However, studies on banana (Musa acuminata) Ca2+-ATPases are very limited. In this study, we identified 18 Ca2+-ATPase genes from banana, including 6 P-IIA or ER (Endoplasmic Reticulum) type Ca2+-ATPases (MaEACs) and 12 P-IIB or Auto-Inhibited Ca2+-ATPases (MaACAs). The MaEACs and MaACAs could be further classified into two and three subfamilies, respectively. This classification is well supported by their gene structures, which are encoded by protein motif distributions. The banana Ca2+-ATPases were all predicted to be plasma membrane-located. The promoter regions of banana Ca2+-ATPases contain many cis-acting elements and transcription factor binding sites (TFBS). A gene expression analysis showed that banana Ca2+-ATPases were differentially expressed in different organs. By investigating their expression patterns in banana roots under different concentrations of Ca2+ treatments, we found that most banana Ca2+-ATPase members were highly expressed under 4 mM and 2 mM Ca2+ treatments, but their expression decreased under 1 mM and 0 mM Ca2+ treatments, suggesting that their downregulation might be closely related to reduced Ca accumulation and retarded growth under low Ca2+ and Ca2+ deficiency conditions. Our study will contribute to the understanding of the roles of Ca2+-ATPases in banana growth and Ca management.Ca For theeostasis . Accordis (ACAs) . Ca2+-ATn plants . The ACAn plants ,7. Moreon plants .Lycopersicon LeECA1 gene was the first Ca2+-ATPase gene cloned from plant. Its expression level increased dramatically under 50 mM NaCl condition [2+-ATPases were continuously discovered in many plants and were found to be widely involved in many cellular and physiological processes of plants. For instance, the Arabidopsis  AtACA9 was reported to be required for normal pollen tube growth and fertilization. Pollens of the ataca9 mutant showed much lower growth potential, higher aborted fertilization frequency and a >80% reduction of seed set [AtACA10 has been reported to play an important role in regulating reproductive development, including adult phase growth and inflorescence development [ateca1-1 mutant displayed Ca2+ deficiency symptoms such as smaller plant size, shorter roots, small yellowish leaves and lack of bolts [2+ conditions (0.2 to 0.4 mM). Similarly, the ateca3 mutant was slightly more sensitive than the wild-type when Ca2+ was omitted from the medium, and the fresh weight of ateca3-1 was significantly lower than that of the wild type plants [The ondition . From thseed set . AtACA10elopment . Moreoveof bolts  under loe plants .2+-ATPases have also been repeatedly found to participate in plant stress responses. The AtACA8 gene is low temperature inducible, and the ataca8 mutant showed increased cold resistance and the higher expression of CBFs, CAMTA and ZAT12 transcription factor genes, as well as cold responsive genes such as UGE2 and GolS3 [Oryza sativa L.), OsACA6 can improve plant salinity and drought stress tolerance by modulating the ROS scavenging pathway and enhancing the expression of stress-responsive genes [Glycine Soja GsACA1-overexpression alfalfa plants exhibited higher tolerance to both carbonate alkaline and neutral salt stresses, and lower levels of membrane permeability and malondialdehyde (MDA) content, higher levels of superoxide dismutase (SOD) activity, proline (Pro) concentration and chlorophyll content under stress conditions [2+ deficiency and toxicity conditions, the expression of Triticum aestivum TaECA1 and TaECA3 significantly increased in both roots and shoots. And the expression levels of TaACA2, 3 and 4 in plants grown under Ca2+ deficiency and toxicity conditions differed significantly from plants grown under control conditions [In addition to their roles in plant growth and development, Cand GolS3 . In riceve genes . Glycinenditions . Under CMusa spp.) is one of the most important fruit crops in the world. As a gigantic herb, the nutrient requirement of a banana tree is huge. However, due to the high temperature and rainy climate characteristic of their cultivation areas, banana trees often suffer greatly from various nutrient deficiencies [2+ is crucial for the growth and development of banana plants, and Ca2+ deficiency will cause malformation or carving of new banana leaves, significantly reduce the accumulation of dry matter in pseudostems and leaves, and inhibit growth of the whole banana plant [2+-ATPases in plant development and stress responses have been demonstrated in many species, but studies on banana Ca2+-ATPases genes is very limited. To provide a basis for future studies and applications of banana Ca2+-ATPases, genome-wide identification of this gene family was performed, their sequence characteristics were studied using a series of bioinformatics analyses, and their expression patterns in different parts of banana  were investigated using transcriptome data. Furthermore, we evaluated the effects of different concentrations of Ca2+ treatments on the growth of banana seedlings by measuring the aboveground part and root fresh weight, root number and root length, and the P, K, Ca, Mg and S contents in root, leaf and pseudostem. Moreover, to reveal the possible roles of Ca2+-ATPases in response to different Ca2+ levels, their expression patterns in banana roots under different concentrations of Ca2+ treatments  were studied using a quantitative real time PCR (qRT-PCR). The results obtained in this study will be helpful for the understanding of the characteristics of the banana Ca2+-ATPase gene family and can provide insights into their roles in banana growth and development and stress responses.Banana  with a molecular weight ranging from 102,943.61 Da to 120,673.88 Da. The theoretical isoelectric point (pI) of Ca2+-ATPase proteins ranges from 5.19 to 8.87, and all of the MaECAs and seven of the MaACAs are acidic proteins, while the other five MaACAs  are basic proteins. Their instability index ranges from 30.90 to 40.33. All of the banana Ca2+-ATPases are hydrophobic proteins with 6~11 transmembrane structures but without signal peptide. Protein subcellular localization prediction results showed that all banana Ca2+-ATPases were plasma membrane-located. In total, 18 Ca2 MaACAs . Accordi2+-ATPase genes are unevenly distributed on nine chromosomes  and no tandem duplication pair (Banana Caomosomes . The mosion pair . 2+-ATPase protein sequences from Banana (18), Arabidopsis (15) and Rice (15), the phylogenetic relationships among Ca2+-ATPases were analyzed  have seven exons. The number of exons in ACA subfamily III genes varies greatly. MaACA6 has one exon, MaACA10 has two exons, MaACA12-2 has 35 exons and the other members have 34 exons. All members in ECA group I have eight exons, and the number of exons of MaECA5 in ECA Group II was 29. The exon-intron structures of banana Caribution b, indica2+-ATPase promoters, high up to 80, while the binding site number for bZIP, HD-ZIP, MYB, MYB-related and Nin-like was only one. In addition, there are significant differences in the TFBS number and distribution in the promoter regions of different banana Ca2+-ATPases. For instance, 59 TFBSs were found in MaECA1, while there is only one TFBS in the promoter region of MaACA2 and MaACA10  were identified in the promoters of 7, 6, 1, 7, 4, 5, 1, 1, 2, 7, 7, 13, 1, 1, 1 and 3 family members, respectively. The binding sites for ERFs were found to be the largest in banana Ca MaACA10 a. Cis-acting element prediction results showed that 21 kinds of cis-acting elements were identified in the banana Ca2+-ATPase promoters, which can be further classified into five categories, including light-responsive, core elements, phytohormone-responsive, stress-responsive and growth and development related elements. Light responsive elements and core box (TATA-box and CAAT-box) are present in all promoters of banana Ca2+-ATPases. Among the phytohormone-responsive elements, the number of elements related to abscisic acid (ABA) and methyl jasmonate (MeJA) was the largest, both containing 15 elements, followed by gibberellin (GA), auxin and salicylic acid (SA) related elements, which contain 12, 11 and 8 elements, respectively. In addition, there are several types of stress-responsive elements on the banana Ca2+-ATPase promoters, including anaerobic induction, low-temperature, drought-inducibility and defense and stress, which account for 10, 10, 8 and 3 elements, respectively. Furthermore, many plant growth and development related cis-elements were also found in the banana Ca2+-ATPase promoters, including meristem expression, zein metabolism, endosperm expression, anoxic specific inducibility, wound responsive element, seed-specific, differentiation of the palisade mesophyll cells, MYB binding site involved in flavonoid biosynthetic genes regulation, and MYBHv1 binding site elements, accounting for 8, 8, 4, 5, 3, 1, 1, 1 and 5 elements, respectively (ectively b.2+-ATPase genes showed divergent expression patterns in different banana parts (MaACA12 (belonging to ACA Subfamily III) was predominantly expressed, followed by MaACA1 (belonging to ACA Subfamily III), MaACA9 (belonging to ACA Subfamily II). In pseudostem, MaACA12 (belonging to ACA Subfamily III) was predominantly expressed, followed by MaECA5 (belonging to ECA Subfamily II), MaACA1 (belonging to ACA Subfamily III). MaACA7 (belonging to ACA Subfamily II), MaACA12 (belonging to ACA Subfamily III) and MaECA5 (belonging to ECA Subfamily II) were highly expressed in corn. MaECA6 (belonging to ECA Subfamily I), MaACA7 (belonging to ACA Subfamily II) and MaECA4 (belonging to ECA Subfamily I) were highly expressed in root. These results suggested that different Ca2+-ATPase family members might function differently in different organs.According to our transcriptome data, the Cana parts . In leaf2+ , the growth-related parameters of river sand cultured banana seedlings were measured  and four MaACAs , using qRT-PCR (MaACA1 in the 0 mM and 1 mM groups were lower than in the 2 mM and 4 mM groups. This indicated that both low Ca2+ (1 mM Ca2+) and Ca deficiency (0 mM Ca2+) suppressed the expression of banana Ca2+-ATPases. The expression levels of MaECA4 and MaACA7 in root of banana plants from the 2 mM group were significantly higher than that in the 4 mM group, but the expression of MaACA1 and MaACA10 were significantly lower than that in the 4 mM group. The expression of MaECA4, MaECA6, MaACA8 and MaACA10 in root of the 1 mM group were significantly lower than that in the 4 mM group. And the expression of MaECA4 was the lowest among all the four groups.We further studied the influences of different concentrations of Ca qRT-PCR . Results2+-ATPase genes from M. acuminata genome, including six MaEACs and 12 MaACAs. The number of banana Ca2+-ATPases was higher than that of Arabidopsis (15) and rice (15) [Arabidopsis, no gene duplication event was observed in the Ca2+-ATPase family [OsACA10 and OsACA11) and one tandem duplication gene pair (OsACA2 and OsACA3) were identified in the rice genome [2+-ATPase gene family was mainly caused by segmental duplications. In this study, we identified a total of 18 Caice (15) , but lesice (15) . Gene duice (15) . In Arabe family . One sege genome . In soybe genome . In our 2+-ATPases are mainly localized in the plasma membrane as well as