receptor activation as a pivotal mechanism; however, effects on α-adrenergic receptors were observed in arteries after β-adrenergic receptor inhibition25. Although these effects were attributed to theodrenaline alone, we did not observe similar effects in human tracheal epithelial cells. These observations are in line with the data reported by Weiterer et al., who reported the exclusive presence of the α1D-adrenergic receptor subtype in murine tracheal epithelium26. However, murine particle transport velocity was independent from α-adrenergic receptor activation. Therefore, we conclude that α-receptor activation might occur in human tracheal epithelial cells following the administration of cafedrine/theodrenaline, but no influence on [Ca2+]i or mucociliary clearance could be detected in light of the available in-vitro data26. The theophylline component of cafedrine and theodrenaline is believed to inhibit PDE, which should lead to the persistence of second messengers, such as cGMP and cAMP, improving cardiac inotropy14. However, only high, clinically irrelevant concentrations of cafedrine/theodrenaline were able to provoke significant inhibition of PDE in human atrial myocardium17. Although we used high concentrations of cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone, we were only able to detect β-adrenergic receptor stimulation because the increase in [Ca2+]i completely vanished in the presence of β-adrenergic receptor inhibition. When other signal transduction cascades were involved, we should have detected a persistent increase in [Ca2+]i. This finding is underlined by the knowledge that PDE inhibition might not influence ciliary beat frequency or mucociliary clearance to a clinically relevant degree, because data remain controversial regarding the alteration of mucociliary clearance following treatment with theophylline29.After the transient peak, during which Ca2+-free buffer solution, [Ca2+]i increased to a significantly lesser degree following administration of cafedrine alone and theodrenaline alone than the increase observed in Ca2+-containing buffer solution. Therefore, extracellular Ca2+ influx contributes to the rise in [Ca2+]i, which is foremost realized through SOCE in non-excitable cells following Ca2+ release from internal stores13. ORAI proteins, which are mediated by stromal interaction molecule proteins, are the most important mediators of SOCE12. Therefore, further experiments elucidating receptor expression and distinct signal transduction pathways should be performed, although their specific inhibition is complicated due to their diverse interactions and multiple targets located on the plasma membrane13. However, because the [Ca2+]i peak in the Ca2+-free buffer solution was comparable to the peak observed in Ca2+-containing buffer solution following the administration of 20:1 cafedrine/theodrenaline, the clinical relevance of SOCE following the administration of cafedrine/theodrenaline in human tracheal epithelial cells remains questionable. Internal stores were depleted to detect the intracellular stores, which released Ca2+ following the administration of cafedrine/theodrenaline. When mitochondrial Ca2+ stores were depleted, [Ca2+]i continued to increase following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone. Therefore, we conclude that cafedrine/theodrenaline does not depolarize mitochondrial membrane potential, which would lead to Ca2+ release from these stores. However, [Ca2+]i peaks following the administration of 20:1 cafedrine/theodrenaline and cafedrine alone were significantly higher than those observed without prior mitochondrial store depletion. This observation confirms the mitochondrial ability to buffer Ca2+ ions via the mitochondrial Ca2+ uniporter when [Ca2+]i exceeds 500 nM30. Interestingly, [Ca2+]i remained higher for the rest of the observation time. Because DNP administration decouples oxidative phosphorylation without altering cytosolic pH, less ATP supplying the ATP-dependent SERCA might be available in these experiments, leading to the observation of persistent [Ca2+]i baseline shift. However, SERCA was still able to handle the transient peak [Ca2+]i by altering [Ca2+]i close to its former baseline value. This finding underlines our conclusion that the elevated peak in [Ca2+]i is foremost triggered by the inhibited mitochondrial Ca2+ capacity and not by a lack of ATP, which might impair SERCA activity.When we used Ca2+]i completely vanished following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone, when RyR were inhibited, we conclude that cafedrine/theodrenaline releases Ca2+ from the ER exclusively by RyR activation. In general, Ca2+ release from the ER is achieved via IP3 receptor or RyR activation31. However, our experiments revealed equal peaks of [Ca2+]i when IP3 receptors were inhibited, and the increase in [Ca2+]i completely vanished after RyR inhibition. Therefore, we conclude that Ca2+ release following β-adrenergic receptor activation depends solely on RyR activation, and IP3 receptor-associated Ca2+ release does not occur in human tracheal epithelial cells following cafedrine/theodrenaline administration.Because the increase in i36. Local increase in [Ca2+]i can be realized through nearby RyR activation, or IP3 receptor activation; however, our data indicate that RyR activation was independent from IP3 receptor activation following cafedrine/theodrenaline administration37. Therefore, alternative RyR activation following β-adrenergic receptor stimulation must be considered in tracheal epithelial cells. β-adrenergic stimulation has been shown to increase RyR-2 activity in cardiac muscle cells via intracellular-mediated Ca2+ and Mg2+ regulation, and receptor phosphorylation38. Furthermore, protein kinase A and cAMP, which are both pivotal messengers following the β1-signal transduction cascade, have been shown to induce Ca2+ release via RyR-2 in cardiac muscles and non-excitable cells, respectively40. In addition, adrenergic receptor signaling increases nicotinic acid adenine dinucleotide phosphate and cyclic adenosine diphosphate-ribose levels, which both activate RyR-2-associated Ca2+ release41. Because RyR-3 is more readily activated by an increase in local [Ca2+]i compared with RyR-2, its activation in tracheal epithelial cells following the administration of cafedrine/theodrenaline can be achieved following RyR 2-associated Ca2+ efflux42. However, as discrepancies in receptor expression between mammals could not be excluded, further studies in human tissues should be conducted to confirm RyR expression. Local distribution, and the RyR-2-to-RyR-3 ratio in tracheal epithelial cells could be evaluated using immunohistochemistry.Consequently, RyR-2 and RyR-3 expression was revealed using RT-PCR in murine tracheal epithelium. While RyR-3 is co-expressed with RyR-1 or RyR-2 in many tissues, RyR 2 has mainly been studied in cardiac muscle cells; however, RyR-2 expression has been demonstrated in smooth muscle cells and non-excitable cells, such as pancreatic acinar cells and kidney epithelial cells2+]i and clinically administered concentrations are much lower; therefore, it is possible that we observed [Ca2+]i kinetics that do not occur when lower concentrations are used in vivo. Third, some concentrations applied to achieve the specific dose–response curves were higher than was warranted by the integrity of the observed cells; therefore, a maximum FURA-2 340/380 ratio following substance administration was set at the last valid observed value. However, concentrations applied