As our understanding of the biologic basis of acute myeloid leukemia evolves, so do the classification systems used to describe this group of cancers. Early classification systems focused on the morphologic features of blasts and other cell populations; however, the explosion in genomic technologies has led to rapid growth in our understanding of these diseases and thus the refinement of classification systems. Recently, two new systems, the International Consensus Classification system and the 5th edition of the World Health Organization classification of tumors of hematopoietic and lymphoid tissues, were published to incorporate the latest genomic advances in blood cancer. This article reviews the major updates in acute myeloid leukemia in both systems and highlights the biologic insights that have driven these changes. Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies characterized by a proliferation of immature cells (blasts). Early classification systems were based primarily on the morphologic features of these blasts, while in the last two decades, the predominant classification systems by the World Health Organization (WHO) have increasingly incorporated immunophenotypic and genetic characteristics to refine these groupings. The last major update to this classification occurred in 2016 with the revised 4th edition of the WHO classification of tumors of hematopoietic and lymphoid tissues [The revised 4th edition WHO classification system (WHO4R) included several genetically defined subtypes of AML, specifically “AML with recurrent genetic abnormalities (AML-RGA)”, a subset of “AML with myelodysplasia-related changes (AML-MRC)”, and “AML with germline predisposition” . Since tRUNX1 mutation. Throughout this article, the ICC classification will be listed before the WHO5 classification based on alphabetical order alone and is not meant to suggest preferential endorsement of either system.These two classifications, the 5th edition of the WHO (WHO5) and the ICC, retain much of the overall structure of the prior AML genetic subtypes, with the persistence of the above three categories based on (1) recurrent genetic abnormalities, (2) myelodysplasia-associated genetic changes, albeit both new classifications recognize specific mutations as myelodysplasia-related, and (3) germline predisposition. While these classifications maintain the same structure as the prior, they include additions, refinements, and removals of entities present in the prior system. This article will review the major updates in the genetically defined groups of AML and discuss the underlying biology of these specifically updated entities. The specific changes to blast thresholds described in each system are outside the scope of this article but are briefly discussed for cases with the AML with defining genetic abnormalities (AML-DGA)”. Within AML-DGA, the WHO5 also includes a placeholder category, “AML with other defined alterations”. The specific entities within these systems are elaborated in The WHO4R system identified a group of AMLs defined by specific gene abnormalities as “AML with recurrent genetic abnormalities (AML-RGA)”. ICC retains this name for the group, while WHO5 renames the group “RUNX1 gene encodes a core binding factor transcription factor that is essential to hematopoietic differentiation and is frequently altered in hematopoietic neoplasms by both germline and somatic alterations, including point mutations and translocation [RUNX1” as the authors felt this alteration did not define a significantly distinct disease entity given the potential for misclassification of “AML with germline predisposition (RUNX1 mutation)” and the close association with progression from myelodysplastic syndromes (MDS) and prior chemoradiotherapy [RUNX1 mutations as myelodysplasia-related in AML, and therefore, cases of myeloid neoplasms with ≥10–19% blasts in the bone marrow are defined as MDS/AML, while cases with ≥20% blasts are defined as AML with myelodysplasia-related gene mutations (RUNX1). The WHO5 would classify cases with ≥20% blasts as AML and provide subtyping based on differentiation in the absence of other specific genetic alterations.The location . Both neotherapy ,6,7,8. TPML::RARA”, “AML with t /MLLT3::KMT2A”, and “AML with inv(3) (q21.3q26.2) or t /GATA2; MECOM(EVI1)” as distinct entities; and also recognizes other leukemias with similar biology but driven by variant fusion partners as “APL with other RARA rearrangements”, “AML with other KMT2A rearrangements”, and “AML with other MECOM rearrangements” [MLLT3::KMT2A” and “AML with inv(3) (q21.3q26.2) or t /GATA2; MECOM(EVI1)” with the more generic driver-translocated groups denoted in the ICC [Several oncogenic drivers were recognized as translocation partners by the WHO4R, with the most common partner as the defining RGA and variant partners listed as variants of the primary entity. ICC continues to recognize the specific entities of “acute promyelocytic leukemia (APL) with t q24.1;q21.2)/;11) p21.;q23.3/ML4.1;q21.2CEBPA gene on the long arm of chromosome 19. This transcription factor is an essential regulator of granulopoiesis, and multiple isoforms are encoded by the CEBPA gene [CEBPA tend to cluster in two regions: In the N-terminus of the protein upstream of the p30 initiation codon and in the C-terminus in the DBD and bZIP domains [The CCAAT/enhancer binding protein α (C/EBP-α) transcription factor is encoded by the BPA gene ,11. Both domains ,11. The  domains . The C-t domains .CEBPA mutations in AML found an improved prognosis in patients with biallelic mutations, with each allele harboring a mutation from each of these clusters [CEBPA.” [CEBPA mutations have been identified in families with inherited risk of AML (WHO4R “AML with germline predisposition (CEBPA mutation)”) [CEBPA mutations is most correlated with C-terminal (aka bZIP) mutations, either as part of a biallelic alteration or alone [CEBPA-mutated AML in the new classification systems have been updated to “AML with in-frame bZIP CEBPA mutations” in ICC and “AML with CEBPA mutation” in WHO5, with the caveat that in WHO5, the acceptable mutations are either biallelic (biCEBPA) or single mutations in the bZIP domain (smbZIP-CEBPA). A large proportion of clinical sequencing in AML is performed using next-generation sequencing, where it is technically challenging to assess the phase of such distant mutations as the N- and C- terminal alterations in CEBPA. Thus, the updated classification, by removing a requirement for biallelic alterations, will clarify which patients belong in this favorable prognostic group.Numerous studies of clusters ,14,15. Tation)”) , and theation)”) ,17. Moreor alone ,19. BecaTP53 gene encodes the tumor suppressor P53 and is the most commonly mutated gene in cancer [TP53 alterations have been well documented to occur in myeloid neoplasms, including MDS and AML, where they act through a dominant-negative or loss-of-function mechanism [TP53 alterations have a very poor prognosis, especially those with multiple simultaneous TP53 alterations [TP53-altered patients have led to numerous updates in the new classification systems.Located on the short arm of chromosome 17, the n cancer ,21. P53 n cancer . TP53 alechanism . Patienterations ,25,26. TTP53” as a distinct subtype, whereas WHO5 has not yet included this as a unique group, despite noting the association of TP53 alterations with very poor outcomes ”, although there are now specific additional genetic alterations recognized as “myelodysplasia-related defining”, and both systems have removed the criteria of morphologic dysplasia for classification in this group  1,2,3,4,3,42,3,4TP53 alterations, 17p abnormalities have been added as defining criteria for myelodysplasia-related AML in both ICC and WHO5. Also common to both systems is the removal of several specific translocations from the summary classification systems released to date [As noted above in the section on  to date ,3,4. ICC to date ,3,4.DNMT3A and TET2 [ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2. As noted above in the section on the RUNX1 mutation, this is also considered myelodysplasia-related in the ICC but not in WHO5 [RUNX1), epigenetics/gene expression , DNA replication/integrity (STAG2), and RNA splicing .The most significant change to this category of myelodysplasia-related AML is the new inclusion of specific somatic mutations in many of the most recurrently mutated genes in MDS ,28,29,30and TET2 ,32,33. T in WHO5 . These gOverall, the changes within this category of AML now emphasize the genetic correlates of the poor prognosis in myelodysplasia-related AML while removing the reliance on morphology to define this group.TP53 alterations, the latter of which were briefly mentioned in the AML-RGA section above. By recognizing several new entities, it is hoped that patients at higher risk for disease will be more closely followed and related donors appropriately screened in cases of allogeneic stem cell transplant.The last broad group of genetically defined AML is associated with germline predisposition. This group was initially included in the WHO4R and has been