endo-membranes. OsACA6-GFP was exclusively observed in the plasma membrane [2+-ATPases were predicted to be located in the plasma membrane, which was consistent with the results reported in some other plant species [2+-ATPases might be closely related to their functions in regulating intracellular Ca2+ homeostasis, which was achieved through transporting Ca2+ into extracellular space or sequestering Ca2+ in the ER and other organelles [2+-ATPase genes and conserved motifs of their encoded proteins in Solanaceae [Medicago sativa [Brassica rapa [2+-ATPase members from the same subfamilies were found to be highly conserved. Moreover, the ACA group is found to be of higher conservation than the ECA group.Previous studies have revealed that plant Camembrane . In addimembrane . In our  species ,10. Protganelles . Similarlanaceae , Medicago sativa  and Brasica rapa , the bancis-acting elements and regulate the expression of a vast number of genes, which is an important epigenetic pathway [2+-ATPase promoters. AP2/ERF, bZIP, C2H2, MYB and WRKY have been reported to play vital roles in regulating the growth, development and stress responses of banana [AtACA8 contains various TFBS responses to low temperature, in consistence, the gene’s expression is responsive to low temperature [2+-ATPase gene expression was regulated by many TFs and responds to a variety of stresses.Transcription factors bind to the specific  pathway . In our f banana ,30,31. Pperature . Thus, i2+  for six months, we investigated the influences of Ca2+ levels on the growth of banana. The banana seedlings grown under 2, 1 and 0 mM Ca2+ showed shorter and less roots compared to plants from the 4 mM group. Consistently, similar results were also reported in wheat [2+ is an essential plant nutrient [2+ deficiency can cause leaf-rot/wilting phenotype in Maize (Zea mays L.) [Brassica oleracea L. var. Capitata), the fresh matter of aerial parts decreased up to 37% in plants under Ca2+ deficiency [2+ deficiency also caused the reduction of plant biomass and root growth in trifoliate (Poncirus trifoliate L.) rootstock seedlings [2+ treatments greatly affected the macro-elements accumulations in banana root, pseudostem and leaf. An excess or deficiency of nutrients in soil will destroy the ion homeostasis in plants, resulting in plant nutritional imbalance and retarded plant growth. Previous studies reported that different concentrations of exogenous Ca2+ treatments could affect the contents of K, Mg, Fe, Mn, Cu and Zn in leaf and the pericarp of pomegranate (Punica granatum L.) [2+ concentration decreased. Furthermore, the P content in banana root, pseudostem and leaf and the S content in banana root increased significantly. It is possible that insoluble Ca3(PO4)2 and CaSO4 will be formed when Ca2+ coexists with PO43− or SO42−. Therefore, the absorption efficiency of P and S in banana will significantly increase after suffering Ca2+ deficiency, resulting in the P and S accumulation increase in banana organs [2+ concentration greatly influenced the Mg and the K accumulations in banana.After treating the banana plants with different concentrations of Cain wheat . It is wnutrient . Previoumays L.) . In cabbficiency . Ca2+ deeedlings . In our atum L.) . In our a organs . In addi2+-ATPase exists in all membrane systems of plant cells, and the expression of Ca2+-ATPase is tissue specific [2+-ATPase family was expressed in at least one organ. MaECA4, 6 and MaACA7, 12 were highly expressed in root, pseudostem, corm, and leaves, which was consistent with the expression of AtACA8 and AtACA10 [MaACA4 and MaACA11 were found to be specifically expressed in banana leaves, and MaECA2 was specifically expressed in pseudocorm, indicating that their functions varied in different organs.Studies have shown that Caspecific . In our 2+-ATPases are considered to be required to maintain homeostatic levels of cytosolic Ca2+ and are widely believed to have roles in abiotic stresses via Ca2+ mediated signaling pathways. For instance, the expression of AtACA8 was up-regulated when plants were under cold stress [AtACA2 and AtACA4 were enhanced under salt stress [2+ condition, the expression levels of some banana Ca2+-ATPase genes, such as MaECA4 and MaACA7, in banana root were both the highest, which was consistent with the previous study [MaECA4, MaECA6, MaACA1, MaACA7, MaACA8 and MaACA10 declined under 1 mM and 0 mM Ca2+ concentration. The down-regulation of these banana Ca2+-ATPase genes indicated that they were responsive to low Ca2+ and Ca2+ deficiency conditions.Cad stress . Similart stress . In the us study , while tMusa acuminata var. DH-Pahang were downloaded from the banana genome database . The HMMER software (version 3.0)  [2+-ATPase proteins using the Hidden Markov Model (HMM) file of Cation_ATPase_C (PF00690)  with e-value ≤ 1 × 10−5. The conserved domain database  was applied for the further confirmation of Ca2+-ATPases. The protein length, molecular weight, isoelectric point (pI) and instability index of banana Ca2+-ATPases were predicted using ExPASy ProtoParam . To know whether their encoded proteins contain signal peptide and transmembrane structure or not, SignaIP 3.0 Server  and TMHMM Server v.2.0  was applied, respectively. For the subcellular localization analysis of banana Ca2+-ATPases, Wolf PSORT  was used. For the self- and pairwise-alignment of Ca2+-ATPase proteins, the website for blast from NCBI  was used using e-value ≤ 1 × 10−10 as criterion. MCScanX (version 0.8)  was used for the gene collinear relationship analysis of the Ca2+-ATPase family members [The gDNA, CDS, and protein sequence files of ge, USA)  was appl members . 2+-ATPases, the protein sequences of Arabidopsis and rice Ca2+-ATPases were downloaded from NCBI . MEGA 6.0  [https://www.evolgenius.info/evolview/, accessed on 10 January 2021).For the phylogenetic analysis of Ca, Japan)  were use, Japan)   was used. The conserved motif analysis of banana Ca2+-ATPases was performed using MEME  [2+-ATPase genes and conserved motifs of their encoded proteins were visualized using TBtools software 3.0  [For gene structure analysis of banana Caer 2022) . Further, China) .2+-ATPase genes were extracted from the banana genome database using TBtools software 3.0., PlantTFDB  and PlantCARE  was used to predict the transcription factor binding sites (TFBSs) and the cis-acting elements on promoters under default parameters, respectively. The 2000 bp sequences upstream of the start codon of each banana CaMusa acuminata cv. Tianbaojiao) seedlings. To facilitate the rooting and growth of tissue-cultured seedlings, ten days’ Hoagland solution (with Ca2+ concentration of 4 mM) hardening [Serendipita indica inoculation [−2s−1 of illumination for 12 h per day. Seedlings were fertilized with Hoagland solution (with Ca2+ concentration of 4 mM) twice a week. Uniform and well-growing river sand cultured ‘Tianbaojiao’ banana seedlings at the five-leaf stage were divided into four groups which were watered with Hoagland solution containing 4 mM, 2 mM, 1 mM and 0 mM Ca2+ twice a week [2+ are shown in The plant materials we used in this study are tissue-cultured ‘Tianbaojiao’ banana  and length (referring to the length of the longest taproot of the plant), fresh weight of aboveground part and root were determined at six months post treatment. For each parameter, at least three replications were made. Moreover, inductively coupled plasma atomic emission spectrometry (ICP-MS AES) was applied for the determination of the P, K, Ca, Mg and S contents in leaf, pseudostem and root of banana seedlings from the four groups. To study the influences of different concentrations of Ca2+-ATPase genes were selected from root, corm, pseudostem, and leaf tissues of banana, and generated the plots by the R package pheatmap (version 1.0.12) . To investigate the expression patterns of banana Ca2+-ATPase genes under the treatments of different concentrations of Ca2+, quantitative real time PCR (qRT-PCR) was used. An RNAprep Pure Plant Kit  was used for the isolation of total RNA from the roots of banana seedlings from the four groups. High quality RNA was used as a template for the cDNA synthesis using PrimeScript™ RT reagent Kit with gDNA Eraser   according to the manual. The generated cDNA was diluted fivefold for subsequent experiments. The PCR reaction conditions used were 95 °C for 30 s; 95 °C for 5 s, and 59 °C for 20 s (40 cycles). Relative gene expression levels were determined using the 2−∆∆Ct method by using MaCAC as an internal reference [The TPM values of Caeference . Statist2+-ATPase genes from M. acuminata, including six MaEACs and 12 MaACAs. The expansion of this gene family was predicted to be caused mainly by segmental duplications. The gene structures of the banana Ca2+-ATPase genes from the same subfamily and motif distributions in their encoded proteins were much more similar, and all the banana Ca2+-ATPase proteins were predicted to be plasma membrane located. A gene expression analysis showed that the expression of banana Ca2+-ATPase genes in different organs varied greatly, and most members showed higher expression under 4 mM and 2 mM Ca2+ treatments, but significantly decreased expression under low Ca2+ (1 mM) and Ca2+ deficiency (0 mM) treatments. Therefore, it was concluded that the decreased expression of banana Ca2+-ATPase genes might be closely related to related to the reduced Ca accumulation and plant growth retardation caused by low Ca2+ and Ca2+ deficiency.In this study, we identified 18 Ca


Besides pro-fibrotic and pro-inflammatory effects, Gq-mediated signaling induces inositol trisphosphate receptor (IP3R)-mediated intracellular Ca2+ mobilization related to delayed after-depolarisations and AF. However, direct evidence of arrhythmogenic Gq-mediated signaling is absent. Methods and results: To define the role of Gq in AF, transgenic mice with tamoxifen-inducible, cardiomyocyte-specific Gαq/Gα11-deficiency (Gq-KO) were created and exposed to intracardiac electrophysiological studies. Baseline electrophysiological properties, including heart rate, sinus node recovery time, and atrial as well as AV nodal effective refractory periods, were comparable in Gq-KO and control mice. However, inducibility and mean duration of AF episodes were significantly reduced in Gq-KO mice—both before and after vagal stimulation. To explore underlying mechanisms, left atrial cardiomyocytes were isolated from Gq-KO and control mice and electrically stimulated to study Ca2+-mobilization during excitation–contraction coupling using confocal microscopy. Spontaneous arrhythmogenic Ca2+ waves and sarcoplasmic reticulum content-corrected Ca2+ sparks were less frequent in Gq-KO mice. Interestingly, nuclear but not cytosolic Ca2+ transient amplitudes were significantly decreased in Gq-KO mice. Conclusion: Gq-signaling promotes arrhythmogenic atrial Ca2+-release and AF in mice. Targeting this pathway, ideally using Gq-selective, biased receptor ligands, may be a promising approach for the treatment and prevention of AF. Importantly, the atrial-specific expression of the Gq-effector IP3R confers atrial selectivity mitigating the risk of life-threatening ventricular pro-arrhythmic effects.Background: Atrial fibrillation (AF) is promoted by various stimuli like angiotensin II, endothelin-1, epinephrine/norepinephrine, vagal