to assess the distinct signal transduction cascades did prove the validity of the calculated dose–response curves in our experimental setting. Fourth, our measurement of [Ca2+]i using the FURA-2 340/380 ratio was not calibrated; therefore, we can only report relative alterations in [Ca2+]i, and no absolute concentrations were measured.Several limitations of our experiments must be acknowledged. First, we used isolated tracheal epithelial cells; therefore, the physiological integrity of a respiratory tract was not preserved, including a lack of basal tissues and cell–cell junctions. Therefore, physiological drug administration via capillary vessels could not be replicated, and atypical entrance  of our tested drugs was preserved. Second, we used high concentrations of cafedrine/theodrenaline, cafedrine, and theodrenaline to achieve alterations in i; therefore, further studies are needed to alter the ability of cafedrine/theodrenaline to change mucociliary clearance in clinical practice.In conclusion, we provide evidence that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone induce the release of CaSupplementary Figure 1.


We performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca2+ signaling in acute cortical brain slices of PDGFRβ-Cre::GCaMP6f mice. We report that mid-capillary pericyte Ca2+ signalling differs from ensheathing type pericytes in that it is largely independent of L- and T-type voltage-gated calcium channels. Instead, Ca2+ signals in mid-capillary pericytes were inhibited by multiple Orai channel blockers, which also inhibited Ca2+ entry triggered by endoplasmic reticulum (ER) store depletion. An investigation into store release pathways indicated that Ca2+ transients in mid-capillary pericytes occur through a combination of IP3R and RyR activation, and that Orai store-operated calcium entry (SOCE) is required to sustain and amplify intracellular Ca2+ increases evoked by the GqGPCR agonist endothelin-1. These results suggest that Ca2+ influx via Orai channels reciprocally regulates IP3R and RyR release pathways in the ER, which together generate spontaneous Ca2+ transients and amplify Gq-coupled Ca2+ elevations in mid-capillary pericytes. Thus, SOCE is a major regulator of pericyte Ca2+ and a target for manipulating their function in health and disease.Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties, such as Ca Pharmacology and imaging of pericytes expressing GCaMP6f uncovers the basis of Ca2+ signaling in mid-capillary brain pericytes. Although they share a common origin3 and similar nomenclature, these cell types have numerous morphological5, transcriptomic7, and functional differences9. For example, aSMCs and ensheathing pericytes fully encircle and exhibit near complete coverage of the endothelial tube, highly express alpha smooth muscle actin (α-SMA), and undoubtedly regulate cerebral blood flow (CBF)10. Mid-capillary pericytes have thin or mesh-like processes that run longitudinal to the vessel, express little to no α-SMA, and their role in controlling CBF in physiological contexts remains poorly defined. Nevertheless, blood flow control aside, mid-capillary pericytes have numerous functions in health and disease, such as blood–brain-barrier regulation13 neuroimmune regulation14, angiogenesis15, glial scar formation16, and suggested stem cell-like properties17. Yet, despite these important functions, the basic physiological properties of brain mid-capillary pericytes, including the mechanisms that regulate their Ca2+ signaling, remain understudied.The mural cells of the brain are multifunctional cells organized in a continuum along the arterio-venous axis of the cerebral vasculature. This continuum is grouped into distinct cell types, which includes smooth muscle cells on penetrating arterioles (aSMCs), pre-capillary sphincters and ensheathing pericytes  of the arteriole-to-capillary transition zone, pericytes of the mid-capillary bed (of mesh and thin-strand morphology – here collectively referred to as mid-capillary pericytes), and venule smooth muscle cells2+, which are termed Ca2+ transients19. Whereas the mechanisms underlying calcium signaling and contraction in aSMCs have been studied in detail (e.g. refs. 26.), investigations into brain pericyte Ca2+ signaling mechanism are only beginning to emerge. In ensheathing-type pericytes, Ca2+ transients can be caused by transmembrane influx through voltage-gated Ca2+ channels (VGCCs) and inositol 1,4,5-trisphosphate receptor (IP3R) signaling that recruits the Ca2+ required for α-SMA-mediated contraction28. Mid-capillary pericytes also exhibit Ca2+ transients with similar properties in vivo and in vitro, which are confined to spatial microdomains and modulated by neuronal activity29. Interestingly, a recent study reported that in resting conditions mid-capillary pericyte Ca2+ transients were only minimally sensitive to the potent L-type VGCC blocker nimodipine but were largely inhibited by the non-selective ion channel blocker SKF-9636518, consistent with a prior study reporting reduced functional expression of VGCCs on distal retinal capillaries30. In addition to non-selective cation TRPC channels, SKF-96365 blocks several other Ca2+ channels that are highly expressed in mid-capillary pericytes such as T-type VGCCs31, and the Orai family of store-operated Ca2+ channels32. Mid-capillary pericytes also express a plethora of Gq-coupled receptors33, which when activated would be expected to trigger Ca2+ release from internal stores via IP3R signaling. Therefore, a more detailed investigation is required to understand the cellular mechanisms underlying Ca2+ signaling in mid-capillary pericytes, their voltage dependence, and the interplay between Ca2+ release from internal stores and transmembrane influx.All mural cells exhibit spontaneous fluctuations in intracellular Ca2+ signaling in brain pericytes. Surprisingly, in contrast to ensheathing type pericytes which we show exhibit Ca2+ transients that are partially dependent on VGCCs, mid-capillary pericyte Ca2+ signals are largely unaffected by inhibition of VGCCs. We show that mid-capillary pericyte Ca2+ transients are, instead, primarily mediated by an interplay between store-operated Ca2+ entry (SOCE) channels and release of Ca2+ from internal stores by IP3Rs and ryanodine receptors (RyRs). Underscoring the potential importance of pericyte SOCE channels, we further demonstrate that they amplify Gq-coupled [Ca2+]i elevations evoked by the potent vasoconstrictor endothelin-1 (ET-1).Here, we utilized pharmacology experiments on acute cortical brain slices from transgenic mice expressing a fluorescent calcium indicator, GCaMP6f, in brain mural cells expressing PDGFRβ  to delineate the mechanisms underlying Ca2+ signals were visualized with confocal imaging in acute cortical slices from PDGFRβ-Cre;GCaMP6f mice. The capillary lumen was labeled with an intravenous (I.V.) injection of RhodamineB-Dextran (70 kDa) and pericyte somas were labeled with the recently described pericyte specific dye TO-PRO-334  by washing out [Ca2+]ex from the perfused artificial cerebral spinal fluid (ACSF). Perfusion of 0 mM Ca2+, 2 mM EGTA solution reversibly depressed the frequency of Ca2+ transients in both the processes and soma of the pericyte -A or thromboxane A2 receptor, ET-1 and U46619 respectively, which increase intracellular pericyte Ca2+ in an IP3-dependent manner. ET-1 and U46619 were still able to evoke Ca2+ transients in Ca2+-free solution  signals generated by ET-1 application were severely blunted when compared to those induced at 