retained in both the ICC and the WHO5, although the new systems recognize a few additional entities and expand previously defined categories  1,2,3,4,43,4,34.SAMD9 and SAMD9L are two adjacent genes located on the long arm of chromosome 7 at 7q21 [SAMD9 mutations are associated with the MIRAGE  syndrome, while SAMD9L mutations are associated with an ataxia pancytopenia (ATXPC) syndrome [SAMD9/SAMD9L mutation, which occurs predominantly through loss of the affected chromosome 7, although it may also occur through a secondary reversion mutation in the mutated gene or uniparental disomy of the wild-type allele in the region of the mutation [SAMD9/SAMD9L allele but may acquire additional somatic mutations leading to progression to MDS and AML  risk stratification based on genetics [In recent years, molecular and genetic alterations have played an essential role in improving the prognosis of AML. Induction therapies  have eliminated leukemic blasts in AML. However, as our understanding of the molecular pathogenesis of the disease has improved, new therapeutic approaches have become crucial, especially in relapsed/refractory AML . At thisgenetics .bold are newly defined in ELN 2022.1. Favorable risk: prognostic entities in -t/RUNX1::RUNX1T1-inv(16) or t/CBFB::MYH1KIT or FLT3 mutations.The risk category remains unchanged, regardless of Mutated NPM1 without FLT3-ITD-NPM1 mutations and adverse risk cytogenetic abnormalities is classified as adverse risk [FLT3-ITD, in patients with NPM1-mutated AML remains undefined.AML with rse risk . The spebZIP in-frame mutated CEBPA-bZIP region of CEBPA have explicitly been associated with a favorable outcome, regardless of whether they occur as monoallelic or biallelic mutations.In-frame mutations within the bold are newly defined in ELN 2022.2. Intermediate risk: prognostic entities in FLT-ITD -FLT3-ITD has diminished in this group due to the lack of standardized measurement techniques [NPM1 mutation status, this group falls into the intermediate-risk category. Midostaurin therapy has significant importance in this group.The importance of the allelic ratio of chniques ,52. Regat/MLLT3::KMT2A-t takes priority over rare concurrent adverse-risk gene mutations.The presence of -Cytogenetic and/or molecular abnormalities not classified as favorable or adversebold are newly defined in ELN 2022.3. Adverse risk: prognostic entities in -t/DEK::NUP214-t/KMT2A rearranged (excluding KMT2A-PTD)-t/BCR::ABL1-/KAT6A::CREBBPt involving the MECOM gene and t associated with KAT6A::CREBBP, have demonstrated a dismal long-term overall survival. Allo-HSCT is a potentially beneficial treatment option in such cases [Furthermore, these adverse-risk cytogenetic abnormalities, such as ch cases ,54.-inv(3) or t/GATA2, MECOM (EVI1)-t/MECOM(EVI1)-rearranged-Monosomy 5 or del(5q); monosomy 7; monosomy 17/abn(17p)Complex karyotype  in the absence of structural abnormalities), monosomal karyotype-Moreover, hyperdiploid karyotypes characterized by multiple trisomies (or polysomies) are now excluded from the complex karyotype classification and the adverse risk group. This reclassification is based on the observation that patients with numerical cytogenetic changes and the absence of structural abnormalities have shown better survival outcomes than those with three or more cytogenetic changes accompanied by structural abnormalities .MutatedBCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSF2ASXL1, -Mutated TP53(Variant Allele Frequency ≥ 10%)-FLT3-mutated AML, midostaurin  is added to intensive chemotherapy. In therapy-related AML and myelodysplasia-related AML, CPX351  is preferred. CPX351, a liposomal formulation of daunorubicin and cytarabine, received approval in 2017 for treating therapy-related AML and myelodysplasia-related AML. For cases with complex karyotypes and TP53 mutations, ongoing research is investigating hypomethylating agents and venetoclax as alternative treatment options [In newly diagnosed AML, intensive chemotherapy is administered if no targetable mutations are present. However, in  options . Gemtuzu options . Venetoc options . In rece options . This ob options .FLT3, IDH1, and IDH2 inhibitors. Currently, menin inhibitors are being evaluated as a treatment option for patients with KMT2A rearrangements or NPM1 mutations, following the same assessment pattern for their efficacy and safety [MLL (mixed-lineage leukemia) translocations involving the KMT2A gene at chromosome 11q23 are present in approximately 5% to 10% of adults with AML [MLL translocations result in the dysregulation and increased expression of homeobox (HOX) genes. These genes are also dysregulated in AML cases with NPM1 mutations and are associated with the self-renewal capacity of hematopoietic stem cells [MEN1 gene, plays a crucial role in the function of KMT2A [KMT2A and is essential for its activity. In recent years, small-molecule inhibitors that interfere with the interaction between KMT2A and menin have been under investigation. Two such inhibitors are SNDX 5613  and KO539 . These inhibitors have shown efficacy in early-phase trials, with SNDX 5613 demonstrating composite complete response rates of 44% among 45 patients with NPM1 or MLL-rearranged AML [The clinical investigation of new therapies and novel combinations plays a vital role in further enhancing the outcomes of AML . Explorid safety ,60,61,62with AML . MLL traem cells . The scaof KMT2A . Menin bnged AML ,66.E-selectin. It has been discovered that uproleselan can disrupt vascular niche-mediated chemoresistance. In the context of cancer, the vascular niche refers to the microenvironment surrounding blood vessels, which can provide a protective environment for cancer cells and contribute to their resistance to chemotherapy [While there have been significant advancements in the management of AML, the outcomes for patients with high-risk diseases remain unsatisfactory. Numerous clinical trials are currently underway to improve these outcomes, evaluating targeted therapies and immunotherapies as potential treatment options. These trials investigate using these therapies as monotherapies or in combination with other treatment modalities. Although most of these therapies are still in the investigational stage and long-term outcomes are not yet fully described, some promising early responses have been observed. In this context, uproleselan (GM-1271) is a drug that functions as an inhibitor of otherapy ,68.do not eat me” signal, which is overexpressed by cancer cells [CD47 is a signaling molecule known as a “er cells . It server cells . This enTIM3. TIM3 acts as an inhibitory checkpoint on immune cells and blasts, regulating immune responses. Significantly, TIM3 is not expressed on normal hematopoietic stem cells. Further research and clinical trials are necessary to understand the efficacy of MBG453 and determine its role in treating AML and high-risk MDS. Nonetheless, these preliminary findings suggest that targeting TIM3 with sabatolimab holds promise as a therapeutic strategy for patients with AML [MBG453, or sabatolimab, is a humanized antibody designed to target a protein called with AML ,72.DART  antibody. Flotetuzumab is designed to bind to CD3ε on T cells simultaneously and CD123 on AML blasts. Doing so redirects the immune system to recognize and eliminate AML cells [In addition to sabatolimab and magrolimab, several other immune therapies are currently under development for treating AML. These therapies include bispecific antibodies and chimeric antigen receptor (CAR) T-cell therapy, targeting various antigens such as CD123, CD33, and CD70 ,74. FlotML cells .Developing bispecific antibodies and CAR T-cell therapies represents a promising field in AML treatment. These therapies aim to enhance the body’s immune response against leukemia cells, potentially improving outcomes for patients with AML. Ongoing research and clinical trials will further evaluate the safety and efficacy of these immunotherapeutic approaches . One chaCRISPR/Cas9 technology to address this issue. One strategy involves editing out the CAR target antigen from a donor allograft, creating a donor graft with a modified antigen profile that is now “leukemia specific”. After Allo-HSCT, CAR T-cells can be administered, explicitly targeting the modified antigen [Researchers are exploring innovative approaches such as genetic editing using  antigen . This apOverall, developing effective and safe immunotherapeutic strategies for AML remains a complex and evolving area of research, and ongoing clinical trials will provide valuable insights into the potential of these innovative approaches.Since the last update to the classification of AML in 2016 with the WHO4R, there has been a huge growth in our understanding of the pathobiology and, in particular, the genomics of this group of diseases. The updated classification systems, while harboring minor differences, both incorporate these changes to refine specific AML entities. Through efforts to more clearly differentiate patients with different disease biology, we gain the ability to better tailor therapies and deliver personalized care in acute myeloid leukemia.