activation, or mechanical stress, all of which activate receptors coupled to G-proteins of the Gα Atrial fibrillation (AF) is the most common cardiac arrhythmia. As well as causing debilitating symptoms, AF is associated with considerable morbidity and increased mortality . Currentq-family (Gq) defined by the α-subunit isoforms Gαq and Gα11. In fact, pro-arrhythmic effects have been consistently demonstrated for the predominant cardiac Gq-coupled receptors, namely the angiotensin II receptor type 1 (AT1 receptor), the endothelin-1 receptor A (ETA receptor), the M3 muscarinic acetylcholine receptor (M3 receptor) and the alpha-1 adrenergic receptor -related fluorescence was measured using confocal line-scan imaging  [2+]), and Ca2+ transients were elicited using electrical field stimulation (1 Hz). Longitudinal scan lines were chosen, and the cellular nucleus visually identified and included when feasible. Line scan images were used to derive cytosolic and nuclear Ca2+ transient release and removal characteristics  [i in intact paced myocytes are expressed as an F/F0 where F represents cellular Fluo-4 fluorescence and F0 is diastolic Fluo-4 fluorescence. Ca2+ waves and Ca2+ spark frequencies were measured during a resting period upon stop of electrical stimulation at steady-state  and manually quantified in a blinded fashion. Ca2+ wave propagation velocity was measured as previously described [2+ spark parameters were analyzed using the automated ImageJ Plugin SparkMaster [2+ content was assessed in longitudinal line scans after Ca2+ spark/wave measurements using Caffeine (20 mM) evoked transients [2+ transients was performed using ImageJ and Liscana (IDL) [All chemicals and reagents were obtained from Sigma-Aldrich  unless noted otherwise. Tyrode solution contained (in mM): 130 NaCl, 4 KCl, 2 CaCligestion . Cells w 515 nm) . Experimansient) . Changesescribed . Ca2+ sprkMaster . SR Ca2+ansients . Analysina (IDL) .t-test, Kruskal–Wallis One Way ANOVA on Ranks, or Chi-square test (dichotomous variables) was used. A p < 0.05 indicates significant statistical difference between groups.All data are presented as mean ± standard deviation. Data analysis was performed in a blinded fashion with respect to genotypes. GraphPad Prism was used for statistical inference and plotting . To test for group differences, student’s q-mediated signaling in AF in vivo, we used a transgenic mouse line with a tamoxifen-inducible, cardiomyocyte-specific Gαq/Gα11-deficiency (Gq-KO) [q-KO and control mice underwent intracardiac electrophysiological studies using a 2F Octapolar catheter inserted via the right jugular vein. The inducibility of AF was determined before and after carbachol-induced vagal activation using a standardized protocol of programmed electrical stimulation [q/G11-KO vs. control mice, with no significant differences between the two groups before or after carbachol-induced vagal activation (q/G11-KO mice before carbachol administration (q/G11-KO mice (27%) (q/G11-KO (23 ± 16 s) than in control mice (89 ± 14 s).In order to define the role of G (Gq-KO) . Gq-KO amulation . Baselintivation . While Astration A,B. Two ce (27%) C. Moreovq-KO mice to elucidate further the role of the Gq pathway for pro-arrhythmogenic Ca2+ release. During electrical field stimulation, cytosolic Ca2+ transient amplitudes were unchanged upon Gq knockout . Diastolic Ca2+ removal measured by assessment of the time-constant TAU  was also unaltered . However, in Gq-KO, time to 50% of maximal Ca2+ release was significantly shortened . In addition, nuclear Ca2+ release, as assessed by peak F/F0 within the nuclear compartment, was significantly reduced in atrial cardiomyocytes from Gq-KO animals  cellular level in Gq-KO. To obtain subcellular Ca2+ release properties, we quantified individual spontaneous Ca2+ spark characteristics in WT and Gq-KO ; however, when corrected for SR Ca2+ content, Ca2+ sparks occurred less often in Gq-KO at a given Ca2+ content as compared to WT. In addition, with increasing SR Ca2+ content, the observed increase of Ca2+ spark frequency was less pronounced in Gq-KO as compared to WT , the endothelin-1 receptor A (ETA receptor), the M3 muscarinic acetylcholine receptor (M3 receptor), thrombin (PAR) receptors and the alpha-1 adrenergic receptor [Greceptor . In partreceptor .q-mediated profibrotic and proinflammatory signaling [2+ leak and delayed after-depolarisations [While arrhythmogenic effects have in part been attributed to Gignaling ,24, direignaling ,26. In tisations ,25,26.q-mediated signaling but equally block all downstream signaling pathways, some of which may even have beneficial effects. Thus, selective inhibition of Gq-mediated signaling may be desirable. This could be accomplished by biased ligands that act as Gq-selective antagonists. In fact, we have recently identified biased AT1-ligands that selectively inhibit Gq-mediated signaling (unpublished data). This angiotensin analog (TRV027) has proven to be well-tolerated and safe in phase II clinical trials in the context of heart failure. It may thus qualify as a suitable candidate for a Gq-targeting AF therapy [However, while indirect evidence from numerous clinical trials indicated that chronic inhibition of AT1 receptor signaling significantly reduces the incidence of AF, large randomized trials failed to demonstrate the beneficial effects of AT1 antagonists on AF ,28,29. A therapy .q-signaling through G-protein-coupled receptors, we have recently shown that this pathway is also mechanosensitive [While many humoral stimuli can activate Gensitive . Mechaniensitive . Howeverensitive . Howeverq form a functional mechanosignaling complex in endothelial cells that may also be operative in cardiomyocytes [3R-mediated Ca2+-signaling as well as NFκB-mediated proinflammatory signaling in response to mechanical forces—both key processes in the pathogenesis of AF. Thus, it is intriguing to speculate that Gq-mediated mechanotransduction is also involved in the arrhythmogenic mechanoelectrical feedback in the context of AF.Interestingly, we recently demonstrated that Piezo1 and Gmyocytes . This coi-coupled M2 muscarinergic receptors that activate G-protein- gated K+ channels, thereby reducing atrial action potential duration and increasing susceptibility to early after-depolarisations as well as reentrant mechanisms [i-mediated signaling downstream of M2 muscarinic receptors, Gq-coupled M3 muscarinic receptors are also expressed in the atria and appear to mediate arrhythmogenic effects of vagal activation to some extent [2+ events. Thus, in light of the data presented here, the relative contribution of Gi-coupled M2 vs. Gq-coupled M3 receptors to vagally dependent AF may have to be reconsidered.Vagotonic conditions are known to promote AF, and in some patients, AF episodes are clearly vagally dependent ,33. Acetchanisms . Howevere extent ,36. Thes3R-mediated Ca2+ release has been shown to also facilitate SR Ca2+ release via sensitization of nearby RyR clusters [q-KO animals showed decreased spontaneous SR Ca2+ release in support of this notion. Interestingly, cytosolic Ca2+ release during excitation–contraction coupling, i.e., Ca2+ transient amplitude, was not significantly affected by Gq knockout, indicating unaltered baseline Ca2+ signaling. Of note, spontaneous Ca2+ release events were reported to be increased in human atrial cardiomyocytes during chronic AF, and enhanced SR Ca2+ leak has been associated with DADs in this setting [2+ leak, e.g., through genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated (CaMKII) RyR2-S2814 phosphorylation, was shown to delay the development of spontaneous atrial ectopy and fully prevent AF in mice [q-mediated signaling altering SR Ca2+ release might represent another piece of the puzzle of IP3R-dependent SR Ca2+ leak causally linked to the development of AF.IPclusters . Atrial  setting . Normali in mice . Gq-medi3R expression in atrial myocytes is 6- to 10-fold higher than in ventricular myocytes [3R-mediated electrophysiological effects on Ca2+ homeostasis are absent in ventricular myocytes [As the use of all currently approved antiarrhythmic drugs is limited by potentially life-threatening ventricular pro-arrhythmic effects, from a translational perspective, it is intriguing that IPmyocytes ,38, and myocytes .2+ transient amplitudes upon Gq knockout, further underscoring the notion of abundant IP3R expression in the nuclear envelope [2+ by IP3R-upregulation, leading to enhanced IP3R-CaMKII-HDAC4 signaling and L-type calcium current downregulation [2+, mediated via the Gq signaling cascade, might, therefore, directly affect gene regulation important for Ca2+ release and SR Ca2+ leak in the setting of AF.We also report a decrease of nuclear Caenvelope . These fgulation . AlteredThe impact of increased calcium release from the sarcoplasmic reticulum on AF inducibility in vivo may be counterintuitive, as it has primarily been regarded as a mechanism of triggered activity-inducing AF rather than an arrhythmogenic substrate sustaining AF. However, our findings are in line with a number of previous studies in which diastolic calcium leak from the sarcoplasmic reticulum was not associated with triggered activity resulting in spontaneous AF events but with an increased AF inducibility by programmed stimulation in mice ,41. Takeq-deficiency demonstrate that Gq-mediated signal transduction promotes arrhythmogenic Ca2+-release and AF in mice. These data suggest that Gq-signaling likely mediates arrhythmogenic effects of angiotensin II, vagal stimulation, mechanical stress, and other stimuli known to promote AF. Thus, targeting the Gq-pathway, ideally using Gq-selective biased receptor ligands, may be a promising approach for the treatment and prevention of AF. Importantly, the atrial-selective expression of the Gq-effector IP3R confers atrial selectivity mitigating the risk of life-threatening ventricular pro-arrhythmic effects.Our combined in vitro and in vivo studies in mice with cardiomyocyte-specific G