5 and 10 min following [Ca2+]ex removal   Fig. S in contr2+]i28. Therefore, we tested the effect of blocking the predominantly expressed pericyte VGCC subtypes, CaV1.2 (L-type) and CaV3.2 (T-type), with nifedipine and Z94436, respectively , a KATP channel opener which hyperpolarizes pericytes, had no effect on transient frequency in processes or the soma  to block neuronal action potentials, which alone had no significant effect on the frequency of mid-capillary pericyte Ca2+ transients, consistent with a previous report18, although resting [Ca2+]i was slightly increased in TTX  required for the generation of Ca2+ transients in mid-capillary pericytes  , and inhibition of TRPC3/6 with GSK-2833503A (10 µM)  robustly reduced the frequency of Ca2+ transients in mid-capillary pericytes in both the soma and processes  rapidly induced an elevation in resting Ca2+ levels  Fig.  and S2. µM) Fig.  and S2, nts Fig.  and S2. ses Fig.  and S2. els Fig.  and S4, y Tables  and S2.F2+ influx through Orai SOCE channels is required for the generation of spontaneous Ca2+ transients in mid-capillary pericytes, store-operated entry and store-release mechanisms are inextricably linked. Therefore, we set out to test the dependence of mid-capillary pericyte Ca2+ transients on store Ca2+ filling and release pathways -plasmic reticulum calcium-ATPase (SERCA), with cyclopiazonic acid  abolished Ca2+ transients  or RyRs with the reversible antagonist tetracaine. Blocking IP3 production with U73122 (25 µM) led a modest reduction in Ca2+ transient frequency in mid-capillary pericyte processes but not soma . Consistent with a functional role of RyRs in mid-capillary pericytes, application of caffeine (10 mM), a RyR potentiator, elevated mid-capillary pericyte Ca2+  for 20 min in Ca2+-free ACSF to deplete ER stores and maximally activate “store-operated” channels. Then, we reintroduced 1.2 mM [Ca2+]ex to the bathing solution while imaging a mid-capillary pericyte . Therefore, we harnessed the potent vasoconstrictor ET-1, which has been previously shown to induce large elevations in mid-capillary pericyte Ca2+18. Indeed, in 1.2 mM [Ca2+]ex, ET-1 triggered a robust and sustained, but highly variable, Ca2+ elevation in mid-capillary pericytes 28, in mid-capillary pericytes, blocking VGCCs (20 µM nifedipine and 2 µM Z944) or TMEM16A (2 µM Ani9) for 20 min had no significant effect on the magnitude of the ET-1 mediated Ca2+ elevation , confirming that ET-1 induced signals are initiated by the Gq-GPCR-PLC pathway  and spatially propagating nature of the observed Ca2+ transients, are consistent with a model in which spontaneous store release through RyRs and IP3Rs is maintained by constitutive SOCE47. Orai channels are ideally suited due to their high Ca2+ selectivity and tight coupling with luminal [Ca2+] in the ER via STIM proteins. Orai Ca2+ influx may additionally be critical for RyR activation by recruiting the intracellular Ca2+ required for calcium-induced-calcium-release (CICR) , a mechanism which classically depends on L-type VGCCs in cardiac muscle49 and in smooth muscle21.Here we demonstrate that spontaneous Ca2+ transients in mid-capillary pericyte processes were independent of L-type VGCCs at rest. However, in slight contrast to our results with 20 µM nifedipine, a minimal decrease in Ca2+ transient frequency within the soma was reported with 100 µM nimodipine18. Here we extend on these results and further show that these transients are not increased by depolarization and are likewise also independent of low-voltage-activated T-type calcium channels (CaV3.2), which pericytes robustly express7. These findings contrast with our initial hypothesis that these transients would require VGCC activity, which was based on our previous finding that [Ca2+]i and Ca2+ transient frequency decreases in mid-capillary olfactory bulb pericytes following local increases in neuronal activity, although on a slower time scale than upstream ensheathing pericytes and SMCs9. While we show that VGCCs are not required for mid-capillary pericyte Ca2+ transients, they highly express both L- and T-type VGCCs7 and it is possible that these channels become inactivated at ~−20 mV when exposed to 60 mM [K+]ex. Therefore, we cannot conclude that these channels are entirely functionally absent, but rather that Ca2+ signaling in mid-capillary pericytes is dominated by other mechanisms, which we report here. Intriguingly, a concurrent study has reported that distal retinal pericytes do show nifedipine-sensitive Ca2+ elevations in response to 60 mM [K+]ex50. However, in contrast to SMCs and ensheathing pericytes, L-type VGCCs were not required for the Ca2+ elevation in retinal mid-capillary pericytes following increased intraluminal pressure50, consistent with our results exhibiting that Ca2+ transients in different subtypes of pericytes exhibit a differential dependence on VGCCs.Our study adds to a growing body of evidence that the mural cells of the cerebral vasculature represent a continuum, with morphological, molecular, and functional heterogeneity along the arterio-venous axis. Our work is largely in agreement with a previous study which found that Ca3R signaling in mediating their Ca2+ transients and [Ca2+]i28. Elucidating the molecular and functional differences between pericyte subtypes, including in the Ca2+ signaling mechanisms described here, remains an important task. While SOCE is universal across cell types, it is more prominent in non-excitable cells, perhaps due, in part, to the inhibition of VGCCs by the essential SOCE proteins STIM1 and STIM252, raising the possibility that elevated STIM-plasma membrane interactions could have inhibitory effects on mid-capillary pericyte VGCCs. Another interesting possibility is differential inhibition by endogenous polyamines, such as spermine, which has been previously suggested to underlie the decreased functional expression of VGCCs in distal capillary pericytes of the retina30. How these signaling differences translate into functional differences remains another key point of future study.Our results in ensheathing pericytes are largely consistent with recent reports that have uncovered prominent roles of depolarization, L-type VGCCs, and Gq-GPCR-IP2+ influx in mid-capillary pericytes occurs primarily via non-voltage-gated channels, this does not exclude a role for pericyte hyperpolarization in functional hyperemia. Several reports on pericyte membrane potential have been made in various preparations from different regions of the CNS, reporting mean resting membrane potentials of between −35 mV and −50 mV50. In pressurized intact retina preparations, pericytes across the vascular arbor are found more depolarized than smooth muscle cells at low pressure (−43 mV vs. −64 mV)50, suggesting pericytes have lower K+ permeability at rest. Opening of KATP channels increases K+ permeability of mid-capillary pericytes, and therefore causes a robust hyperpolarization39, which can then be propagated to upstream mural and endothelial cells39. Retrograde hyperpolarization is a robust phenomenon in the microvasculature which rapidly closes VGCCs on upstream SMCs and ensheathing pericytes, thereby decreasing intracellular Ca2+ and dilating these contractile cells to increase local cerebral blood flow55. Interestingly, we found that blocking KATP channels with glibenclamide led to a decrease in the frequency of Ca2+ transients, consistent with a decreased driving force for Orai mediated Ca2+ influx. This result suggests that KATP channels may be open at rest in pericytes from our