TET2 promoter. 5hmC levels in the TET2 promoter could represent TET2 activity resulting from mutations in DNA demethylation genes and other factors. The novel genes and key signaling pathways associated with aberrant 5hmC patterns could add to our understanding of DNA hydroxymethylation and highlight the potential therapeutic targets in AML. Our results identify a novel 5hmC-based AML classification system and further underscore cfDNA 5hmC as a highly sensitive marker for AML.Epigenetic abnormality is a hallmark of acute myeloid leukemia (AML), and aberrant 5-hydroxymethylcytosine (5hmC) levels are commonly observed in AML patients. As epigenetic subgroups of AML correlate with different clinical outcomes, we investigated whether plasma cell-free DNA (cfDNA) 5hmC could categorize AML patients into subtypes. We profiled the genome-wide landscape of 5hmC in plasma cfDNA from 54 AML patients. Using an unbiased clustering approach, we found that 5hmC levels in genomic regions with a histone mark H3K4me3 classified AML samples into three distinct clusters that were significantly associated with leukemia burden and survival. Cluster 3 showed the highest leukemia burden, the shortest overall survival of patients, and the lowest 5hmC levels in the  Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is characterized by genetic and epigenetic heterogeneity . DespiteTET2, IDH1, or IDH2 mutations [A global decrease in 5-hydroxymethylcytosine (5hmC) levels has been widely observed and closely correlates with poor prognosis in AML ,3,4,5. 5utations . AML patutations . HMAs tautations ,9,10,11.utations ,13, 5hmCDNA methylation status can be used to classify AML patients by genetic features and clinical outcomes ,15,16. AMost studies of 5hmC in AML report 5hmC at global levels due to current technical limitations ,3,4,5. ATET2 promoter. The classification of AML by cfDNA 5hmC is a new approach for guiding therapeutic treatment decisions in AML. Our findings identify key genes and signaling pathways that are associated with aberrant 5hmC levels in AML and may have significant implications for therapeutic targeting in AML.To delineate region-specific 5hmC distribution and assess the role of cfDNA 5hmC in AML, we performed genome-wide 5hmC profiling in plasma cfDNA from 54 AML patients using the highly specific nano-hmC-Seal method. We used an unbiased clustering approach and found that 5hmC levels in regions marked with H3K4me3 classified AML samples into three distinct clusters. These clusters were significantly associated with leukemia burden, overall survival (OS), and 5hmC levels at the The study included blood samples from 54 patients diagnosed with AML  that werg for 10 min at 4 °C. Plasma cfDNA was extracted from 1 mL of plasma using the QIAamp Circulating Nucleic Acid Kit  following the manufacturer’s instructions. We assessed cfDNA quantity and quality using the Qubit Fluorometer with dsDNA HS Assay Kit  and Bioanalyzer 2100 with Agilent High Sensitivity Assay Kit . We constructed the 5hmC library as previously described [https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 1 May 2020). We trimmed adaptors and low-quality reads using Trimmomatic version 0.39. We mapped high-quality reads to the reference genome (GRCh38) using the bowtie2 version 2.4.5 with the end-to-end mode. Reads with a mapping quality score ≥20, insert size <600 bp, up to one ambiguous base, and <3 mismatches were retained. We removed PCR duplicates using the SAMtools version 1.16.1.We isolated the plasma by centrifuging whole blood at 1350× escribed . Brieflyescribed . Brieflyhttps://cran.r-project.org/package=pheatmap (accessed on 14 September 2020). Clusters were defined based on the dendrogram of the heatmap.We normalized raw gene read counts using counts per million (CPM), and 29,423 genes remained in reads with CPM ≥ 3 in more than 10% of the samples. We obtained histone ChIP-seq peak data for histone marks of H3K4me1, H3K4me3, H3K27ac, and H3K36me3 in common myeloid progenitor (CD34+) and populations from the Encyclopedia of DNA Elements project. High-quality reads were counted into histone-enriched regions and gene bodies (RefSeq) using featureCounts from Subread package version 2.0.0 [http://gepia.cancer-pku.cn/detail.php (accessed on 14 July 2021) [https://cran.r-project.org/package=eulerr (accessed on 8 March 2023)). The ggplot2 R package was used for data visualization.To identify differentially hydroxymethylated regions (DhMRs) among the clusters, differential hydroxymethylation analysis controlling for the effects of age and sex was performed using the R/Bioconductor package DESeq2 (v1.34.0) . Regionsly 2021) . Venn dihttps://cran.r-project.org/web/packages/survminer/index.html (accessed on 26 July 2021). A log-rank test was used to evaluate the statistical significance of OS between the groups. Multivariate Cox regression analysis was performed using the Survival R package . Comparisons between the groups were analyzed using the Wilcoxon Rank Sum test. An FDR value <0.05 was considered significant.We performed plotting and statistical tests using R language version 4.1.1. Kaplan–Meier curves were used to display OS, and survival curves were generated using the survminer R package (cis-regulatory enhancer elements) [p = 1.2 × 10−6) or cluster 2 , H3K4me3 (in promoters), H3K4me1 and H3K27ac (in lements) , we analp = 1.7 × 10−5) and cluster 2 (p = 0.0056) compared to specimens from cluster 1  and cluster 3 (p = 3.3 × 10−5) had significantly lower 5hmC levels in the TET2 promoter  or 2  was closely correlated with gene expression , and we promoter A. Intereectively A. The saobserved . These rp = 0.97; hazard ratio (HR) 0.97; 95% confidence interval (CI): 0.2–6.38; p = 0.0063; HR 6.2; 95% CI: 1.4–27.9) and cluster 2  database. In cluster 2, of the 1506 DhMR-related genes presented in the GEPIA database, 883 genes (58.6%) exhibited differential expression at the mRNA level . SimilarTo determine the functional impact of aberrant 5hmC distributions, we analyzed the signaling pathways in DhMR-linked genes in clusters 2 and 3. A total of 151 pathways (86.3% of cluster 2 and 61.1% of cluster 3) were shared by both clusters, indicating these were important pathways for leukemogenesis . Many ofPrevious studies have revealed that DNA methylation patterns can segregate AML patients into subtypes associated with diagnosis and prognosis ,15,16. ATET2 promoter was more relevant than mutations in DNA demethylation genes for assessing TET2 activity. In addition to AML samples with a mutation in TET2, samples in cluster 3 showed enriched IDH1, IDH2, or WT1 and were significantly associated with reduced 5hmC levels in the TET2 promoter. In AML, reduced 5hmC levels were observed in patients with or without a mutation in epigenetic modifiers, including TET2, IDH1, IDH2, and WT1, suggesting that other genes or posttranslational changes also regulated 5hmC levels [TET2 promoter likely represent reduced TET2 activity resulting from mutations in DNA demethylation genes and other factors. From a clinical perspective, 5hmC analysis is easier to assess relative to gene expression or other analytes. Compared to the assessment of simple mutations in TET2, IDH1, IDH2, and WT1, 5hmC analysis is a promising new approach for guiding therapeutic management. For example, patients with decreased TET2 activity may benefit from treatment with a hypomethylating agent and vitamin C (ascorbate) [Our results indicated that the 5hmC level in the C levels ,31,32,33corbate) ,32,34.WT1 promoter in AML samples without a mutation in TET2, IDH1, IDH2, or WT1. WT1 is a transcription factor that recruits TET2 to DNA, enabling promoter demethylation [WT1 result in a global reduction in 5hmC levels comparable to that seen in AML patients with TET2, IDH1, and IDH2 mutations [WT1 is overexpressed in the majority of AML patients and is considered an oncogene [WT1 in most AML samples was consistent with previous reports [WT1 in AML.Interestingly, we found increased 5hmC levels in the hylation . Loss-ofutations ,36. HoweCDK6 [ZMYND8 [ARHGAP26 [GLI3 [Consistent with a global reduction in 5hmC levels in AML, we found more H3K4me3-associated DhMRs with reduced 5hmC levels compared to increased 5hmC levels. H3K4me3 involves transcriptional initiation and transcriptional pause-release and elongation and is closely associated with gene expression . Our finCDK6  and ZMYN [ZMYND8 , and redARHGAP26  and GLI326 [GLI3 . These rOur classification of AML using 5hmC revealed distinct dysfunctional epigenetic pathways related to gene regulation. As the majority of signaling pathways with aberrant 5hmC levels were shared by clusters 2 and 3 and involved in cell proliferation, targeting these pathways may help inhibit leukemia cell proliferation. Signaling pathways that are specific for each cluster may be associated with the leukemia burden and provide potential drug targets for patients with a specific 5hmC pattern. For example, PPAR signaling was significantly enriched in cluster 2. PPARs are critical sensors and regulators of lipids, and their excessive expression can be related to cell growth and survival in several malignancies, including leukemia . PPAR moUsing cfDNA 5hmC to classify AML patients has distinct advantages, given its high tissue and cancer specificity, which may mitigate interference from cfDNA derived from non-cancerous cells ,3. HowevHere, we have shown how plasma cfDNA 5hmC is a powerful approach for classifying AML patients. Cluster 3 was significantly and independently associated with prognosis in AML. A deeper understanding of how novel genes and critical signaling pathways influence DNA hydroxymethylation in AML could allow the optimization of therapeutic targets for AML patients. The distinct genetic composition of these clusters sheds light on potential therapeutic approaches for AML patients. Our results further underscore the importance and relevance of cfDNA 5hmC as a marker in AML.