Saccharomyces cerevisiae, as well as in any eukaryotic organism, depend on various transporters and channels located on both the plasma and intracellular membranes. The activity of these proteins is regulated by a number of feedback mechanisms that act through the calmodulin-calcineurin pathway. When exposed to hypotonic shock (HTS), yeast cells respond with an increased cytosolic calcium transient, which seems to be conditioned by the opening of stretch-activated channels. To better understand the role of each channel and transporter involved in the generation and recovery of the calcium transient—and of their feedback regulations—we defined and analyzed a mathematical model of the calcium signaling response to HTS in yeast cells. The model was validated by comparing the simulation outcomes with calcium concentration variations before and during the HTS response, which were observed experimentally in both wild-type and mutant strains. Our results show that calcium normally enters the cell through the High Affinity Calcium influx System and mechanosensitive channels. The increase of the plasma membrane tension, caused by HTS, boosts the opening probability of mechanosensitive channels. This event causes a sudden calcium pulse that is rapidly dissipated by the activity of the vacuolar transporter Pmc1. According to model simulations, the role of another vacuolar transporter, Vcx1, is instead marginal, unless calcineurin is inhibited or removed. Our results also suggest that the mechanosensitive channels are subject to a calcium-dependent feedback inhibition, possibly involving calmodulin. Noteworthy, the model predictions are in accordance with literature results concerning some aspects of calcium homeostasis and signaling that were not specifically addressed within the model itself, suggesting that it actually depicts all the main cellular components and interactions that constitute the HTS calcium pathway, and thus can correctly reproduce the shaping of the calcium signature by calmodulin- and calcineurin-dependent complex regulations. The model predictions also allowed to provide an interpretation of different regulatory schemes involved in calcium handling in both wild-type and mutants yeast strains. The model could be easily extended to represent different calcium signals in other eukaryotic cells.Calcium homeostasis and signaling processes in  HTS consists in a sudden variation of the osmotic pressure, due to a consistent dilution of the solution concentration to which the cell is exposed. Following a HTS, water flows into the cell, causing an increase in cell volume and turgor pressure that might induce the cell burst. To avoid cytolysis, yeasts have evolved mechanisms to sense and respond to HTS, by rapidly triggering at least three different mechanisms: 1) the cytosolic Ca2+ concentration is transiently increased; 2) the osmolyte glycerol is released to the medium in order to relieve osmotic pressure; 3) phospholypase C hydrolizes PIP2 generating the major second messengers, diacylglycerol (DAG) and inositol--tris-phosphate (IP3). In mammalian cells, DAG is well-known as a protein kinase C activator, while IP3 is involved in triggering the release of Ca2+ from intracellular compartments such as endoplasmic reticulum or Golgi  and mechanosensitive channels (MS); 2) the vacuolar transporters Pmc1 and Vcx1; 3) the Ca2+-binding messenger protein calmodulin (CaM), and the Ca2+-CaM-dependant phosphatase calcineurin (CaN). The model is formalised as a system of coupled Ordinary Differential Equations (ODEs), and it can be conceptually divided in two modules: a biophysical module, describing the physical properties of yeast cells , and a biochemical module, describing the changes in the concentration of protein and molecules involved in Ca2+ signaling upon HTS, and including the feedback regulation via the calmodulin-calcineurin pathway.The mathematical model presented in this work was defined by integrating well-established experimental evidences with plausible hypotheses on the functioning of HTS response. The main components of the model comprise: 1) the Ca2+ pulse. The rapid recovery of the basal Ca2+ levels in the cytosol primarily involves Pmc1, differently than in other signalling processes where massive amounts of Ca2+ enter the cytoplasm requiring Vcx1 to engage in the recovery  is composed of Cch1 and Mid1. Cch1 is a homolog of the pore-forming channels , while Malian α1 . Cch1 an+ uptake , store-o2+ entry , ER stre+ uptake , and hyptake -related calcium channels  organelle is critical to its functions; in S. cerevisiae it is maintained at 10 μM  subfamily identified , and calcineurin. Calcineurin is a Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase, and represents the major Ca2+ signaling effector  is a highly conserved and ubiquitous Catrations , which ttrations . The numtrations , whereastrations . During effector . Calcinetor Crz1 , and at ger Vcx1 .2+ channels which mediate a rapid increase of the cytosolic Ca2+ concentration due to both Ca2+ influx and Ca2+ release from intracellular stores, which in turn is often the signal that triggers release of osmolytes, reducing osmotic gradients and helping volume regulation  . In somegulation . In addi2+ increase  can be depleted by activation of phospholipase C (PLC), producing diacylglycerol (DAG) and the diffusible molecule Ins  P3 (IP3), which in mammals is a fundamental signaling molecule. In budding yeast, as in all eukaryotic cells, different phosphoinositide species are generated in a compartment-specific manner and, hence, can be regarded as distinct markers for each organelle  P2 with a mechanism dependent on Plc1 but independent on the extracellular Ca2+ concentration. This process liberates IP3, which is then rapidly phosphorylated in IP6. In addition, another phosphoinositide, PtdIns4P, and is rapidly synthesized and then progressively consumed in the next minutes -P2 could influence the activity of some channels. Flc2 is the best candidate, since it resides on plasma membrane and contains a putative lipid-binding domain. Channels localized on Golgi or ER could also be influenced by changes in phosphoinositides abundances: PtdIns4P transient increase could regulate Golgi channels, since PtdIns4P is the signature lipid of this organelle. Some Ca2+ regulation seems to be at stake in HTS because PLC1 gene deletion, which abolishes PtdIns--P2 depletion and IP6 production, causes a greater increase in calcium influx after HTS compared to a wild type strain  cytosolic Ca2+ pulse  triggerea2+ rise . Moreovechannels . In yeasmembrane .cch1Δ mutants, lacking a functional HACS channel on the plasma membrane, responded to HTS with a higher calcium peak at all [Ca2+]ext considered, suggesting a negative regulatory role for this protein during HTS. Mutants in other known influx pathways, on the other hand, and had a response similar to the wild-type. Calcium levels are affected in calcineurin mutants, suggesting that this Ca2+-dependent effector shapes the calcium signal during HTS through dephosphorylation of target transporters and/or by modulating their long-term expression. In addition to display altered resting calcium levels, mutants lacking calcineurin respond to HTS with a dramatically reduced peak. Flc2 was found to be involved in the HTS-induced calcium response, since deletion of FLC2 increases the initial rate of the Ca2+ increase compared with wild-type, suggesting an inhibitory role of this protein on the channel that is activated by the HTS. Based on the experimental evidences described above, a model is proposed that includes only the essential players in the HTS-induced calcium response. In non signaling conditions, Ca2+ enters the cell through HACS channel and other unidentified influx pathways. A still unidentified mechanosensitive calcium channel is located on the plasma membrane and activated by the increased membrane tension caused by HTS. In addition, this channel appears to be negatively regulated by Flc2. Since the elevation of [Ca2+]cyt is transient, some intracellular transporters must restore the steady-state levels of cytosolic Ca2+. This signal attenuation is probably performed by the Golgi-localized Pmr1, together with vacuolar-localized Pmc1 and Vcx1.The HTS response was also measured for mutants lacking proteins known to be involved in calcium signaling and homeostasis. S. cerevisiae was defined on the basis of available experimental evidences  of mechanosensitive channels was thus formally defined as:P is turgor pressure, PMS is the value of turgor pressure at which Popen is equal to 0.5, and gMS is a slope parameter.Stretch-activated channels were modeled differently: they can be viewed as pores, whose opening probability depends on membrane tension. In particular, the opening probability of mechanosensitive channels was shown to follow a Boltzmann distribution . For thej is then given by:k is a rate parameter and Δc is the Ca2+ concentration gradient across the membrane.Since ions pass through the channel pore down their electrochemical gradient, we assume that calcium ions flow according to their concentration gradient. The calcium flux https://www.ebi.ac.uk/biomodels/MODEL2112030001.The model was simulated with COPASI (version: 4.19) , using tUnknown parameters were estimated with COPASI , using tcyt], measured in different experimental conditions in 2+ concentrations. It is worth mentioning that, in the parameter estimation process, we took into account the well known fact that the intracellular calcium levels are kept in a narrow range despite wide variations in external conditions, thanks to calcium buffering and sensing and feedback mechanisms  can be defined as the sum of an osmotically active volume (water volume) Vos and an osmotically inactive volume (solid volume) Vb:Vb assumed to be constant. The extracellular volume, Vex, is much greater than the volume occupied by the cell. Therefore, for the sake of simplicity, we assume that Vex = V (0) ⋅ 1,000, where V (0) is the initial cell volume.Assuming that volume changes are only due to water flow and not to solute flow, the total cell volume Lp is the hydraulic conductivity (in dm⋅MPa−1⋅s−1), A is the cell surface area (in dm2) and P is the intracellular hydrostatic pressure exerted on the cell wall—i.e., the turgor (in MPa)—which equilibrates ΔΠ under steady-state conditions n and s denote non-permeable and permeable solutes, respectively), while the dimensionless parameter σ is the reflection coefficient, which depends on the solute permeability  of the solute, ks is the membrane solute permeability (in dm⋅s−1), R is the gas constant (in J⋅mol−1⋅K−1) and T is the temperature (in K)  . Since tcPCRTΔc, where Δc is the concentration and cPC is a conversion factor relating concentrations in M to pressures in MPa. Thus, c is substituted with [Osme] − [Osmi], where [Osme] and [Osmi] are extracellular and intracellular concentration (in μmol⋅L−1) of osmotically active molecules, respectively. If P0 and the initial external osmolarity Osme (0) as:The van’t Hoff law can be used to express the osmotic pressure in terms of concentration of osmotically active molecules: ΔΠ = Glye] is the extracellular glycerol concentration. The initial extracellular glycerol concentration ([Glye](0)) is assumed to be 1,000 times lower than the initial intracellular glycerol concentration  = [Glyi](0)/1,000), providing a gradient for glycerol efflux from the cell. All concentrations are expressed in μmol⋅L−1.Both intracellular and extracellular osmolarities are the sum of concentrations of permeable solutes (glycerol) and non-permeable solutes. The extracellular osmolarity is thus:Glyi] is the intracellular glycerol concentration, Vos and Vos(0) are, respectively, the cytosolic volume and the cytosolic volume at t = 0. Concentrations are expressed in μmol⋅L−1, while volumes are expressed in μm3.The intracellular osmolarity, accounting for cell volume variation, is defined as:P is proportional to a relative change in cell volume:ϵ is called volumetric elastic modulus, or Young’s modulus (in MPa).The water potential gradient maintained by all cells across their membrane is balanced by a hydrostatic pressure called turgor. In walled cells, turgor pressure causes the cell membrane to exert a force on the cell wall, which expands due to its elasticity. The elastic-theory of turgor pressure states that the change in turgor pressure V0 as the volume when turgor becomes zero, the turgor pressure can be expressed as a function of volume:The dependence of turgor pressure on volume