brain slices, potentially due to increased adenosine tone which can open KATP channels via A2A receptors38. Although modest increases in [K+]ex to 10 mM were previously suggested to decrease Ca2+ transient frequency via KATP dependent hyperpolarization18, unlike Kir2.x channels, KATP channels are not known to be activated by [K+]ex. If KATP channels were indeed open at rest, as appears to be the case in our study, even modest increases in K+ would depolarize pericytes via these open K+ channels. Therefore, an alternative interpretation of these results could be that blocking KATP channels in their experiments blocked K+ induced depolarization rather than hyperpolarization, and thereby reduced driving force for Ca2+ entry via non-voltage-gated channels, consistent with our findings. Recordings of mid-capillary pericyte membrane potential changes in response to modest changes in K+ concentration are therefore needed to properly interpret these differences.Although our data suggests Ca56. Whereas ensheathing type pericytes dilate actively and independently from the arteriole10, it remains to be determined whether the mid-capillary diameter increase is a purely passive process or has an active component mediated by mid-capillary pericytes. Interestingly, under pathological conditions such as Alzheimer’s disease and stroke, even mid-capillary pericytes are found to be constricted57, and prolonged optogenetic activation of ChR2 on cortical mid-capillary pericytes has been shown to locally constrict the capillaries that they contact on a slow timescale58. Likewise, increases in intraluminal pressure lead to mid-capillary pericyte depolarization in the retina, and are followed by a delayed constriction50. Although these studies suggest that mid-capillary pericyte membrane depolarization is correlated with increased rigidity and tone of the capillary bed, a causal relationship is lacking. Given that our data suggests that mid-capillary pericyte Ca2+ influx is dominated by voltage-independent channels, depolarization would be expected to reduce this Ca2+ influx and subsequent store filling, due to a decrease in driving force, as in other non-excitable cells. This is consistent with a recent report showing that hyperpolarization of mid-capillary pericytes by application of pinacidil increased pressure-induced Ca2+ influx50.During neurovascular coupling, ensheathing pericytes of the capillary-arteriole transition zone (which robustly express α-SMA) dilate rapidly, whereas the pericytes of the mid-capillary bed (low or negative for α-SMA) increase their diameter more slowly2+ elevation mediated by the vasoconstrictor ET-1. The magnitude of the ET-1-induced Ca2+ responses when applied in normal ACSF were highly variable, which could reflect heterogeneous expression of the endothelin-A receptor and/or SOCE proteins in mid-capillary pericytes. ET-1 is a central molecule to pericyte pathology, which is released in ischemia59, and which is elevated downstream of Amyloid-β oligomers to constrict pericytes in Alzheimer’s disease57. In ensheathing pericytes (1–3rd branch order), the ET-1 evoked [Ca2+]i increase was also recently shown to require an amplification step, but via Ca2+ activated Cl- channel mediated depolarization and L-type VGCC activation28. Our results indicate a similar amplification process in mid-capillary pericytes, but with a clear molecular divergence in the mechanisms mediating ET-1 evoked Ca2+ influx. As Ca2+ is a ubiquitous signal transduction molecule throughout biology, mediating an array of cellular functions, including gene transcription and contraction, SOCE channels may therefore play an important role in numerous pericyte functions and contribute to their dysfunction in disease.Finally, we show here that SOCE is required to amplify the mid-capillary pericyte Ca2+ signaling. Our pharmacological modulation of SOCE with non-selective (SKF-96365 and 2-APB) and relatively selective (GSK-7975A and IA65) compounds is consistent with the highly Ca2+ selective family of Orai channels  as the molecular identities underlying pericyte SOCE. However, TRPC channels have been shown to interact with and contribute to SOCE in certain conditions61, although this concept is heavily debated62. While our results show that TRPC3/6 are not major contributors, we cannot completely rule out participation of the TRPC1/4/5 subfamily to pericyte SOCE.Genetic tools will ultimately be required to uncover the precise molecular identities underlying mid-capillary pericyte Ca3R and RyR channels in mid-capillary pericytes will similarly require genetic tools to elucidate. While the IP3Rs are broadly dispersed throughout cell types, the distribution of RyR isoforms is more segregated, with RyR2 constituting the dominant variant in smooth muscle cells63, making it the most likely variant to be functional in pericytes. Interestingly, RyRs have been classified as functionally absent in most64  pericytes of different tissues, including ensheathing type pericytes in the retinal CNS vasculature27. It remains to be determined whether the functional expression of RyRs in cortical CNS mid-capillary pericytes represents a difference across brain regions and/or between pericyte subtypes.The molecular identities of the IPOur study was conducted solely in the brain slice preparation, which is a highly useful tool for probing cellular signaling mechanisms, as pharmacological compounds can be delivered at a controlled concentration and cellular architecture is kept largely intact. However, there are important limitations when compared to in vivo settings: there is no blood pressure and myogenic tone, arterioles and capillaries are collapsed, oxygen concentration (95%) is supraphysiological, and the slice surface has undergone mechanical damage. Translation of this work to in vivo experimentation is an important future direction.67 were crossed with Ai95(RCL-GCaMP6f)-D reporter mice (Jackson Laboratory) to obtain PDGFRβ-Cre; GCaMP6f-floxed double transgenic mice. Male and female mice aged P28-P163 were used in experiments.All procedures conformed to the guidelines of the Canadian Council on Animal Care and were approved by the “Comité de déontologie sur l’expérimentation animale” (CDEA) of the Université de Montréal . PDGFRβ-Cre miceN-Methyl-d-glucamine, 3 KCl, 25 NaHCO3, 7 MgCl2-6H2O, 1 NaH2PO4-H2O, 20 Glucose, 2.4 Na-pyruvate, 1.3 Na-ascorbate, 1 CaCl2-H2O. Then, 300 µm thick coronal cortical slices were cut with a Leica VT 1200S Vibratome. Slices were transferred to a custom chamber with artificial cerebral spinal fluid (ACSF) containing (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO3, 1.5 MgCl2-6H2O, 1.3 NaH2PO4-H2O, 10 Glucose, and 1.2 CaCl2 at 36 °C for 10 min. Slices were then recovered in the chamber at room temperature until use. The fluorescent dye TO-PRO-3 has previously been shown to robustly label mid-capillary pericytes in fixed tissue34, and we have adapted this for imaging in acutely prepared live brain slices. Prior to use, slices were incubated in 1 µM TO-PRO-3 diluted in ACSF for 20 min at room temperature to label and identify the pericyte soma. All solutions used were continuously gassed with 95% O2 and 5% CO2.Prior to slicing, mice were put into deep anesthesia with isoflurane and 50 µL of Rhodamine B isothiocyanate-Dextran  was injected retro-orbitally to label the vessel lumen. Mice were euthanized by decapitation. Following extraction from the skull, brains were placed into ice-cold NMDG-based slicing solution containing (in mM): 120 2+-free ACSF, CaCl2 was omitted from