RyR2 oxidation promotes the formation of disulfide bonds between two cysteines on neighboring RyR2 subunits, known as intersubunit cross-linking. However, the large number of cysteines in RyR2 has been a major hurdle in identifying the specific cysteines involved in this pathology-linked post-translational modification of the channel. Through mutagenesis of human RyR2 and in-cell Ca2+ imaging, we identify that only two cysteines (out of 89) in each RyR2 subunit are responsible for half of the channel’s functional response to oxidative stress. Our results identify cysteines 1078 and 2991 as a redox-sensitive pair that forms an intersubunit disulfide bond between neighboring RyR2 subunits during oxidative stress, resulting in a pathological “leaky” RyR2 Ca2+ channel.The most common cardiac pathologies, such as myocardial infarction and heart failure, are associated with oxidative stress. Oxidation of the cardiac ryanodine receptor (RyR2) Ca Oxidation of ryanodine receptor calcium channels play a critical role in the onset of many cardiac diseases. Here, authors identify specific amino acids that cause ryanodine receptor malfunction during oxidative stress. In cardiac myocytes, Ca2+ released from the sarcoplasmic reticulum (SR) through the type 2 ryanodine receptor (RyR2) is essential for initiating a robust myocardial contraction2. Consequently, defects in RyR2 regulation cause contractile dysfunction and decreased cardiac output. The most common cardiac pathologies with fatal outcomes, such as myocardial infarction and heart failure, are associated with abnormal Ca2+ regulation3. Pathological alterations during those conditions commonly involve substantial structural and functional remodeling of RyR2 by oxidative stress7. It has been shown that reactive oxygen species (ROS) can directly activate RyR2-mediated Ca2+ release in the form of diastolic spontaneous Ca2+ sparks9 and Ca2+ waves10, causing arrhythmias and depletion of SR Ca2+ load. Oxidative stress also causes the dissociation of an important regulator of RyR2 function, calmodulin (CaM)13. As a result, RyR2 oxidation has a significant impact on Ca2+ signaling and contraction in infarcted and failing hearts16.Calcium ion  is associated with an intersubunit dynamic23. Thus, we hypothesized that intersubunit cross-linking should significantly impact RyR2 function and Ca2+ signaling. In agreement with our hypothesis, we found that in ventricular myocytes from healthy hearts, the degree of SR Ca2+ leak induced by oxidative stress positively correlated with the level of RyR2 cross-linking22. Moreover, a significant level of RyR2 cross-linking has been detected in ventricular myocytes from failing hearts7.In our previous studies, we discovered a novel posttranslational modification (PTM) of RyR2 induced by oxidative stress. As a common mechanism of channel assembly, the RyR2 complex is formed by four identical subunits. We have shown that oxidative stress can promote the formation of disulfide bonds between cysteines on neighboring RyR2 subunits, also known as intersubunit cross-linking2+ release in skeletal muscle fibers24. It has been suggested that cysteine 3635 in RyR1 is the most important residue responsible for redox regulation of the channel26. It has been shown that cysteine 3635 can be oxidized during oxidative stress to form a disulfide bond28. We re-evaluated the functional significance of the corresponding cysteine 3602 in RyR2 and showed that this residue is not involved in channel cross-linking nor Ca2+ leak activation by oxidative stress13. It appears that despite ~70% homology between RyR1 and RyR2, these channels exhibit important structural and functional differences, particularly in redox regulation. In the end, the specific mechanisms of redox regulation and the redox-sensitive residues within RyR2 remain largely unknown.The effect of oxidative stress on the channel’s structure and function is better described for the skeletal muscle type 1 RyR isoform (RyR1). Historically, the oxidation of thiol groups in RyR1 by reactive sulfhydryl agents has been linked to increased SR CaIn this study, we identify cysteines 1078 and 2991 as a critical redox-sensitive pair that can form a disulfide bond between neighboring RyR2 subunits during oxidative stress, resulting in “leaky” RyR2.29. Changes in the ER luminal [Ca2+] ([Ca2+]ER) were measured with the genetically encoded ER Ca2+ sensor R-CEPIA1er in HEK293 cells expressing human RyR2  due to spontaneous hRyR2 activation followed by SERCA-mediated Ca2+ reuptake  caused a global ER Ca2+ release and [Ca2+]ER depletion. The completeness of [Ca2+]ER depletion by caffeine was validated by the SERCA inhibitor thapsigargin (10 µM). Since the application of thapsigargin did not cause additional [Ca2+]ER depletion, we used the caffeine output as Fmin during signal normalization  with increasing concentrations of the thiol oxidant diamide. Western blot analysis revealed that increasing concentrations of diamide caused the disappearance of the hRyR2WT 560 kDa monomeric band and promoted the formation of cross-linked oligomers. This effect could be reversed by treating cell samples with the reducing agent DTT  that caused the maximal cross-linking  also resulted in 50.1 ± 1.4% (n = 72) depletion of [Ca2+]ER (or ER Ca2+ load) in cells that express hRyR2WT tagged with GFP  structure (residues 1-547), which includes three domains 32. The NTR of RyR2 has been shown to play an important role in the channel’s tetramer formation and regulation of the channel’s function36. It has also been characterized as a “hotspot” for the hRyR2 mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT)38, suggesting its important role in channel gating. Based on the analysis of the NTR domains orientation in the available RyR2 cryo-EM models40, we decided to focus on domains A and B (residues 1-409), which harbor 9 of 10 NTR cysteines  and 43.5 ± 10.7%  of hRyR2ΔAB monomers from cross-linking at 25 and 50 µM diamide, correspondingly  decline in ER Ca2+ load caused by increased ER Ca2+ leak through hRyR2WT. However, in cells expressing hRyR2ΔAB, the same diamide concentration caused only 11.1 ± 1.3% depletion of [Ca2+]ER, yielding a relative protective effect of 79% . hRyR2ΔA showed substantial resistance to cross-linking similar to the one observed for the hRyR2ΔAB construct  and 21.8 ± 8.3%  of the hRyR2ΔA monomeric form after the corresponding treatment with 25 and 50 µM diamide , none of them prevented the channel’s cross-linking  loop on one RyR2 subunit and cysteine 1078 of the P1-SPRY2 linker on another subunit . Western blot analysis revealed that the hRyR2C1078S mutation substantially prevented cross-linking caused by diamide  of hRyR2C1078S monomers from cross-linking at 25 µM and 53.4 ± 7.4%  at 50 µM diamide  decline of ER Ca2+ load in cells expressing hRyR2WT and 24.3 ± 2.0% (n = 40) in cells expressing hRyR2C1078S  caused the 46.9 ± 2.0% decline (n = 27) of ER Ca2+ load for hRyR2WT and 28.5 ± 2.0% (n = 36) for hRyR2C1078S . Since the higher diamide concentration only slightly increased the ER Ca2+ load depletion, these results suggest that the most functionally significant disulfide cross-linking occurs at 25 µM diamide (by oxidizing cysteine 1078), while higher diamide concentrations oxidize other hRyR2 cysteines without a substantial increase in ER Ca2+ leak.Our recent data demonstrated that RyR2 intersubunit cross-linking positively correlated with RyR2 activation by oxidative stress2WT Fig. . The app78S Fig. . Since w78S Fig. . Thus, tC2991S). Western blot analysis showed that the C2991S mutation significantly prevented hRyR2 cross-linking, protecting 48.7 ± 7.3%  of its monomeric form at 25 µM diamide and 72.3 ± 2.3%  at 50 µM diamide  depletion of ER Ca2+ load for hRyR2WT and 25.0 ± 2.8% (n = 28) for hRyR2C2991S. The treatment with 50 µM diamide caused 47.0 ± 1.9% (n = 40) depletion of ER Ca2+ load for hRyR2WT and 28.9 ± 1.9% (n = 48) for hRyR2C2991S . The level of protection was similar to those seen for the hRyR2C1078S mutant, suggesting a common mechanism for the redox resistance in hRyR2C1078S and hRyR2C2991S mutants. Thus, cysteines 1078 and 2991 are likely involved in the hRyR2 intersubunit disulfide formation.To explore whether cysteine 1078 forms the intersubunit disulfide bond with cysteine 2991, we mutated cysteine 2991 to serine . When expressed in HEK293 cells, the hRyR2C1078S/C2991S mutant demonstrated significant protection against hRyR2 intersubunit cross-link induced by diamide. The protection of hRyR2C1078S/C2991S monomers from cross-linking was in line with the one seen for hRyR2C1078S and hRyR2C2991S mutants: 53.7 ± 7.6%  at 25 µM diamide and 53.1 ± 3.3%  at 50 µM diamide  decline in ER Ca2+ load for hRyR2WT and 24.3 ± 1.6% (n = 44) for hRyR2C1078S/C2991S  for hRyR2WT and 28.3 ± 1.9% (n = 27) for hRyR2C1078S/C2991S . This level of protection against the cross-linking was similar to those seen