can be deduced by integration:It is known that glycerol efflux and synthesis is tightly regulated according to environmental conditions . Howeverμmol⋅L−1), d is the diluting factor, t is the simulation time instant (in s), toff (in s) is the instant when dilution is applied, and tm is the mixing time (in s) that regulates the speed of dilution.Hypotonic shock is applied to the cell by diluting the medium with distilled water, thus decreasing its osmolarity. This dilution is modeled as follows:2+ concentration depends only on dilution factor and mixing time :js are Ca2+ fluxes (in nM⋅s−1) across calcium channels and transporters.The rate of change of the Ca2+, while Vcx1 and Pmc1 are responsible for its sequestration into the vacuole. Ca2+ enters the cell through the plasma membrane-located HACS channel and other transporters whose molecular identities are yet unknown  . Indeed, unknown . Here, t2+ in cells challenged with HTS is caused by the opening of a MS channel on the plasma membrane (jMS. The equations describing the fluxes are:Popen is the opening probability of the MS channel (see below), kCch1 and kMS are rate parameters (in s−1), vs. are rate constants (in nM⋅s−1), and all other ks are Michaelis constants (in nM).Experimental evidences strongly suggest that the increase of cytosolic Camembrane . This caP is the cell turgor pressure in MPa (see PMS is the turgor pressure (in MPa) at which Popen is equal to 0.5, and gMS is a slope parameter (in MPa).The opening probability of MS channels follows a Boltzmann distribution . MS chan MPa see , PMS is2+ ions with high cooperativity , CaMt is the total calmodulin concentration (in nM), −4⋅s−1 and s−1, respectively).Inside the cytosol, yeast calmodulin binds three Carativity :CaM+3Ca2+-bound calmodulin. By the law of mass action we can state that:−2⋅s−1 and s−1, respectively).Calcineurin, a protein phosphatase, is activated upon binding with the Ca2+, the signal dissipation observed in the successive seconds must be the result of either an increased activity of one of the intracellular transporters or a feedback inhibition on the MS channels. Since no positive regulation is known for any of the relevant transporters, in the present model the latter mechanism is assumed. This assumption is supported by circumstantial evidence suggesting that the yeast MS channel interacts with a homologous of TRP proteins .Following HTS, calcium ions enter the yeast cell through MS channels located on the plasma membrane. After the initial rise in Caproteins . TRP proproteins . It is aproteins . The fol2+ measurements from HTS experiments conducted by S. cerevisiae cells were grown in YPD medium, which has an estimated osmolarity of 0.26 Osm/L , and the cch1Δ by setting to zero the rate parameter kCch1. However, these parameter settings were not sufficient to reproduce the behavior displayed by the three mutants. Thus, we performed additional parameter estimations exploiting available experimental time traces of the mutant strains  the feedback inhibition on the HACS channel, mediated by calcineurin (b) the influx pathway that was called “IN” . Extended versions of the model could be useful for understanding the role of known channels and transporters, suggesting novel putative regulatory mechanisms and gaining new insights on Ca2+ signaling that can be further investigated experimentally.As extensively described, the simulations of our model are in agreement with the experimental data, suggesting that the cellular components included in the model and their interactions are sufficient to explain the biological data in our hands. The model was defined on the basis of experimental evidences from the literature, and the parameter estimation process was performed against data from both the wild-type and mutant cells, in order to maximise the prediction’s reliability. The model presented in this work could be extended to include the response to several other stimuli known to elicit a calcium signal in yeast . Such ex2+ an essential signaling ion, proteins involved in its homeostasis are increasingly studied as potential targets of antifungal drugs (2+ signaling could be used to predict the outcomes of the inhibition of a particular protein on the calcium physiology of fungal cells, thus helping to identify the best drug targets. Moreover, such models could be used to study in silico the relationships between pathogenic fungi and human cells (2+ signaling is well conserved among fungi (S. cerevisiae would require only slight modifications to describe what happens in other, pathogenic fungi.Being Caal drugs . A modelan cells , accelerng fungi , a modelFinally, as a future development, we plan to perform a large-scale sensitivity analysis of the model, in order to determine the most relevant parameters governing the response to hypotonic shock. Global sensitivity analysis requires a massive amount of independent simulations, which can lead to a huge computational effort . In orde


Ca2+ dynamics have been studied widely in cellular monocultures, yet organs in vivo comprise heterogeneous populations of stem and differentiated cells. Here, we examine Ca2+ dynamics in the adult Drosophila intestine, a self-renewing epithelial organ in which stem cells continuously produce daughters that differentiate into either enteroendocrine cells or enterocytes. Live imaging of whole organs ex vivo reveals that stem-cell daughters adopt strikingly distinct patterns of Ca2+ oscillations after differentiation: enteroendocrine cells exhibit single-cell Ca2+ oscillations, whereas enterocytes exhibit rhythmic, long-range Ca2+ waves. These multicellular waves do not propagate through immature progenitors (stem cells and enteroblasts), of which the oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates Ca2+ oscillations in all cell types – even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. Our findings establish that cells adopt fate-specific modes of Ca2+ dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently.Cytosolic Ca Summary: Live imaging of the Drosophila intestinal epithelium reveals that stem cells and their terminal progeny exhibit distinct, fate-specific Ca2+ oscillations that are paced independently of each other. In exciractions . Large-snetworks .2+ dynamics have been studied widely in cell culture, investigations in tissues and organs – particularly those that are non-excitable – have been more limited. Studies in Drosophila demonstrated intercellular Ca2+ waves that traverse large tissue domains and might depend on actomyosin organization  and quantified the frequency of oscillations.To examine Ca GCaMP6s  and jRCa GCaMP6s , hereaftell type A–C, we eS system  under thprotocol  preprint2+ waves that travel through R3 enterocytes. We also observed Ca2+ dynamics in enterocytes in the posterior region of the midgut, but not consistently. We did not observe any Ca2+ dynamics in the anterior (R2) region, which might reflect a physiological difference in this region of the gut, or which might be a consequence of dissection or ex vivo culture. We used the GAL4 driver midgut expression 1 or mex1 (mex-GAL4) to label enterocytes and focused the remainder of our experiments on R3.The R3 region of the midgut, which is responsible for acid secretion, attracted our attention due to the consistent appearance of rapid, multicellular Ca2+ oscillations in single cells from each cell type over time. Enterocytes in the R3 region are subdivided into acid-secreting copper cells (CCs) and interstitial cells (ICs)  , which wls (ICs) B.2+ dynamics with a mean oscillation frequency of 31±8.9 mHz (s.e.m.) per gut. By comparison, copper cells primarily displayed Ca2+ spikes at a frequency of 5.2±0.9 mHz per gut -expressing] of 15±4.3 mHz per gut, approximately twice that of progenitors , 7.1±2.6 mHz per gut. Finally, the robust Ca2+ oscillations we observed in progenitors are consistent with prior reports , with the longest wave we observed covering approximately five cells. These Ca2+ waves, which exhibited a multitude of dynamic patterns, such as propagating along a single trajectory, splitting and colliding, did not have an obvious point of origin. Multi-enterocyte waves traveled almost exclusively through interstitial cells and rarely through copper cells. GCaMP signals appeared repeatedly in the same cell over time as part of multi-directional waves that travelled through the tissue (Movie 1), individual interstitial cells spiked, on average, every 23 s, leading to an average frequency of 44±1.4 mHz.Unexpectedly, we found long-range Cae midgut A. We obse tissue B. In thi2. Based on the fluorescence intensity, we estimated cell outlines and identified copper cells based on their unique cell shapes and signals  and the signal traveled in both proximal-distal and distal-proximal directions.To assess whether individual waves traveled along recurring paths, we followed GCaMP signals in selected regions of a single midgut A–D, whic signals B; inters signals . The wav2+ oscillations in distinct cell types are coupled, we used the orthogonal driver systems LexA/LexAop . In combination with mex-GAL4, we could visualize Ca2+ dynamics directly in enterocytes and progenitors, simultaneously (Movie 4).To directly test whether CaA/LexAop  and GAL4GAL4/UAS  to expre drivers , in whic2+ dynamics in interstitial cells and progenitors. Characteristic oscillations of ∼24 mHz were observed for interstitial cells in the GCaMP channel and wide signal elevations (20 s) in the RCaMP channel; these were comparable to observations when using a single-driver system . With time lag 0, which corresponds to the Pearson correlation, the enterocyte-to-progenitor cross correlations for the four direct enterocyte-progenitor pairs in the ROIs  have absolute values <0.2 for all four direct pairs , indicating weak correlation. When all non-zero time lags are considered, the cross correlations for direct enterocyte-progenitor pairs never exceed an absolute value of 0.32 . These values do not support the hypothesis that progenitor and enterocyte oscillations are correlated.To quantitatively assess whether Caour ROIs E that coTable S4 reports all the correlation values for all direct and non-direct pairs for time lags up to 12 s.We also considered cross correlations between enterocyte-progenitor pairs in which the enterocyte and the progenitor are in different ROIs. GAL4/UAS and LexA/LexAop systems to simultaneously visualize Ca2+ dynamics in enteroendocrine cells (pros-GAL4>UAS-GCaMP) and progenitors (esg-LexA>LexAOp-jRCaMP). Similar to enterocytes and progenitors, we observed that Ca2+ oscillations in enteroendocrine cells and progenitor cells were independent of each other (Movie 5).We also used the combination of the 2+ signaling is regulated by a major gap junction protein  .Gap junctions are intercellular channels that allow direct transfer of small molecules and ions. Thus, cells sharing gap junctions are electrically coupled. For example, in the regenerative basal layer of the skin epithelium, directed Ca protein  preprintne (CBX)  to the i2+ oscillations for all cell types, but the consequence to cytosolic Ca2+ levels differed depending on the cell type. Although interstitial cells exhibited weaker GCaMP signals with CBX treatment, copper cells exhibited substantially