the ACSF and 2 mM EGTA was added. For 60 mM K+ ACSF, equimolar NaCl was replaced with KCl. Slices were perfused with TTX for 10–20 min prior to imaging. For 60 mM K+ experiments, slices were perfused with 60 mM K+ for 10-20 min prior to application of VGCC blockers.All salts were obtained from Sigma-Aldrich. Please refer to Table 2+ imaging. Ensheathing pericytes in a subset of explicitly labeled experiments were identified based on GCaMP fluorescence almost fully enwrapping the vessel and were definitively confirmed to be within 3 branch orders of the penetrating arteriole. Mid-capillary pericytes were selected for all other experiments on the basis of: (1) Morphology: thin-stand or mesh morphology in which processes clearly did not fully enwrap the vessel (notably different than those found on first 3 branches), (2) being positive for TO-PRO-3 labeling, and 3) the lack of an arteriole detected within 4 branches of the capillary using the Rhodamine B signal. In several cases for mid-capillary pericytes they could not be traced back 4 branches and an arteriole was not visible in the field of view, in these cases only criteria 1 and 2 were used. To measure calcium transient frequency over time, 50 s image acquisitions were made every 3–4 min in frame scanning mode at a 2 Hz sample rate. To record SOCE experiments and [Ca2+]i rises evoked by agonists, a single acquisition was made in frame scanning mode at a 2 Hz sample rate. Slices were perfused with ACSF at 2 mL/min and were kept at 34 ± 2 °C during experiments.Imaging was performed with a Zeiss LSM 510 laser scanning confocal microscope with a 40X water immersion objective lens (0.8NA). Pericytes in cortical brain slices were located by TO-PRO-3 and GCaMP6f co-localization and association with a Rhodamine B labeled vessel. GCaMP6f was excited with a 488 nm LED laser and was detected after passing through a 505–530 nm bandpass filter. TO-PRO-3 and Rhodamine B were excited with a 633 nm and 543 nm HeNe laser respectively, which were generally turned off during Caimregdemons  and imwarp MATLAB functions . Hand-drawn regions of interest (ROI) separated pericyte somas from processes. The Astrocyte Quantification and Analysis (AQuA) MATLAB tool35 was used for unbiased event-based Ca2+ transient analysis. A Gaussian filter was applied to the images (σ = 2), minimum event size was set to (pixels): 5/pixel size (µm), and detected events occurring in the same frame separated by (in pixels) 1/pixel size (µm) were merged. Event threshold parameters were set to reliably detect the majority of pericyte Ca2+ signaling events with minimal detection of false events outside the cell boundaries. Identical analysis parameters were used for all time series of a given cell and experiment. In experiments in which movies were collected at differing timepoints and average data was shown, a linear interpolation between data points was performed. Ca2+ transients following agonist application (ET1 or U46619) in Ca2+ free solution ; U46619 (78.86 ± 12.53 s); ET-1 in GSK-7975A (149.1 ± 7.841 s). To compare ET-1 responses at 5, 10, and 30 min in Ca2+-free solution .Between frame XY-plane translational movement and within frame distortion were removed using a custom non-rigid alignment MATLAB algorithm, based on the ion Fig.  or GSK-7ion Fig.  were onlls Fig. S and S2, t test was conducted. For paired data with three groups, a one-way repeated measures ANOVA was conducted, which if significant, was followed by Tukey’s or Šidák’s post-hoc test . For all other statistical comparisons normality was first assessed with a Shapiro–Wilk normality test. If this test failed, statistics were calculated by a Kruskal–Wallis non-parametric test, followed by Dunn’s multiple comparisons test.For all paired data with two groups a two-tailed paired Students’ Software used in the study is open source.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary DataSupplementary Data 1Supplementary Movie 1Reporting Summary


In cardiomyocytes, Ca2+ not only regulates excitation–contraction coupling, but also mitochondrial metabolism and oxidative stress signaling, thereby controlling the function and actual destiny of the cell. Understanding the mechanisms of mitochondrial Ca2+ uptake and the molecular pathways involved in the regulation of increased mitochondrial Ca2+ influx is an ongoing challenge in order to identify novel therapeutic targets to alleviate the burden of heart failure. In this review, we discuss the mechanisms underlying altered mitochondrial Ca2+ handling in heart failure and the potential therapeutic strategies.Heart failure is a serious global health challenge, affecting more than 6.2 million people in the United States and is projected to reach over 8 million by 2030. Independent of etiology, failing hearts share common features, including defective calcium (Ca Heart failure (HF) is a complex clinical syndrome characterized by the inability of the heart to pump blood at a rate and with adequate force to meet the metabolic demands of the body. This condition can result from various underlying causes, including damage to the heart muscle (such as from a heart attack), hypertension, valvular heart disease, or other cardiac conditions. HF can affect either the left, right, or both ventricles of the heart and can manifest as systolic dysfunction (impaired ability of the heart to contract and eject blood) or diastolic dysfunction (impaired ability of the heart to relax and fill with blood). The hallmark symptoms of HF include shortness of breath, fatigue, fluid retention (edema), and reduced exercise tolerance. It is a chronic condition that requires ongoing management and treatment to improve the patient’s quality of life and reduce morbidity and mortality. HF shows a steadily growing prevalence and remains the leading cause of death in developed countries ,2. Altho2+) and reactive oxygen species (ROS) are generally considered the main transduction signals linking the SR and mitochondrion and permit them to adapt their responses to external stress in a highly regulated manner [2+ dyshomeostasis, altered bioenergetics, changes in mitochondrial respiration, increased oxidative stress, and impaired energy production and consumption [2+ uptake due to dysregulation in several calcium cycling proteins and increased SR Ca2+ leak through type-2 ryanodine receptors (RyR2) resulting in reduced intracellular Ca2+ stores required for cardiomyocyte contraction and increased baseline cytosolic Ca2+ [2+ uptake follows, which contributes to mitochondrial Ca2+ overload, dysfunction, and oxidative stress [The heart consumes a significant amount of energy supplied almost entirely by mitochondrial ATP production derived from oxidative metabolism . Nearly d manner ,38,39,40sumption ,42,43,44lic Ca2+ ,48,49,50e stress . Mitochoe stress ,54,55,562+ overload in inducing oxidative stress during HF and highlight the potential therapeutic targets as well as new outlooks for future studies.In this review, we summarize the recent evidence demonstrating the crucial role of mitochondrial CaThe heart primarily depends on mitochondrial oxidative metabolism to fulfill approximately 95% of its energy demands , with a 2+ ions are essential to this process. Indeed, increased cytosolic Ca2+ levels with elevated cardiac contractions allow Ca2+ to enter the mitochondria and stimulate pyruvate dehydrogenase, α-ketoglutarate, and isocitrate dehydrogenase to regenerate reducing equivalents [2+ entry into the mitochondria, as in HF, may be deleterious, leading to mitochondrial Ca2+ overload, increased ROS production, and oxidative stress [2+ overload during HF are discussed in the following sections.Increased ATP consumption in cardiomyocytes requires additional reducing equivalents, and Caivalents , as wellivalents . Howevere stress . The fun2+ stored and released within cardiomyocytes in EC coupling. During the depolarizing phase of the cardiac action potential, Ca2+ enters the cardiomyocyte through voltage-activated L-type Ca2+ channels and triggers RyR2 to open and release Ca2+ from SR Ca2+ stores through calcium-induced calcium release (CICR), raising cytosolic Ca2+ levels about ten-fold to ~1 μM [2+ then activates specific proteins of the contractile apparatus and induces contraction of cardiac cells [2+ is removed from the cytosol via three primary mechanisms: type-2a SR Ca2+-ATPase (SERCA2a) transport, sarcolemmal Na+/Ca2+ exchanger (NCX) extrusion, and mitochondrial Ca2+ uptake [Cardiac muscle contraction is regulated beat-to-beat by Cato ~1 μM . Ca2+ thac cells ,69,70. S+ uptake ,73,74,752+ cycling leading to significant increases in cytosolic baseline Ca2+ has been observed [2+ channels in failing compared with non-failing human ventricles was confirmed, which resulted in an excessive calcium influx into the cytosol [2+ leak, was also shown in human and mice cardiomyocytes of failing hearts [+/Ca2+ exchanger (NCX) in extruding cytosolic calcium was demonstrated in a mouse model of heart failure [2+ and potentially increase Ca2+ entry into the mitochondria, causing a detrimental mitochondrial Ca2+ overload in cardiomyocytes [During HF, the widespread dysregulation of Caobserved . First,  cytosol ,78. Moreg hearts . Further failure . Finally failure ,81. Thesmyocytes ,83.2+ dyshomeostasis [2+ cycling proteins [2+ overload [2·−) and resulting in an SR Ca2+ leak [2+ and mitochondrial Ca2+ overload, leading to ROS formation and oxidative stress. The signaling cascades associating the inflammatory response to SR/mitochondrial dysfunction in HF still remain to be elucidated.Inflammation has emerged as a major pathophysiological feature of HF progression, regardless of etiology. Numerous pro-inflammatory signaling molecules are produced and released in HF, initiating vicious cascades involving oxidative stress, mitochondrial dysfunction, myocardial weakened contractility, and, importantly, Caeostasis ,85. Elevproteins ,85. TNF-proteins ,87. Seveoverload ,87. Incroverload ,89. Finaoverload ,85. Wania2+ leak . The ele2+ influx occurs primarily via the mitochondrial Ca2+ uniporter (MCU), whereas Ca2+ efflux occurs mainly via the mitochondrial Na+-Ca2+—Li+ exchanger (mNCLX) [2+ channel in the mitochondrial inner membrane that facilitates Ca2+ transport down its electrochemical gradient without coupling Ca2+ transport with the movement of other ions [2+ uptake channel is a large holocomplex consisting of MCU as the pore-forming unit and multiple regulatory proteins, including the essential MCU regulator (EMRE) [Mitochondrial Ca (mNCLX) ,92,93,94her ions ,108,109.r (EMRE) ,112,113,r (EMRE) ,123,124,r (EMRE) ,127,128,r (EMRE) ,131,132.r (EMRE) . These rr (EMRE) . Howeverr (EMRE) ,135; howr (EMRE) .2+ leads to excessive Ca2+ entry into the mitochondria and causes mitochondrial Ca2+ overload, which is detrimental to the cardiomyocyte [2+ uptake causes mitochondrial Ca2+ overload and oxidative stress, MCU modulators can be an interesting therapeutic option to rescue the subsequent detrimental signaling cascades observed in HF [2+ uptake during acute stress can be reduced by the deletion of the MCU gene or by the MCU blocker, Ru360 [2+ levels remain unchanged [2+ uptake [2+ uptake with different levels of sensitivity to cytosolic Ca2+. Possible mediators of MCU-independent mitochondrial Ca2+ uptake include the Ca2+/H+ exchanger Leucine Zipper And EF-Hand Containing Transmembrane Protein 1 (Letm1) [2+ sensitivity has been shown [d~20–30 μMol) and, as such, resting cytosolic Ca2+ can be insufficient to trigger MCU activity [2+ concentration in cardiomyocytes is, as well as the difference between basal cytosolic Ca2+ in normal versus failing cardiomyocytes when associated with a defective RyR2 leak or reduced SERCA2a activity. Nonetheless, is basal cytosolic Ca2+ in HF sufficient to enter mitochondria and cause Ca2+ overload? This dilemma represents an important question that needs to be clarified by future investigations.During HF, increased cytosolic Caomyocyte ,83,137. ed in HF ,145,146.r, Ru360 ,148, whinchanged ,135. Fur+ uptake . These f (Letm1) ,155,156  (Letm1) ,162,163.en shown  to be quactivity ,165,166.2+ homeostasis [2+ uptake has to be balanced with extrusion in homeostatic conditions, they hypothesized that the key channel responsible for mitochondrial efflux Ca2+, mNCLX [2+ handling [2+ efflux by 38%, displayed an attenuated infarct size after MI or I/R injury, improved cardiac function, and reduced superoxide generation [2+ overload in HF is due to enhanced Ca2+ uptake or repressed extrusion has yet to be further elucidated. Furthermore, the following queries need to be investigated: does increased mNCLX expression/activity affect cytosolic Ca2+ levels by increasing mitochondrial Ca2+ extrusion? Moreover, what are the long-term outcomes of enhanced mNCLX activity in mice? Will those mice be more vulnerable to arrhythmias due to cytosolic Ca2+ overload? We believe that the long-term outcomes of this enhanced mNCLX activity are deteriorated when cardiac disease is associated with leaky RyR2 channels. Although, these hypotheses remain to be verified, recent studies by Garbincius point towards the deleterious effect of mNCLX cardiomyocyte-specific overexpression in mice. Indeed, survival was reduced when mNCLX cardiomyocyte-specific overexpressing mice were subjected to severe neurohormonal stress with angiotensin II and phenylephrine [Recently, Luongo and collaborators revealed a novel mechanism, independent of MCU, responsible for mitochondrial Caeostasis . Since m+, mNCLX ,167, woulephrine .2+ when the Ca2+ concentration is below a threshold of 200 nM [2+ release channels RyR2 and/or 1,4,5-trisphosphate receptors (IP3Rs) [2+ concentrations (10–100 µM) are required to activate Ca2+ uptake into isolated mitochondria; however, in intact cardiomyocytes, the cytosolic Ca2+ concentration after Ca2+ release from the SR remains less than 10 µM, yet Ca2+ is still internalized by mitochondria [It is well established that MCU does not transport Caf 200 nM . However (IP3Rs) ,170,171.chondria ,173.2+ concentrations in microdomains between mitochondria and the SR. For example, RyR2 Ca2+ release during systole creates microdomains of high, localized Ca2+ concentrations (~30 µM) in the vicinity of the mitochondrial membrane, leading to mitochondrial Ca2+ uptake [2+ transfer puzzle that mostly concern the complexity of this highly regulated process, which involves two dynamic organelles. Due, in part, to the low Ca2+ affinity of the MCU, we believe that the formation of Ca2+-rich microdomains alone is not sufficient to allow Ca2+ to enter the mitochondria, especially in the presence of competing sarcolemmal NCX and SERCA2a pumps with a higher Ca2+ affinity and faster uptake rate.Such a discrepancy between isolated mitochondria and intact cells was partially resolved by the finding of high cytosolic Ca+ uptake ,182,183.2+ to transfer to the mitochondria. This hypothesis was recently investigated and two major, non-mutually exclusive interactions between the SR and mitochondria were identified. The functional, tight coupling between the SR and mitochondria