for the single cysteine mutants  concentrations  play a particularly important role in the functional response of the channel to oxidative stress. Mutation of one of those cysteines substantially protected against hRyR2 cross-linking and ER Ca2+ released through RyR2 plays a central role in initiating a robust myocardial contraction. Consequently, defects in RyR2 function can lead to contractile dysfunction in a variety of cardiac pathologies. It has been shown that redox-dependent PTMs of RyR2 contribute to abnormal Ca2+ regulation during myocardial infarction and heart failure7. In the canine model of chronic heart failure, disulfide formation within the RyR2 complex has been characterized as the major redox modification responsible for “leaky” RyR2, abnormal Ca2+ regulation, and arrhythmias5. We previously showed that oxidative stress could promote the formation of disulfide bonds between two neighboring subunits of RyR2 through a PTM known as intersubunit cross-linking22. We found a strong positive correlation between the level of RyR2 cross-linking and an elevated SR Ca2+ leak22. Moreover, a substantial portion of RyR2 in cardiomyocytes from failing hearts exists in the cross-linked form7. Despite its clinical significance, the redox-sensitive sites involved in RyR2 cross-linking have not yet been identified.In the mammalian heart, SR Ca41, we identified two candidates for the channel’s intersubunit disulfide formation at neighboring subunits of RyR2: cysteines 1078 and 2991 , CPVT mutation, and the presence of CaM or the Rycal drug ARM21041  would bind to C1078 and stabilize the linker in the favorable conformation for further interaction with C2991. In the next step, C1078 would create a disulfide bond with C2991, releasing the reduced form of oxidant . The disulfide bond formation between two RyR2 subunits highlights the essential functional role of the intersubunit interface comprised of the P1-SPRY2 linker from one subunit and the HD2 loop from another subunit. In the previous structural study of the RyR2R2474S CPVT mutant41, ARM210 has been shown to bind to the P1 domain, stabilizing the intersubunit interface between the beginning of the P1-SPRY2 linker (preceding C1078) from one subunit and the HD2 loop (including C2991) from another subunit, reverting the primed state of the mutant channel to the closed state. In the proposed model, the stabilization of the intersubunit interface occurs through the interaction between D1070 on one RyR2 subunit and H2995 on another, with the possible involvement of H1071 and R298841. The intersubunit disulfide formation between C1078 and C2991 would likely destabilize this interface, shifting the channel’s structure to a “leaky” conformation.By analyzing the recently published cryo-EM structures of hRyR2991 Fig. . We foun2+]ER dynamics in HEK293 cells expressing hRyR2 and SERCA2a Ca2+ pump, we showed that the mutations of cross-linking cysteines (C1078S or/and C2991S) substantially ( ~ 50 %) protect against a depletion of [Ca2+]ER induced by oxidative stress. Since the level of oxidative stress that induced almost complete hRyR2WT cross-linking (at 50 μM diamide) did not affect the function of SERCA2a29 and endogenous ion channels/pumps in our cell model system, the depletion of ER Ca2+ load was chiefly mediated by increased ER Ca2+ leak via hRyR2. It is well known that oxidation of RyR2 is commonly associated with increased channel activity and ER/SR Ca2+ leak (reviewed in refs. 45). In the present study, we discovered that oxidation of only two cross-linking cysteines was responsible for half of the channel’s functional response to oxidative stress. It appears that defective intersubunit dynamics due to RyR2 intersubunit cross-linking plays a key role in RyR2 dysfunction during oxidative stress.By analyzing ER, R-CEPIA1er was excited with the 543 nm line of a He-Ne laser, and fluorescence was measured at > 580 nm. For measurements of [Ca2+]ER load-leak balance, 2D images (512 × 512 pixels) were collected every 5 s at a scanning speed of 6 ms/line. The cells were treated with corresponding diamide concentration for at least 15 min until the complete equilibration of [Ca2+]ER levels. At the end of each experiment, caffeine (10 mM) was applied to induce complete depletion of [Ca2+]ER. Then, Fmax was estimated by applying 2 µM ionomycin, escin 0.005%, and 10 mM Ca2+. The changes in [Ca2+]ER were presented normalized according to [Ca2+]ER = (F-Fmin)/(Fmax-Fmin). To obtain the change in [Ca2+]ER caused by oxidative stress, Ca2+ wave peak values were averaged before and after diamide application, and the relative change was calculated.For functional CaP < 0.05. Peak analysis, statistical analysis, and graphical representation of averaged data were carried out on Origin 2021b SR2 software .All data are presented as means ± SEM of n measurements. Obtained 2D images were analyzed using ImageJ software . Groups were compared using the two-sided Student’s t-test for unpaired data sets or one-way ANOVA followed by Tukey’s post-hoc test where appropriate. Differences were considered statistically significant at The cryo-EM models of RyR2 were obtained from RSCB protein data bank. The structural analysis was performed using PyMOL 1.7.0.3, WinCoot 0.9.8.1 and AREAIMOL program from CCP4i package (v. 8.0.007). PyMOL 1.7.0.3 was used to create graphical representations of the RyR2 structural models.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Ca2+ plays many diverse roles including carrying electric current, driving electrogenic sodium–calcium exchange (NCX) particularly when Ca2+ is extruded across the surface membrane causing depolarization, and activation of enzymes which target organelles and surface membrane proteins. Heart function is also influenced by Ca2+ mobilizing agents  acting on intracellular organelles. Lysosomal Ca2+ release exerts its effects via calcium/calmodulin-dependent protein kinase II to promote SR Ca2+ uptake, and contributes to arrhythmias resulting from excessive beta-adrenoceptor stimulation. A separate arrhythmogenic mechanism involves lysosomes, mitochondria and SR. Interacting intracellular organelles, therefore, have profound effects on heart rhythms and NCX plays a central role.Rhythms of electrical activity in all regions of the heart can be influenced by a variety of intracellular membrane bound organelles. This is true both for normal pacemaker activity and for abnormal rhythms including those caused by early and delayed afterdepolarizations under pathological conditions. The influence of the sarcoplasmic reticulum (SR) on cardiac electrical activity is widely recognized, but other intracellular organelles including lysosomes and mitochondria also contribute. Intracellular organelles can provide a timing mechanism (such as an SR clock driven by cyclic uptake and release of CaThis article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’. An influence of intracellular organelles on these channels and transporters requires a functional linkage between them. It will be argued below that electrogenic sodium–calcium exchange (NCX) plays a key role in this linkage .2+, probably involving intraluminal Ca2+ concentration reaching a critical level to control this release), and/or can provide an intracellular store for Ca2+ involved in signalling mechanisms. The release of Ca2+ from intracellular organelles is central to the control of most if not all cell functions m) and cytosolic [Ca2+] in the nanodomain between SR and mitochondria m during high Ca2+ transients [2+]m and the voltage across the inner mitochondrial membrane ΔΨm. During a period of prolonged quiescence when cytosolic Ca2+ concentration is stable at around 100 nM, the [Ca2+]m is slightly higher. The local cytosolic Ca2+ concentration can reach as high as 10 µM when the cell is stimulated to fire Ca2+ transients accompanying APs. However, initiation of APs after a period of quiescence causes only a slow steady rise in [Ca2+]m to reach a new stable level over a period of many minutes [2+ transients and leads to a slow further rise in [Ca2+]m. It, therefore, appears that the frequency and amplitudes of Ca2+ transients determine [Ca2+]m and consequently ATP production.Mitochondria are a major source of ATP which is essential for cell functions, and mitochondria are abundant in the heart ,172. It  tissues ,175. In ial Ca2+ . Indeed ansients ,176,177.chondria ,175. Ca2ansients ,175. ATP minutes ,175. Bet2+]m is stable, and the behaviour of mitochondria is matched to function this is clearly not the case (as discussed above) under conditions in which there is a substantial increase in ROS and opening of mPTP. In addition there are other conditions in which mitochondrial mechanisms appear to contribute to disturbances of electrical activity including generation of DADs [Although under ‘normal’ conditions [Ca of DADs ,179, and of DADs . Further of DADs .2+ release. It has been argued that it is hard to see how evolutionary pressures would result in lysosomal Ca2+ release via TPC1 channels only having harmful effects on mitochondria under conditions of ischaemia-reperfusion [2+ release via TPC1 channels on mitochondrial function which is yet to be explored experimentally. Another issue concerns whether the distribution of TPC1 and TPC2 channels on the lysosomal membrane is uniform or whether TPC1 and TPC2 are arranged to face their targets. Evidence also supports the proposal that the relative expression of TPC1 and TPC2 channels varies with the developmental stage in the endolysosomal system [2+ content. This would allow separate activation of different sets of related organelles carrying either TPC1 or TPC2. These questions remain for future study.In the context of the major themes of this review it may be questioned how NAADP can exert effects on SR that are beneficial (at least until stimulation becomes excessive) while effects on mitochondria are harmful. The physiological conditions for the two effects are dramatically different but this may not be the only factor underlying the difference in effects. This topic has been discussed extensively elsewhere . It remaerfusion . It is cl system . On the . 