higher GCaMP signals   .2+ dynamics in the midgut of adult Drosophila, we found that differently fated cells in the midgut R3 region exhibited characteristic patterns of Ca2+ oscillations. Performing live imaging of midguts ex vivo, we identified propagation of Ca2+ waves through networks of interstitial cells and characterized Ca2+ oscillations in enteroendocrine cells and progenitors. Employing orthogonal expression of red and green Ca2+ sensors, we demonstrated that the Ca2+ dynamics of enterocytes and progenitors are paced independently of one another. We also found that the Ca2+ dynamics of progenitors and enteroendocrine cells are paced independently. Our work demonstrated that whole-organ inhibition of gap junctions eliminated dynamic Ca2+ responses in all three cell types and led to an accumulation of Ca2+ ions in copper cells. Thus, gap junction inhibition potentially disrupts organ-scale Ca2+ homeostasis.Examining Ca2+ dynamics that differ from their mother cells and from each other. This finding implies that as stem-cell progeny undergo differentiation, they adopt fate-specific modes of Ca2+ pacing. Ca2+ signal integration remains an intriguing open question due to its pleiotropic and ubiquitous nature. One possibility is that unique oscillatory Ca2+ patterns are interpreted by downstream effectors to subsequently activate different cellular processes; another is that the Ca2+ dynamics support the physiological functions of the specific cell types.An intriguing aspect of our results is that differentiated cell types in the same epithelium, despite being derived from the same progenitor cell population, exhibit Ca2+ oscillations in stem cells can influence division rate  and, to a lesser extent, innexin2 (Inx2)  is the predominant innexin in progenitor and enteroendocrine cells  ; single-ne cells . Future 2+ dynamics within a heterogeneous population of stem and differentiated cells in an adult tissue. Our observations lay the groundwork for using this highly tractable genetic model to investigate the roles of spatiotemporal Ca2+ changes within signal transduction, organ renewal and stem cell differentiation.In summary, our findings establish a novel model for studying cell-type-specific Ca20XUAS-IVS-NES-jRCaMP1b-p10 (BL63793), 20XUAS-IVS-GCaMP6s (BL42746), 13XLexAop2-IVS-NES-jRCaMP1b-p10 (BL64428) and P{ST.lexA::HG}SJH-1  from the Bloomington Stock Center, Indiana University, IN, USA. esg-GAL4 was obtained from the Department of Drosophila Genomics and Genetic Resources (DGGR), Kyoto Institute of Technology, Japan. The following stocks were gifts: mex-GAL4 , esg-GFP[KI]/CyO  and pros-GAL4   . The comFlies were fed a diet of standard cornmeal molasses food at 25°C or room temperature. Flies were collected 0–24 h post eclosion, placed in vials with males and shifted to 25°C with 12 h light on and 12 h light off. The flies were fed a diet of standard cornmeal molasses food supplemented with yeast paste  and the food vials were changed every 1–3 days. Experiments were performed on female flies, 4–7 days post eclosion.esg-LexA, esg-LexA>LexAop-jRCaMP1b was co-expressed with esg-GAL4>UAS-his2b::CFP and esg-GFP in two separate experiments. Comparison between esg-LexA>LexAop-jRCaMP1b and esg-GAL4>UAS-his2b::CFP has shown that esg-LexA>LexAop-jRCaMP1b almost always colocalizes with esg-GAL4>UAS-his2b::CFP . In general, esg-LexA>LexAop-jRCaMP1b exhibits a weaker signal. esg-LexA>LexAop-jRCaMP1b also colocalized with esg-GFP[KI] (esg-GFP) and similarly, the signal is stronger in esg-GFP  and then bathed in whole-organ ex vivo culture medium with 10 µg/ml isradipine  to decrease muscle contractions. The guts were then transferred in a droplet to #1.5 coverslips coated with poly-L-lysine  with 120 µm spacers  and sealed with 250 µm-thick polydimethylsiloxane sheets . The assembled view of the ex vivo midgut mount for inverted microscopy is illustrated in Fig. S3.Female flies were briefly anaesthetized on carbon dioxide and then placed on ice in Eppendorf tubes to induce a chill coma. Guts were dissected in room temperature in adult hemolymph-like (AHL) medium  and the tissues were incubated in this medium for 15 min prior to imaging.For experiments with the gap junction inhibitor, carbenoxolone  was added to the ex vivo midgut culture was used as previously described  BioImage Archive under the accession number S-BSST849. Movies were captured with cycle time of 4.7 s or faster. Movies were included in data analysis . Confocal stacks were acquired with a analysis E only ifz-stacks into maximum projection movies in Fiji. We identified cells of interest manually and then tracked the mean fluorescence intensity of cells using the Active Contours (min) and peaks were identified using custom codes in MATLAB 2019b (available upon request) with the aid of the Signal Processing Toolbox. Peaks were manually verified.Movie stacks were imported into Fiji  using thContours  plugin iContours . We plotTables S2 and S3, per midgut. The average oscillations are also presented if only cells exhibiting at least one oscillation are included in the analysis.The average oscillations in mHz for the different cell types are presented in i and pi stand for the enterocyte and progenitor intensity at timepoint i, respectively, and h is the time lag. The quantities µE and µp refer to the mean intensity of enterocytes and progenitors over the entire time series. We use the time series data presented in To calculate cross correlations between enterocyte (EC) and progenitor time series data, we used the following formula for cross correlations between two time series with time lag h:


Ischemic heart disease (IHD) is a high-risk disease in the middle-aged and elderly population. The ischemic heart may be further damaged after reperfusion therapy with percutaneous coronary intervention (PCI) and other methods, namely, myocardial ischemia–reperfusion injury (MIRI), which further affects revascularization and hinders patient rehabilitation. Therefore, the investigation of new therapies against MIRI has drawn great global attention. Within the long history of the prevention and treatment of MIRI, traditional Chinese medicine (TCM) has increasingly been recognized by the scientific community for its multi-component and multi-target effects. These multi-target effects provide a conspicuous advantage to the anti-MIRI of TCM to overcome the shortcomings of single-component drugs, thereby pointing toward a novel avenue for the treatment of MIRI. However, very few reviews have summarized the currently available anti-MIRI of TCM. Therefore, a systematic data mining of TCM for protecting against MIRI will certainly accelerate the processes of drug discovery and help to identify safe candidates with synergistic formulations. The present review aims to describe TCM-based research in MIRI treatment through electronic retrieval of articles, patents, and ethnopharmacology documents. This review reported the progress of research on the active ingredients, efficacy, and underlying mechanism of anti-MIRI in TCM and TCM formulas, provided scientific support to the clinical use of TCM in the treatment of MIRI, and revealed the corresponding clinical significance and development prospects of TCM in treating MIRI. Cardiovascular disease (CVD) is one of the leading causes of death worldwide. As the most common CVD, ischemic heart disease (IHD) has the highest morbidity and mortality rate . Therefo+/H+ and Na+/Ca2+; and using adenosine receptor agonists, magnesium, statins, and angiotensin receptor antagonists to attenuate MIRI. However, acting on a single aspect is considered the main cause of unsatisfactory therapeutic outcomes of the aforementioned medicines. In contrast, traditional Chinese medicine (TCM) potentially offers a novel avenue for MIRI treatments based on their advantages of being multi-component and multi-target.The mechanism of MIRI is linked to multiple factors, and so far, research has mainly focused on oxidative stress , inflammSalvia miltiorrhiza Bge. (or “Danshen”) (Panax notoginseng (or “Sanqi”)   and Pana“Sanqi”) , which hBlood reperfusion is an essential process during the treatment of myocardial ischemia, but it may also further aggravate damage or destroy cardiac function, inducing severe arrhythmias or even leading to sudden death . The sevReperfusion therapy improves myocardial blood supply, but it is accompanied by a complex series of pathophysiological responses, such as oxidative stress, inflammatory response, calcium overload, and mitochondrial dysfunction, of which the key driving factor that causes reperfusion injury is oxidative stress .Oxidative stress is a consequence of imbalanced redox systems in the body, which lead to the accumulation of oxygen radicals and a decrease in the activity of antioxidant enzymes, triggering lipid peroxidation and causing cell damage. During myocardial ischemia, there is an insufficient supply of oxygen and ATP cannot be supplied adequately, impairing the ability of the myocardium to clear free radicals. When blood supply is restored by reperfusion, the large accumulation of oxygen radicals leads to lipid peroxidation, producing malondialdehyde (MDA). MDA triggers cytotoxicity and affects the mitochondrial respiratory chain complex and key enzyme activities within the mitochondria . In addi2+) are intracellular secondary messengers involved in maintaining cellular physiological functions. Ca2+ overload is both a result of myocardial damage and a cause of further damage. Upon myocardial ischemia, the myocardial membrane structure is damaged, and the membrane becomes more permeable to Ca2+. Increasing entry of Ca2+ from the extracellular space, following a concentration gradient, leads to myocardial Ca2+ overload. Cells switch from aerobic to anaerobic respiration during myocardial ischemia, which increases lactate production and decreases intracellular pH. Activation of the Na+–H+ exchanger leads to a marked increase in intracellular Na+ concentration. Reperfusion rapidly restores extracellular pH, resulting in a significant difference in intra- and extracellular pH, causing a large inward flow of Na+ through the Na+–H+ exchanger and an excess of intracellular Na+, triggering a reversal of the 2Na+–Ca2+ exchanger, and resulting in intracellular Ca2+ overload  and creatine phosphate (CP) in the myocardium, resulting in an excess of non-phosphorylated purines in cardiomyocytes. The non-phosphorylated purines enter blood vessels and subsequently block the generation of ATP . After rThe inflammatory response is also an important part of MIRI. The membrane structures of myocardial endothelial tissue are damaged during MIRI and attract large numbers of neutrophils into the tissue or adhere to the myocardial vascular endothelium. The accumulation of neutrophils can lead to the release of IL-6, TNF-α, and IL-1β, causing an inflammatory response and inducing microcirculatory disturbances, which can lead to myocardial injury .Cell death is also a consequence of ischemia–reperfusion injury during MIRI. Cell death pathways include autophagy, ferroptosis, pyroptosis, and apoptosis, which are regulated by a variety of signaling pathways that affect the state of the cell .Autophagy is a process of phagocytosis and degradation that maintains cell homeostasis. It