is attributed to the inter-organelle tether protein involving mitofusin2 (MFN2) [2+ overload observed in HF is discussed below.Thus, a very close proximity between the two organelles seems to be necessary to allow Ca2 (MFN2) ,185,186 2 (MFN2) ,187,188.Recently, the purification of subcellular fractions corresponding to endoplasmic reticulum (ER)–mitochondria contacts, referred to as mitochondria-associated membranes (MAMs), led to the identification of proteins enriched in those membrane domains ,190,191.2+ transfer to the mitochondria. The lack of MFN2 to tether the ER to mitochondria resulted in a significant reduction in mitochondrial Ca2+ uptake, without any impairment of mitochondrial capacity to uptake Ca2+. This impaired uptake reflects an increased distance between the two organelles and, therefore, a limited generation of Ca2+ microdomains. Therefore, MFN2-KO cells provide experimental proof for the importance of Ca2+ microdomains between the ER/SR and mitochondria, a theory postulated by Rizzuto and Pozzan almost 30 years ago [2+ signaling in the myocardium. This is consistent with a requirement for SR–mitochondrial Ca2+ signaling through microdomains to maintain a competent bioenergetic feedback response in cardiomyocytes [MFN2-KO cells were used to investigate how tethering between the ER and mitochondria would impact Caears ago ,197,198.myocytes .2+ signals through inter-organelle microdomains, where the signaling factor is prevented from diffusing into the cytosol. However, as mitochondria are the main source of ATP in cardiomyocytes and the SR is the main source of Ca2+, it is reasonable to postulate the existence of a physical, dynamic interaction that facilitates direct Ca2+ exchange between the two organelles. Increased beat-to-beat SR Ca2+ release can then be rapidly sensed by the mitochondria, inducing mitochondrial Ca2+ uptake, stimulating mitochondrial dehydrogenases, increasing the production of NADH for the ETC, and increasing ATP production according to the physiological demand . This suggests that these channels do not play a role in the malfunctioning of Ca2+ regulation in ventricular cardiomyocytes [2+ overload in HF and the leakage of Ca2+ from RyR2 channels [In the same vein as the close proximity between the SR and mitochondria described above, recent studies have identified several proteins enriched at the SR–mitochondria interface, highlighting the emerging role of inter-organelle interactions within the cell ,201. Onechondria . Specifichondria . The funachinery . This fiachinery . Howeverachinery ,203. Furmyocytes . Interesmyocytes . On anotchannels . This dichannels .2+ overload, the precise quantity of Ca2+ taken up by mitochondria with each heartbeat in both normal and failing hearts remains uncertain. Furthermore, there is an ongoing debate regarding whether the mitochondrion can function as a primary reservoir for Ca2+ ions [2+ homeostasis is manipulated [2+ in various animal models has led to a mitochondrial phenotype resembling that observed in heart failure. This phenotype includes compromised ATP generation, heightened mitochondrial oxidative stress, and, ultimately, the death of cardiomyocytes [Despite the important progress achieved in the investigation of the pathophysiologic implications of mitochondrial Caa2+ ions ,206,207.ipulated ,111. Indmyocytes ,209.2+ fluxes between the ER and mitochondria [2+ leak was not investigated in these models.Other proteins facilitating a physical connection between the ER and mitochondria have been proposed, including PDZ-domain-containing 8 (PDZD8) ,211, prochondria ; however2+ levels lead to the production of mitochondrial reactive oxygen species in various diseases, including heart failure [2·−) and hydroxyl radical (·OH), as well as non-radical species, such as hydrogen peroxide (H2O2) and hypochlorite (OCl−) [2+ stimulates the Krebs cycle, resulting in the generation of NADH and FADH2, both of which provide electrons to the electron transport chain (ETC). As electrons progress through a series of redox reactions along the ETC, a proton gradient is established, facilitating ATP production, as described earlier [2O2) by superoxide dismutase (SOD), with SOD2 being the primary mitochondrial isoform [2O2 is then removed by the antioxidant systems of peroxiredoxin (Prx) and glutathione peroxidase (Gpx) in the mitochondria [2·− generation from complex I of the ETC caused excessive formation of H2O2 and ·OH, which became deleterious when their production overwhelmed detoxification [2+ overload. This overload further amplifies mitochondrial oxidative stress, propagating a vicious cycle.Numerous studies have shown that excessive mitochondrial Ca failure ,68. ROS  failure , protein failure , and nuc failure . This cae (OCl−) ,221. In  earlier . Dependi earlier ,224,225. isoform ,227. Mitchondria ,229. In fication ,231. Sinfication ,232. In 2+ leak through the hyperactivity of RyR2 [2+ leak not only augments cytosolic Ca2+ concentrations by depleting Ca2+ stores but also increases mitochondria Ca2+ overload, which further produces ROS [Increased mitochondrial oxidative stress during HF, in human patients as well as in animal models ,234, is  of RyR2 . As HF p of RyR2 . Increasuces ROS  and wors2O2, RyR undergoes covalent disulfide cross-linking between the N-terminal domains of the four neighboring protomers, adopting an open state conformation and generating an SR Ca2+ leak [2+ leak through the channel [Many cysteine residues in RyR can be subjected to oxidative post-translational modification; however, the exact nature of these modifications for available cysteines and how they affect the activity of the channel remains to be determined. Recent work suggests that, in the presence of Ha2+ leak . We and  channel ,236,237. channel ,240,241 2+ have been implicated as a key factor for the development of age-related cardiac disorders, HF, and atrial fibrillation [2+ release (SOICR) by raising the activation threshold and delaying the termination of Ca2+ release. Furthermore, the C3602A mutation markedly increased fractional Ca2+ release and diminished the inhibitory effect of N-ethylmaleimide on Ca2+ release, but had no effect on the stimulatory action of 4,4′-dithiodipyridine on Ca2+ release [2+ release. Likewise, Nikolaienko et al. reported that this particular residue did not participate in inter-subunit cross-linking and played a limited role in RyR2 regulation by calmodulin during oxidative stress [RyR channels comprise several surface-exposed cysteine residues with free thiol groups prone to oxidation by reactive oxygen and nitrogen species. Oxidized cysteines that render RyR2 channels leaky to Caillation . Assessiillation . On the illation  in RyR1  release . Moreovee stress . Based oe stress  and our e stress , we mappThe other hyper-reactive cysteines C36, C253, and C315 are located in the N-terminal domain (NTD) of RyR1, while only C36 is conserved in RyR2. The N-terminal β-trefoil domains form the central cytosolic vestibule mediating inter-subunit interactions in the resting state of the channel. Since these interactions are disrupted during channel activation, it has been proposed that NTD is involved in the allosteric regulation of channel gating in both RyR1 and RyR2. Importantly, NTD is a known disease hot spot for pathogenic CPVT mutations weakening the RyR2 stability of the closed state ,249,250.2+ release. The RY1&2 domain is highly dynamic and its oxidation at C917 may contribute to the local regulation of this region.The hyper-active cysteine C822 is located in the