9 (a)2+ mobilizing agent with diverse actions throughout the body [2+ mobilizing agent was presented above in the context of activation of lysosomal Ca2+ release to result in modulation of SR and mitochondrial function. Further discussion of these cardiac actions can be found in [NAADP is widely recognized as a Cathe body ,22,151. found in ,30 and [found in . A recenfound in . (b)2+-mobilizing agent which promotes Ca2+ release from endoplasmic reticulum via an action at RyR2s in many cell types [2+ transients accompanying APs, without an effect on triggering L-type Ca2+ currents and without an effect on the amount of Ca2+ loaded into the SR as assessed from the Ca2+ release in response to a high concentration of caffeine [2+ release from the SR. This interpretation was supported by Prakash et al. [cADP-ribose is a Call types ,151. In caffeine . It was h et al. , and earh et al. ,184 and h et al. .2+ release from the SR cannot cause a maintained effect on the amplitude of Ca2+ transients because of a compensatory mechanism in which the amount of Ca2+ in the SR declines to exactly match the effect of enhanced release [2+ with little effect on the threshold for cytosolic Ca2+ [2+ release from the SR via RyR2s by a cytosolic action which may be related to FKBP12.6, a protein associated with RyR2 [2+ spark frequency, SR Ca2+ content was reduced by caffeine since spark amplitude declined, but spark amplitude remained unchanged with cADP-ribose [2+ release from the SR at a range of concentrations [2+. Caffeine may, therefore, have a diastolic effect to promote SR Ca2+ loss which is not shared by cADP-ribose. 8-amino-cADP-ribose blocked the effect of photoreleased cADP-ribose on Ca2+ transients accompanying APs, but did not block the response to caffeine [2+ transients accompanying APs was maintained and did not show compensatory changes with time [2+ transients accompanying APs, but for observations over several minutes cADP-ribose was without effect on SR Ca2+ content while NAADP caused a substantial increase. The above evidence supports substantial differences between the actions of cADP-ribose and caffeine.The above conclusion remains controversial. On the basis of observations with caffeine, and related theoretical arguments, it has been asserted that all agents acting at RyR2s to increase Ca release . The explic Ca2+ , but seelic Ca2+ . cADP-riith RyR2 . There mith RyR2 . AnotherP-ribose . Caffeintrations , and higcaffeine . The effith time . Both cAAn important aspect of the actions of cADP-ribose was that the effects were markedly temperature dependent so that actions at room temperature were negligible while substantial effects occurred close to body temperature .2+ release via RyR2s in experiments in planar lipid bilayers [2+ spark frequency in permeabilized ventricular myocytes from WT mice but not in FKBP12.6 knockout mice. cADP-ribose displaced FKBP12.6 from mouse cardiac SR vesicles. Evidence in rat pancreatic islets [Questions remain about the exact mechanism of action of cADP-ribose at RyR2s. Early observations show that cADP-ribose did not increase Cabilayers –193 and c islets , arteriac islets ,196 and c islets  also supc islets .2+ transients accompanying APs under conditions in which the SR Ca2+ load and Ca2+ currents were unchanged [2+ transients.FK506 also increased the amplitude of Canchanged . These o2+ release from the SR, there might be additional indirect effects resulting from the consequent rise in cytosolic Ca2+, perhaps in localized regions. In cardiac homogenates and permeabilized cells the major effect of cADP-ribose was to increase Ca2+ uptake into the SR by SERCA [2+ transients accompanying APs were increased when cADP-ribose was applied either by patch pipette or photorelease in guinea pig ventricular myocytes. In each case there was no increase in SR Ca2+ assessed from the response to a high concentration of caffeine . In rat ventricular myocytes permeabilized with saponin, application of cADP-ribose for 30 s and 3 min increased Ca2+ spark frequency without a change in SR Ca2+ content (assessed from the response to caffeine). There was also no change in spark amplitude or decay time providing further independent evidence that SR Ca2+ content was not increased [2+. There was also a quickening of spark decay time which was taken to result from increased activity of SERCA. This may have arisen as a secondary consequence of a prolonged increase in spark frequency with possible effects on PLB/SERCA [Even if effects of cADP-ribose over the first few minutes were simply on RyR2s to promote Caby SERCA . Followiby SERCA . Again tncreased . HoweverLB/SERCA .2+ transients associated with APs by a cytosolic action at RyR2s without an increase in SR Ca2+ content [2+ uptake. Under physiological conditions, cADP-ribose probably acts in concert with NAADP, when the enhanced uptake of Ca2+ into the SR resulting from NAADP-mediated effects will complement the actions of cADP-ribose on Ca2+ release via RyR2s In the section above on DADs, it was mentioned that 8-amino-cADP-ribose, an antagonist of cADP-ribose, suppressed arrhythmias brought about by high levels of beta-adrenoceptor stimulation ( I(d)3 is a Ca2+ mobilizing agent of major importance in the heart. Evidence concerning its actions is very well described elsewhere [3 can provoke Ca2+ release from the SR [3 exerts particularly important effects in atrial myocytes where expression of type II IP3 receptors is much higher than in ventricular myocytes. IP3 acts on junctional SR beneath the plasmalemma to increase the amplitude of Ca2+ transients accompanying APs [3 on short term function of ventricular myocytes were minor in comparison to those in atrial cells, but long-term effects occur in both cell types as a result of changes in protein synthesis following nuclear actions of IP3 [IPlsewhere  and onlym the SR . IP3 exeying APs . Effectss of IP3 .3 in atrial myocytes [3 pathway, and could cause arrhythmias [3R [Further experiments showed functional effects of IPmyocytes ,206. Endmias [3R .3 pathway to increase the amplitude of Ca2+ transients accompanying APs, and in experiments on cat atrial myocytes the actions involved endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) [2+ transients seemed to depend on a novel signalling pathway involving IP3 and Ca2+-stimulated adenylyl cyclases, AC1 and AC8, which were first described in heart by Mattick et al. [3 increased the amplitude of Ca2+ transients accompanying APs, and the effects were suppressed by the AC inhibitor MDL12,330A and by the PKA inhibitor H89. It was suggested that IP3 caused Ca2+ release from the SR which acted on neighbouring Ca2+-stimulated AC1 and AC8 to provoke actions via the cAMP/PKA pathway. Under the conditions of the experiments the NO signalling pathway seemed not to be a major contributor since effects of photoreleased IP3 on Ca2+ transient amplitude were little if at all affected in the presence of L-NAME to inhibit eNOS or ODQ to inhibit guanylyl cyclase [Alpha-1 adrenoceptors also activate the IPide (NO) . In guink et al. . Photore cyclase .3 signalling in pacemaker function by actions that involve Ca2+ handling by the SR [There also appears to be a role for IPy the SR ,209,210.3 actions in the heart can be found in [Reviews of IPfound in –213 and found in .. 