is a key regulator of ischemia/reperfusion (I/R) injury and is believed to play important roles in the heart during I/R, which can be activated by several stimuli including nutrient starvation, glucose deprivation, caloric restriction, oxidative stress, and brief episodes of ischemia and reperfusion to eliminate damaged mitochondria. Autophagy prevents damaged mitochondria from releasing harmful ROS, decreases apoptosis, and helps maintain mitochondrial homeostasis and cell life activities through the related pathways, mediated by the mammalian rapamycin target protein (mTOR) . Under t2+ overload during reperfusion promotes CaMKII, which phosphorylates Beclin 1 at Ser90, subsequently promoting the ubiquitination of Beclin 1 at the K63 site to cause excessive autophagy and cell death  or gasdermin E (GSDME). ROS could activate the NOD-like receptor protein 3 (NLRP3) inflammasome and then induce the activation of caspase enzymes, which is an important process of MIRI-mediated pyroptosis . On the Ferroptosis is an iron-dependent, non-apoptotic form of cell death that is characterized by iron overload and lipid peroxidation. Ferroptosis is critical for the pathogenesis of MIRI. In the transplant model, ferrostatin-1 (FER-1) has been found to protect the heart from MIRI by reducing the levels of hydrogen peroxide-arachidonic acid-phosphatidylethanolamine, which is an intermediate product that mediates iron death; in this way, FER-1 reduces the death of cardiomyocytes and myocardial fibroblasts. At the same time, iron death specifically promotes the adhesion recruitment of neutrophils to coronary endothelial cells through the TLR4/TRIF/IFN-1 signaling pathway, which aggravates heart damage . Meanwhi“Myocardial ischemia-reperfusion injury” is not defined in the theory of Chinese medicine, but based on its lesion location  and clinical manifestations , MIRI is classified into “chest paralysis,” “heartache,” “palpitation,” and other categories . ChineseSome scholars believe that the etiology and pathogenesis of acute myocardial ischemia are caused by the organ’s dysfunction and qi–blood–body fluid generation, resulting in the turbidity of phlegm. Phlegm turbidity directly attached to the vein wall, resulting in abnormal qi–blood circulation, blood stasis, phlegm–blood stasis, and heart vessel blockage stasis, hinders the operation of meridians and collaterals and thus causes microcirculation disorders . After sThe blood and oxygen supply are cut off during myocardial ischemia, which leads to myocardial dystrophy and causes varying degrees of insufficiency of the heart qi. The relationship between qi and blood is one of mutual sustenance, interdependence, and mutual use, and blood has the role of moistening and carrying qi.If there is insufficiency of the heart qi for a long time, blood vessels may show restenosis symptoms due to poor blood flow and venous stasis. Suddenly, large amounts of blood await delivery to distal tissues during reperfusion, instead increasing the burden on the heart, depleting the qi of the heart and leading to reperfusion injury . Qi defiThe myocardial tissue will lead to myocardial qi deficiency due to ischemia and hypoxia; however, qi deficiency of the heart results in slow blood flow. Therefore, after reperfusion, the heart is unable to deliver blood quickly to the distal tissues for use, resulting in blood stasis in the distal vessels. In this case, the myocardium becomes deficient in qi and blood, which is called “pulse knotting and palpitations” in Chinese medicine, just like reperfusion arrhythmia in Western medicine .TCM plays an important and indispensable role in preventing and treating MIRI. Some Chinese patent drugs have shown significant myocardial protection in MIRI. The chemical components of TCM are structurally diverse and have multiple pharmacological effects. Single Chinese herbs and their chemical constituents, TCM formulas, and TCM-based prescriptions may have therapeutic efficacy against MIRI ,2. ClassSalvia miltiorrhiza, also known as “Danshen” in China, was primarily recorded in “Shen Nong Ben Cao Jing.” The Chinese traditional herb “Danshen” refers to the dry roots and rhizomes of Salvia miltiorrhiza Bge., which is famous for its effects in promoting blood circulation and removing blood stasis; it has been extensively applied for many years to treat various diseases, especially coronary heart diseases and cerebrovascular diseases, either alone or in combination with other Chinese plant-based medicines , which is achieved by providing reducing equivalents to scavenge ROS  is present in relatively higher concentrations and is one of the most effective components being applied to CVD treatment. Many studies have provided evidence for the remarkable effect of TA on MIRI. For example, TA has been reported to improve cardiac function by inhibiting collagen I, collagen III, tissue inhibitors of metalloproteinases (TIMP-1), and galectin-3 mRNA expression and enhancing matrix metallopeptidase (MMP)-2 mRNA and protein expression to inhibit myocardial fibrosis and improve myocardial remodeling in rats with I/R-induced heart failure . Some st pathway . Moreove pathway . Dihydroenge ROS . Furtherenge ROS .Other components of Danshen have also been found to effectively treat MIRI. Salvianolate is a class of hydrophilic components, among which magnesium lithospermate B is a prominent one and has been shown to improve cardiac function. Salvianolate was reported to significantly inhibit cardiomyocyte apoptosis by regulating apoptosis-related proteins such as Bcl-2, Bax and active caspase-3, indicating the potential therapeutic ability of salvianolate against MIRI . MagnesiIn addition, the polysaccharides of Danshen have a variety of biological activities such as anti-inflammatory, antioxidant, and immunomodulatory actions . HoweverPanax notoginseng, or Sanqi, was first discovered and recorded in ancient China. Sanqi was conventionally used for stopping bleeding, promoting blood circulation, and pain relief, as recorded in “Ben Cao Gang Mu.” Modern studies have suggested that Sanqi is widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases, owing to its significant effects on cerebrovascular dilation, vessel resistance reduction, and vessel endothelial function improvement.It has been more than 600 years since Panax notoginseng saponins (PNS) are one of the major active components in Sanqi. It has been reported that PNS can effectively improve MIRI by reducing oxidative stress, reducing calcium overload, suppressing neutrophil activation and adherence, improving metabolism, increasing anti-apoptotic activity, and modifying endothelial functions of blood vessels  has been increasingly recognized to benefit the heart. Rg1 (5 mg/kg/h) could prevent myocardial injury caused by I/R, and the mechanism may be related to inhibiting myocardial apoptosis and regulating energy metabolism  pathway . The com pathway . More sp pathway . Wang et factors . As mostAstragalus membranaceus is a flowering medicinal plant. “Huangqi,” in China, is specifically referred to as the dried root of Astragalus membranaceus (Fisch.) Bge. Modern studies have confirmed that astragalus has a series of effects attributed to its rich chemical composition, including neuroprotection, anti-fatigue, renal protection, hepatoprotection, anti-cancer, and cardiac protection  are another main active ingredient of Astragalus membranaceus. ASP could reduce intracellular ROS and inhibit apoptosis, and these effects were achieved by the downregulation of the expressions of caspase-3 and Bax and the upregulation of the expression of Bcl-2 both in vivo and in vitro  and hanbaicalin and their aglycones, which are baicalein and wogonin (WG), respectively  is associated with inflammation-related pathways such as signaling through MAPK, Akt, NF-κB, and JAK-STAT . HoweverRheum palmatum L., Rheum tanguticum Maxim. ex Balf., or Rheum offcinale Baill. and has also been shown to have a protective effect on the heart. Emodin (Emo) is an anthraquinone compound extracted from Rheum, the root rhizome of which was primarily used to treat constipation and other gastrointestinal problems. Pharmacological properties of Emo have been demonstrated as anti-inflammation, platelet aggregation inhibition, immune regulation, and anti-tumor  (Paeonol (Pae) is a bioactive compound derived from the root bark of the Moutan Cortex. Studies have demonstrated the outstanding antioxidant effect of Pae in multiple pathogenetic processes, implying that Pae is a potential option for treating oxidative stress-induced diseases. A study by  (SIRT1) . SIRT1 i (SIRT1) . In the An intravenous infusion of Pae 15 min before a ligation operation on rats was found to improve the cardiac function in I/R-injured myocardium and reduce cardiac I/R-induced arrhythmias and mortality . Pae inhHypericum perforatum L. Flavonoid compounds have been proven to contribute to the treatment of CVD (Hyperoside (Hyp) is a kind of flavonoid compound extracted from many plants, such as t of CVD . Some ret of CVD . Recentlt of CVD . Numerou2+ overload and inhibiting apoptosis  . Subtypepoptosis . Wang suErigeron breviscapus (vant.) Hand-Mazz. A pharmacological study has shown that Bre not only has beneficial effects on cerebrovascular vessels but also inhibits platelet aggregation and oxygen radicals (Breviscapine (Bre) is a flavonoid compound extracted from the plant radicals . The sturadicals . mTOR isradicals , which wCoptis chinensis (“Huanglian”). Ber exhibits anti-inflammatory, antibacterial, and anti-tumor activities  is a kind of alkaloid extracted from various kinds of herbs, including tivities . The carpression .Crataegus pinnatifida. According to modern pharmacology, generally, flavonoid compounds extracted from Crataegus pinnatifida leaves have certain benefits such as increasing coronary blood flow, preventing myocardial ischemia, and exhibiting antioxidant properties (in vivo and H/R injury in vitro (Vitexin (VT) is a bioactive flavonoid component of dried leaves from operties . It has operties . More imin vitro . Accordiin vitro .Rhodiola rosea , Xianhecao (Agrimonia pilosa Ledeb.), and Hongjingtian (Rhodiola crenulata). In addition to Danshen and Sanqi , the other three herbs also show outstanding cardioprotective effects against MIRI. Psoralen is the major bioactive component of Ficus simplicissima Lour. and contributes to anti-coagulation and immunoregulation. Agrimonia pilosa Ledeb. has been proved to show great anti-inflammatory effect. Rhodiola crenulata is a well-known herb with an antioxidant property ] and Wuzhaolong (the root or branch of Ficus hirta Vahl.), respectively. The new formulation was found to alleviate MIRI in laboratory conditions, which was consistent with clinical observations, and the underlying mechanism was considered to be linked to an increase in SOD activity and a decrease in TNF-α and MMP-9, underpinning the antioxidant and anti-inflammatory effects of Yiqi Huayu decoction  and ventricular fibrillation (VF) in the Huanglian Jiedu decoction group were significantly lower; at the same time, the MDA concentration was significantly decreased, and the SOD activity and NO concentration were significantly increased .Furthermore, Shenmai injection , Si-MiaoAccording to TCM theory, the main