SPRY1 (SPIa kinase and ryanodine receptor) domain, C917 in the tandem-repeat domain RY1&2, and C1582 in the SPRY3 region of RyR2. The SPRY domains represent important protein–protein interaction scaffolds in the periphery of the cytosolic shell of RyR. Interestingly, C1582 is located near the calstabin-binding site. Its oxidation can be important to modulating calstabin binding to the channel, which is critical for stabilizing the closed state of RyR and reducing spontaneous Ca2+ release by RyR2. Interestingly, helical domain 1 in the BSol1 domain is where C2402, C2572, and C2577 are located, and it exhibits a domain–domain interface with NTD. The disruption of NTD–Bsol interactions leads to inappropriate channel activation [The hyper-active cysteines C2402, C2572, C2577, and C3158 are located in the bridging solenoid (Bsol) of RyR2, another highly flexible and dynamic region of the protein. BSol encompasses the helical domains BSol1, BSol2, and BSol3. Between the first two is the RY3&4 domain that contains the PKA phosphorylation site (S2808) important for the adrenergic regulation of Cativation ,252. Sim2+ leakage [Nonetheless, through the application of genetic techniques to induce specific truncations and modifications in channel residues and domains, a recent study pinpointed that only two cysteine residues within each RyR2 subunit were accountable for nearly half of RyR2’s functional response when exposed to oxidative stress, specifically cysteine 1078 within the Ry1&2-SPRY2 linker and cysteine 2991 in the BSol loop function as a redox-sensitive pair. They engage in the formation of inter-subunit disulfide cross-links between adjacent subunits when confronted with oxidative stress, ultimately leading to an aberrant RyR2 Ca leakage . This fi leakage .2+ leakage due to oxidative stress [2+ leak.Additionally, as previously discussed, while the previous research has pointed to cysteine 3635 in RyR1 or its equivalent, cysteine 3602, as the most hyperactive redox-sensitive residue, Nikolaienko et al. demonstrated that this particular residue did not contribute to channel cross-linking or the activation of Cae stress ,253. In It has been observed that RyR channels self-organize into higher-order clusters forming checkerboard-like arrangements in the terminal cisternae of SR membranes. Peripheral domains likely involved in mediating clustering interactions between RyR channels are SPRY1, RY1&2 domains, and the C-terminal Bsol region ,256. Imp2+/calmodulin-dependent protein kinase II (CamKII), both known to be redox sensitive, have been shown to phosphorylate RyR2 at different sites and promote RyR2 Ca2+ leak. PKA and CamKII regulate RyR2 function and play an important role in regulating cardiac contractility and arrhythmogenesis [2+ leak, which in turn reduces cardiac contractility and ultimately furthers HF progression. Chronic PKA activation in HF is caused by the hyper-adrenergic state observed in HF patients, as well as in animal models of HF [RyR2 is a large scaffold on which several regulatory proteins and enzymes can be assembled. Among them, two major regulatory kinases, protein kinase A (PKA) and Caogenesis ,268,269.ls of HF .2+ leak, which may lead to mitochondrial Ca2+ overload and dysfunction. To date, however, the exact physiological relevance of this ROS-dependent activation of PKA under physiological and pathological conditions, especially in comparison to adrenergic-dependent activation, remains mostly unknown.Furthermore, the type-I negative regulatory subunits of PKA are also subject to oxidation by ROS . The oxi2+/CaM autonomous activity due to T287 auto-phosphorylation [2+ uptake in the heart by phosphorylating the MCU, with subsequent impacts on mPTP activation and programmed cell death [2+ uptake, respiration, or ROS emission in isolated cardiac mitochondria, nor in isolated cardiac myocytes [Since CamKII has also demonstrated autonomous activation following the oxidation of its regulatory domain, the role of CamMKII oxidation in cardiovascular disease, including HF, has garnered significant attention . The Andrylation . Recentlll death . Howeverll death . These dmyocytes .2+ uptake into the SR, which may increase mitochondrial Ca2+ overload and further ROS production [In addition to RyR2, PKA, and CamKII, SERCA2a has also been shown to experience dysregulation following oxidation by ROS. By treating cardiomyocytes with various reactive oxygen species, several studies have demonstrated decreased SERCA2a Caoduction ,278,279.2+ triggers the opening of the mitochondrial permeability transition pore (mPTP), a non-specific pore in the inner membrane of the mitochondria [c and SMAC/Diablo into the cytosol and, eventually, apoptosis [2+ overload, production of ROS, depletion of ATP and ADP, and increases in inorganic phosphate [Elevated mitochondrial matrix Cachondria ,281, conchondria . Thus, wpoptosis ,284,285.hosphate , all of 2+ handling, mitochondrial dysfunction, and oxidative stress have been linked to the development and progression of HF, independent of etiology. Therefore, the SR and mitochondria may serve as two of the most promising therapeutic targets for HF [2+ handling, as well as its relation to altered cytosolic Ca2+, appears pivotal. Several strategies targeting mitochondria or the SR, including small molecules, peptides, and antioxidants that have been tested in preclinical and clinical studies in HF, are summarized in As previously discussed, defective Cas for HF ,290,291.2+ overload and subsequent oxidative stress, thereby controlling the function and fate of cardiomyocytes. The impairment of RyR2 Ca2+ release has emerged as a major mechanism underlying HF and arrhythmogenesis as a consequence of genetic mutations and/or stress-mediated RyR2 post-translational modifications, loss of channel stability, and impairment of coupled gating.As mentioned above, RyR2 is an essential player in several functions of cardiomyocytes, from EC coupling to the activation of numerous pathways, gene expression, as well as mitochondrial Ca2+ leak by preventing calstabin2 depletion without blocking the channel pore [A family of compounds known as Rycals, a small, orally available molecule, prevent RyR2 Canel pore ,247. Pronel pore ,293. Thinel pore .2+ homeostasis, known as the major mechanism in cardiomyopathies, arrythmias, and death.SS31, a potent antioxidant peptide with the ability to penetrate cell membranes, is located within the inner mitochondrial membrane, where it exhibits protective properties for mitochondria ,295. Exp2+ reservoirs. They accumulate Ca2+ ions during cytosolic Ca2+ elevations in cardiomyocytes. Elevations in intramitochondrial Ca2+ levels trigger multiple enzymes within the mitochondrial matrix. These enzymes, in turn, boost ATP generation, modify the patterns of intracellular Ca2+ signaling across both space and time, and assume a pivotal role in instigating mitochondrial oxidative stress and pathways leading to cell death. It is clear that, in HF, an excessive SR Ca2+ leak, mainly through RyR2 channels, plays a crucial role in the pathophysiology of mitochondrial Ca2+ accumulation and dysfunction and constitutes a feedback loop of alterations between the SR and mitochondria, which contribute to the impairment of cardiac function. Future investigations should focus on breaking this feedback loop by simultaneously targeting the SR leak and mitochondrial Ca2+ overload/oxidative stress using RyR2 stabilizing drugs and mitochondrial antioxidants.Beyond their crucial role as an energy source, cardiac mitochondria serve as Ca