102+ release to enhanced SR Ca2+ uptake, and in effects mediated by the calcium mobilizing agents cADP-ribose and NAADP. cADP-ribose is thought to act at RyR2s, though this remains controversial. Since CaMKII can phosphorylate RyR2s such phosphorylation might perhaps be a requirement for cADP-ribose action, but additional work is needed to test this proposal. CaMKII was thought to be necessary for cADP-ribose actions in pancreatic islets [2+ content following NAADP-induced Ca2+ release from lysosomes  [Previously well-known actions of CaMKII were summarized and discussed in an early section of this review. In subsequent sections evidence has been presented for the involvement of CaMKII in the signalling pathway linking lysosomal Cac islets . The obsc islets . In addithan SR) ,147,148.As outlined above, the arrhythmogenic effects of high levels of beta-adrenoceptor stimulation include actions of NAADP and cADP-ribose following an increase in their synthesis by CD38, and CaMKII is expected to be involved in the effects of both these calcium mobilizing agents during arrhythmias resulting from excessive beta-adrenoceptor stimulation. This evidence must be taken into account to provide a full explanation of the contribution of CaMKII to arrhythmogenic mechanisms. It is also possible that these more recently discovered mechanisms might contribute to the linkage of PKA and CaMKII effects discussed in .. 112+ concentrations with subsequent Ca2+ extrusion, although there may be additional roles for ion channels activated or inhibited by cytosolic Ca2+.The evidence presented here provides a compelling case for the influence of intracellular organelles on electrical activity recorded across the surface membrane. The operation of NCX plays a central role in linking events within the cell to changes in surface membrane electrical activity. Most frequently NCX causes depolarizations resulting from increases in subsarcolemmal Ca2+ released from the SR can contribute to EADs, and is crucially important for the generation of DADs and alternans. Under ‘normal’ physiological conditions, the SR plays a role in the timing of the natural pacemakers in the SA and AV nodes.The SR exerts a dominant influence in initiating arrhythmogenic events under pathological conditions in all regions of the heart. Ca2+ mobilizing agent, NAADP is synthesized by CD38 and acts on lysosomes to release Ca2+ via TPC2 channels into a nanodomain between the lysosome and SR membranes. Evidence supports the hypothesis that Ca2+ released into this nanodomain activates CaMKII leading to phosphorylation of PLB and enhancement of Ca2+ uptake into the SR by SERCA. This results in an increase in the amplitude of Ca2+ transients accompanying APs. In the heart the synthetic enzyme, CD38, is located at or close to the SR. Synthesis of NAADP and therefore its effects are enhanced following beta-adrenoceptor stimulation. Excessive stimulation of this pathway, whether acute or chronic, is arrhythmogenic. A separate pathway involving lysosomes is a dominant influence in arrhythmias caused by reperfusion after a period of ischaemia. In this case NAADP causes Ca2+ release from lysosomes via TPC1 channels into a nanodomain between lysosomes and mitochondria, and the arrhythmia results from an interaction between the three intracellular organelles, lysosomes, mitochondria and SR. A protective effect of PKAIalpha has been identified which is thought to result from inhibition of Ca2+ release via TPC1 channels in the lysosomal membrane. Mitochondria are also involved in other arrhythmogenic events.Lysosomes can also have important effects on cardiac function. The Ca2+ mobilizing agents, cADP-ribose and IP3 can contribute to the generation of arrhythmias under appropriate conditions. cADP-ribose is also synthesized by CD38, with production enhanced following stimulation of beta-adrenoceptors. Excessive stimulation again causes arrhythmias which can be suppressed by an antagonist of cADP-ribose. cADP-ribose is thought to act on RyR2s via FKBP12.6, but this remains controversial.In addition to NAADP, two other CaCaMKII plays a pivotal role in coordinating the functions of different intracellular organelles and their interactions with the surface membrane.The evidence presented here strongly supports the proposal that intracellular organelles play diverse roles in controlling the timing mechanisms which determine heart rhythms and initiate cardiac arrhythmias.


Mitochondrial Calcium Uniporter (MCU) mediates the mitochondrial Ca2+ uptake. MCU is a heterooligomeric complex with a pore-forming component and accessory proteins required for channel activity. Though MCU regulation by MICUs is unequivocally established, there needs to be more knowledge of whether divalent cations regulate MCU. Here we set out to understand the mitochondrial matrix Mg2+-dependent regulation of MCU activity. We showed Mrs2 as the authentic mammalian mitochondrial Mg2+ channel using the planar lipid bilayer recordings. Using a liver-specific Mrs2 KO mouse model, we showed that decreased matrix [Mg2+] is associated with increased MCU activity and matrix Ca2+ overload. The disruption of Mg2+dependent MCU regulation significantly prompted mitochondrial permeability transition pore opening-mediated cell death during tissue IR injury. Our findings support a critical role for mMg2+ in regulating MCU activity and attenuating mCa2+ overload.Calcium (Ca The simultaneous interplay of the on-and-off mechanisms maintains the intracellular Ca2+ (iCa2+) transients7. Such a fine regulation is mediated by mitochondria that can decode and transduce iCa2+ signals into an energy output (ATP) to match energetic cellular demand1. The Ca2+ selective channel, the Mitochondrial Ca2+Uniporter (MCU), precisely controls mitochondrial Ca2+ (mCa2+) uptake and is driven by the considerable electrical gradient (Δψm) across the inner mitochondrial membrane (IMM)11. MCU functions as a hetero-oligomeric complex after self-association and interaction with its regulators19. Under physiological conditions, MCU-mediated mCa2+ uptake regulates bioenergetics by increasing the supply of reducing equivalents to the electron transport chain and increasing F1-F0 ATP synthase activity22. Reciprocally, in ischemia and reperfusion (I/R), dysregulation of mCa2+ cycling is prominent and results in either necrosis or apoptosis through mCa2+ overload28. Thus MCU-mediated mCa2+ uptake must be tightly regulated to support both cellular energy demand and also prevent cell death.Calcium  that binds Mg2+/Ca2+ with ~mM affinity. The binding of Mg2+/Ca2+ to MRAP destabilizes MCU, shifts the self-association equilibrium to monomer, and attenuates mCa2+ uptake33. A seminal work from Foskett’s group showed the Ca2+ that permeates through the channel pore to bind the MRAP region and close the channel despite Ca2+ binding to MICU1/234. Also, mutating the two key aspartic acid residues (D131 and D147) to alanine abolished matrix [Ca2+] dependent channel inhibition, validating divalent cation binding site encompassing D131 and D147 could account for Ca2+ (and Mg2+) dependent MCU inhibition34. Though matrix Ca2+ was shown to inhibit MCU channel activity, the weak binding affinity for divalent cation for MRAP is well suited to the high Mg2+ levels of the mitochondrial matrix (0.5–1.5 mM). Because proteins structurally sensitive to cations have evolved affinities close to the concentration range in the compartment where they function, we anticipate mitochondrial matrix [Mg2+] to regulate MCU channel activity tightly in addition to Ca2+.Though many Ca2+] dependent regulation of MCU-channel activity. Using biochemical, lipid bilayer, and patch-clamp recordings, we show Mrs2 as the authentic mammalian mitochondrial Mg2+ channel. Using our newly developed liver-specific mouse model, we illustrate the loss of Mrs2 to drastically alter the matrix [Mg2+]. The decreased matrix [Mg2+] was associated with increased MCU activity and matrix Ca2+ overload. The disruption of Mg2+-dependent MCU activity also significantly prompted mitochondrial permeability transition pore opening (mPTP). We employed a hanging-weight system for liver ischemia/reperfusion (IR) to assess the loss of Mg2+-dependent MCU regulation and mPTP-mediated cell death during tissue IR injury. Our findings support a critical role for mMg2+ in regulating MCU activity and attenuating mCa2+ overload.Here we set out to understand the matrix [Mg2+] regulates MCU channel activity, we recorded MCU Ca2+ currents (IMCU) in mitoplasts isolated from HeLa cells. We used varying [Mg2+]  in the patch-clamp pipette solution that filled the mitochondrial matrix and measured IMCU. MCU currents were large with pipette solutions containing 0 and 1 mM Mg2+, whereas they were inhibited by ~30 and 65% at 5 and 10 mM Mg2+ . In contrast, it preserved the interaction with negative regulators (MICU1 and MCUb)  . Togethercomplex –1c.2+ transporter in Saccharomyces cerevisiae mitochondria36. The electrophysiological studies showed yeast Mrs2 (sMrs2) to form an Mg2+- selective channel (155 pS) and not permeable to Ca2+ 36. Though the core/pore component of the mMg2+ influx machinery was defined in yeast, the physiologic relevance of this observation in higher-order systems remains understudied. A recent study