pathological mechanisms of MIRI are mainly qi deficiency, blood stasis, and phlegm turbidity. Therefore, herbs and compound prescriptions, with the effects of benefiting qi and nourishing Yin, activating blood stasis, dispelling phlegm, and dispersing nodules, are commonly used in clinical practice to prevent and treat MIRI; good therapeutic effects have been achieved. As research on Chinese herbal medicines and compound drugs continues to deepen, not only will the main active components that exhibit myocardial protective effects be discovered, but also the main mechanisms of myocardial protection, including scavenging of oxygen free radicals, anti-oxidation, inhibition of the inflammatory response, and inhibition of the apoptosis of cardiomyocytes. Whether single herbal medicine, a group formula, or an extracted component of a certain herbal medicine, research on multi-pathway, multi-system, and multi-target effects of Chinese herbal medicines is still insufficient. In addition, there is a gap between animal models and clinical practice, and there are few models of TCM evidence; the application of TCM lacks the guidance of the discriminatory treatment system and cannot reflect the advantages of discriminatory treatment. Therefore, it is important to combine modern medicine and technology with traditional Chinese medicine theories of diagnosis and treatment to explore pathological mechanisms of Chinese medicine in order to prevent MIRI and innovate Chinese medicine prescriptions and medications.Salvia miltiorrhiae, Panax notoginseng, and Scutellaria Baicalensis have been highlighted for their efficacy in alleviating myocardial injury through anti-oxidative, anti-inflammatory, anti-apoptotic, and autophagy regulatory effects via the Notch signaling pathway, the JAK2/STAT3 pathway, the PINK/Parkin pathway, and the PI3K/Akt pathway. TCM formulas have been widely used in the clinical treatment of a variety of CVD by adding and subtracting medicine components to achieve disease prevention.As reviewed above, it could be concluded that TCM has made great contributions to the treatment of MIRI. Their benefits in promoting blood circulation and removing stasis, clearing heat, and detoxification are key aspects against MIRI. CVD treatment has been a hot topic worldwide that has drawn great interest for decades. Reperfusion injury is a key pathological process in many cardiac diseases and remains a major problem in the medical field. Though a range of Western chemical drugs have been proposed to potently target the causes of CVD, undoubtedly, some chemical drugs can cause severe side effects, and the uses of some of these drugs are also limited by their high costs. In contrast to chemical drugs, TCM provides novel approaches, targeting MIRI due to their characteristics of multi-component, multi-target, and fewer side effects. In particular, the complex chemical composition of TCM formulas allows a single herb or formula to target multiple signaling pathways, highlighting that the components can improve cardiac functions synergistically. The unique advantages of TCM leave it a great space for development. For example, the additive beneficial effects of the combined treatment of TCM and Western medicine are worth further exploration. The beneficial effects are expected to be more remarkable if the dosages of TCM and chemical drugs are well balanced.Despite the advantages of TCM in treating MIRI, evaluation of the efficacy and safety of herbs and formulas still lacks a systematic method; therefore, advanced technology for TCM assessment is expected to be developed using a variety of research models. Establishing global criteria for evaluating the performances of TCM contributes to popularizing TCM worldwide. Generally speaking, further research on TCM ingredient pharmacokinetics and pharmacodynamics and the most optimal dosages is necessary to provide a clearer blueprint for future TCM development, encouraging the production of more TCM-based treatments targeting MIRI.


Central diabetes insipidus (CDI) is an uncommon complication of acute myeloid leukemia (AML). Patients present with polyuria either preceding or at the time of diagnosis of AML. We describe the case of a 36-year-old male who presented with a subacute onset of polyuria, polydipsia, nocturia, and fatigue. After an extensive workup, he was diagnosed with AML/CMML (chronic myelomonocytic leukemia) with a normal karyotype with DNMT3A, CBFB, and PTPN11 mutations. Further workup of the polyuria with a water deprivation test helped confirm the diagnosis of CDI along with MRI findings suggestive of hypophysitis. In this report, we analyze the clinical workup for AML and CDI and report the possibility of extramedullary leukemic infiltration associated with DNMT3A mutation, which has been reported as one of the mechanisms in the existing literature. We also discuss other theories hypothesized to cause CDI in AML patients with abnormal karyotypes. Our patient progressed to AML from CMML-2 after a cycle of decitabine, with confirmed gingival and presumed central nervous system (CNS) involvement. He is in minimal residual disease (MRD)-negative complete remission after induction with a CNS-active acute lymphoblastic leukemia-2 regimen. He also received double umbilical cord blood transplantation, conditioned with cyclophosphamide, fludarabine, thiotepa, and total body irradiation (TBI) of 4 Gy. This was complicated by engraftment syndrome for which he is currently being managed. CDI of the patient was corrected by desmopressin administration. Pituitary involvement has been reported in 0.6% of leukemia cases; however, because pituitary involvement and the presence of diabetes insipidus are not necessarily correlated, the true prevalence of leukemia-associated central diabetes insipidus is unknown . DiabeteμL with 32% neutrophils and 17% blasts in peripheral blood), anemia (hemoglobin: 8.9 g/dl), and a normal platelet count. He was admitted and started on hydroxyurea 500 mg daily, and allopurinol 100 mg daily. A bone marrow aspirate (BMA) and biopsy were performed, and he was discharged on the same day. However, because of increased bleeding from the bone marrow site, he returned to the hospital the day after. The BMA/biopsy results were consistent with chronic myelomonocytic leukemia (CMML-2)/acute myeloid leukemia (AML) with normal karyotype with DNMT3A, CBFB, and PTPN11 mutations. After the course of hydroxyurea, the patient was started on decitabine. He also received continuous intravenous hydration with isotonic fluids and was noted to have a urine output amounting to 12–16 litres per day. The polyuria and polydipsia in conjunction with hypernatremia raised concerns for DI. At baseline, he had a serum osmolality of 297 mOsm/kg, a urine osmolality of 99 mOsm/kg, and the urine sodium was less than 20 mEq/L. The patient underwent a water deprivation test along with administration of one dose of desmopressin acetate (0.05 mg) following which his urine osmolality was found to be 260 mOsm/kg establishing a diagnosis of CDI. Further workup with an MRI (magnetic resonance imaging) of the brain showed focal nodular enhancement at the proximal pituitary infundibulum with a measured value of 0.4 cm with slight effacement of the infundibular recess. The pituitary gland also demonstrated heterogeneous enhancement, and these findings were collectively suggestive of hypophysitis. Additionally, there was a loss of the normal posterior pituitary high-intensity signal on a pre-enhanced T1WI which reflects decreased storage of vasopressin that has a T1-shortening effect in patients with CDI. He was treated with desmopressin and symptomatically improved in the coming months. His urine output during his initial inpatient admission amounted to almost 13-14 liters/day (while receiving maintenance intravenous fluids). With desmopressin, fluid restriction, and diligent follow-up by endocrinologists, his urine output decreased to 5-6 liters/day. He reported improvement in nocturia and polydipsia with the treatments. He also received pituitary radiation for secondary prophylaxis.This is the case of a 36-year-old healthy male who presented with subacute onset of excessive fatigue, polydipsia, polyuria associated with nocturia, and cold intolerance. On initial workup, his urinalysis was normal with no signs of infection. His complete blood count was significant for leukocytosis , exposure to ionizing radiation, chemotherapeutic agents , certain RNA retroviruses, and secondary AML in the context of a prior history of myelodysplastic syndromes [The combination of AML and DI is unique, and the diagnosis of DI is mostly made either before the diagnosis of AML or at the time of diagnosis of AML , 6. DI wTypically, a bright spot is seen on T1-weighted MRI, due to vasopressin that is stored in the posterior pituitary. It is not necessary for all the patients with central diabetes insipidus (CDI) and AML to have MRI findings; however, patients can have a loss of the neurohypophysis (posterior pituitary) bright spot, which is suggestive of hypothalamic-pituitary dysfunction . AlthougAccording to the report of Müller et al, out of 18 cases that were evaluated cytogenetically, 89% had abnormal chromosome 7, 44% had abnormal chromosome 3, and 39% had abnormalities of both chromosome 3 and 7 .It is hypothesized that monosomy 7 may lead to abnormal expression of the neutrophil migration gene which causes neutrophil chemotaxis, in turn causing infiltration of the posterior pituitary leading to DI , 9. FurtBased on a study, 90% of ADH is bound to platelets, and only 10% is free in the plasma. This is consequential to ADH uptake by platelets from plasma , 11, 12.A study by Xu et al. explored the possibility of extramedullary leukemic infiltration mediated by TWIST1 in patients with DNMT3A mutation (which was seen in our patient); however, a definite causal relationship is still unclear .Cytogenetic abnormalities can be detected in approximately 50% to 60% of newly diagnosed AML . ClinicaAnalogously, CDI with AML is an unfavorable association and some reports have documented poor clinical outcomes , 6, 16. From a leukemia standpoint, he progressed to AML from CMML-2 after a cycle of decitabine, with confirmed gingival and presumed central nervous system (CNS) involvement. He is in minimal residual disease (MRD)-negative complete remission after induction with a CNS-active acute lymphoblastic leukemia-2 regimen with high dose cytarabine, mitoxantrone, and intrathecal methotrexate. Most recently, he received double umbilical cord blood transplantation, conditioned with cyclophosphamide, fludarabine, thiotepa, and total body irradiation (TBI) of 4 Gy. This was complicated by engraftment syndrome for which he is currently being managed.Endocrine manifestations can be seen in patients with hematologic malignancies. They can be a direct consequence of the malignancy or because of certain chemotherapeutic agents. Their recognition is crucial for prompt management. CDI can be one of the manifestations of AML and carries both therapeutic and prognostic implications. CDI can be managed with desmopressin as seen with our patient. The role of intrathecal chemotherapy and radiation therapy in the pituitary needs to be explored further in adult patients with this presentation.