from Madesh and colleagues showed mammalian Mrs2 (mMrs2) to form a selective pore for lactate-mediated mMg2+ uptake37, authenticating Mrs2 as the conserved mMg2+ transport machinery from yeast to mammals. Though mMg2+ influx machinery was defined in mammals, the electrophysiological properties of mMrs2 are not established. To show whether mMrs2 forms an Mg2+-selective channel, we purified mMrs2 from HEK293 cells  was initially considered the primary Mg93 cells . The hige states –g. We ree states –1j. Sing by Co2+ –j. The cvoltages . Fig. 1i12. HEK 293 cells stably expressing Mrs2-Flag were permeabilized with digitonin (40 μg/ml). Digitonin-permeabilized cells were incubated with mastoparan or alamethicin to elicit outer or inner mitochondrial membrane permeabilization. OMM permeabilization (with mastoparan) was confirmed by the cytosolic appearance of cytochrome C  were generated using CRISPR/Cas9 strategy. Mrs2fl/fl mice were crossed with Albumin-Cre to allow germline deletion specifically in the liver (Mrs2Δhep) . The gen2+ pulse –3i. The 2+ pulse . Loss of2+ pulse  and 3l.2+ in the presence of stimuli, we then asked whether loss of Mrs2 alters cytosolic Mg2+ (cMg2+) dynamics. Mrs2fl/fl and Mrs2Δhep hepatocytes were loaded with MagGreen-AM and TMRM. cMg2+ and mMg2+ dynamics were measured simultaneously after stimulating the cells with 10 μM glucagon and 2 mM Mg2+ . Confocal measurements show Mrs2Δhep hepatocytes to have delayed cMg2+ clearance  compared to Mrs2fl/fl, confirming mitochondria play a critical role in regulating cellular Mg2+ dynamics. We performed mitoplast patch clamp recordings to verify whether loss of Mrs2 ablates mitochondrial Mg2+ current (IMrs2). Loss of Mrs2 significantly ablated IMrs2 validating Mrs2 as an authentic mammalian mMg2+ channel  and 1 μM hexamine cobalt(III)chloride. Hexamine cobalt(III)chloride, not Ru360, abolished IMrs2, confirming the identity of IMrs2 recordings as Mrs2 currents .Because mitochondria can accumulate and release Mg channel  and 3n. 2+ in regulating MCU-mediated mCa2+ uptake, we used our permeabilized cell system to perform simultaneous measurements of mCa2+ uptake and Δψm38 (2+ pulse (10 μM) was added, and the rate of mCa2+ uptake was measured as a function of the decrease in bath Ca2+ fluorescence. Mrs2Δhep cells could rapidly clear extramitochondrial Ca2+ pulse, indicating increased MCU activity (IMCU) in Mrs2Δhep mitoplasts  in HEK293 cells to alter MCU activity . Previously we showed the binding of Mg2+ to the MRAP region to destabilize and shift the self-association equilibrium of MCU to monomer33. Consistent with our previous finding, FPLC analysis showed a heterogenous population of MCU complex (both low and high molecular weight) in HEK 293 NegShRNA cells, whereas MCU assembled as a supercomplex in Mrs2 KD cells . Collectively, these results suggest the loss of mMg2+ to stabilize the MCU complex and promote channel activity.To validate the role of mMgnd Δψm38  and 4b. activity –4e. Mito2+ at the resting state. Permeabilized cells loaded with Fura-FF tetra potassium salt were treated with Ru360  and CGP . After baseline measurement, an uncoupler CCCP was added to depolarize the mitochondrial membrane . Mrs2Δhep rapidly cleared the 1 μM Ca2+ pulse, whereas the channel gatekeeping function remained intact in Mrs2fl/fl . Additionally, the bath Ca2+ before adding Ca2+ pulse was lower in Mrs2Δhep compared to Mrs2fl/fl . Also, the bath Ca2+ after CCCP addition was significantly higher in Mrs2Δhep than Mrs2fl/fl , confirming the loss of Mrs2 to activate MCU in the low-[iCa2+] regime. We asked whether the loss of Mrs2 alters MCU/MICU1 interaction to activate MCU in the low-[iCa2+] regime. The immunoprecipitation with Flag antibody pulled down MCU and MICU1, indicating MCU/MICU1 complex to be intact in Mrs2 KD cells .We next asked whether increased MCU activity overloads matrix Camembrane . Though x [Ca2+]  and 4j. rs2fl/fl  and 4j. 2+]i regime despite stable MCU/MICU1 interaction, we asked whether the increased mCa2+ uptake is strictly MCU-mediated. We stably expressed MCUΔDIME mutant in HEK293 NegShRNA and Mrs2KD cells. MCUΔDIME expression significantly decreased MCU-mediated mCa2+ uptake in both control and KD cells  is the enzyme that catalyzes the irreversible conversion of pyruvate to acetyl-CoA. PDH is regulated through the alterations in the phosphorylation state of the enzyme by PDH kinase (PDHK) and PDH phosphatase (PDHP). The major regulators of PDHP are the divalent cations Mgrs2fl/fl  and 5b.  control  and 5b.Because we saw opposing results with PDH activation and ATP levels, we assessed the oxygen consumption rate (OCR) in control and Mrs2 KO/KD cells (fl/fl and Mrs2Δhep in the presence or absence of Oligomycin (ATP synthase and hydrolysis inhibitor) or BTB06584 (selective inhibitor of ATP hydrolysis). Both Oligomycin and BTB treatment depolarized/normalized the Δψm in Mrs2Δhep. Conversely, oligomycin treatment hyperpolarized the Mrs2fl/fl mitochondria, and BTB had no effect . Because the reversal of ATP synthase depletes cellular ATP and activates the mitochondrial permeability transition pore (mPTP)41, we next asked whether increased matrix Ca2+ overload and ATP reversal make Mrs2Δhep susceptible to mPTP opening. We simultaneously measured calcium retention capacity (CRC) and Δψm in Mrs2fl/fl and Mrs2Δhep. Mrs2Δhep exhibited decreased CRC associated with an early Δψm collapse , triggering channel closure46. We generated loss-of-function (Mrs2ΔGMN) and gain-of-function (Mrs2ΔMBS) mutant constructs  injury by hanging weight method. Using this method, we saw the portal triad immediately occluded by hanging the weights over the poles, causing the blood supply to the left, median, and caudate lobes of the liver to be interrupted. Successful occlusion was confirmed by visual inspection of pale blanching in the ischemic lobes  , and increased congestion in Mrs2Δhep compared to sham and Mrs2fl/fl IR injured mice , magnesium (Mg2+) is the abundant intracellular divalent cation. It is an essential co-factor in the machinery replicating, transcribing, and translating genomic information52. As a structural cofactor, Mg2+ stabilizes the ribosome, lipid membranes, and nucleic acids53. It is pivotal in metabolic networks and signaling cascades where it regulates enzyme activity, especially those requiring ATP56. Significantly Mg2+ alters the electrophysiological properties of ion channels55 and can also affect the binding affinity of Ca2+ to specific Ca2+- binding proteins61, thus altering the iCa2+ dynamics and signaling62. Most cell types’ total [iMg2+] is ~20 mM64. The free [Mg2+] is typically in the range of ~0.5–1.0 mM, and a considerable proportion of the iMg2+ pool exists as ATP-bound Mg2+ 64. Remarkably, the free [iMg2+] is 100 fold below the electrochemical potential, theoretically indicating a tight regulation of iMg2+ entry37. Regulation of iMg2+ homeostasis occurs via numerous Mg2+ transport machinery on the plasma membrane, including the ubiquitous MagT1, ACDP, TRPM7, SLC41A1, and tissue-specific TRPM6, as well as cyclin M2 and cyclin M479. Though these findings have enhanced the knowledge of the occurrence of significant Mg2+ fluxes in either direction across the plasma membrane of mammalian cells following metabolic or hormonal stimuli, little is known about the organellar mobilization of Mg2+. Nuclei, mitochondria, and endoplasmic or sarcoplasmic reticulum (ER/SR) compartmentalize iMg2+.Next to potassium (K2+ (mMg2+) is known to significantly impact the metabolic state93, mitochondrial Ca2+ (mCa2+) homeostasis97, and apoptosis98. Mitochondria can accumulate and release Mg2+ in response to metabolic stimuli, thus representing a critical iMg2+ store100. Mitochondrial RNA splicing 2 (Mrs2) was identified as the primary Mg2+ transporter in yeast mitochondria36 and formed a selective pore for lactatemediated mMg2+ uptake in mammals39, authenticating Mrs2 as the conserved mMg2+ transport machinery from yeast to mammals. Though Mrs2 has been shown as the transport machinery of mammalian mitochondria, Mrs2 has not been characterized. Here using the purified mammalian Mrs2 protein and the state-of-the-art planar lipid bilayer electrophysiological measurements, we characterized Mrs2 as the authentic mammalian mitochondrial Mg2+ channel  to be critical for Mrs2 regulation and function.Mitochondrial Mg channel . Using b channel . Recent 2+ transients; a fast uptake of Ca2+ followed by a slower and more gradual uptake108. Studies show increasing extramitochondrial Mg2+ ([Mg2+]out) to have a differential effect on these two modes of mCa2+ transients. Early initial velocity studies suggest Mg2+ be a competitive inhibitor for mCa2+ uptake where it induces sigmoid kinetics, and the degree of sigmoidicity increases with an increase in [Mg2+]out without altering the maximal activity of the uniporter108. However, subsequent studies on kinetics reported a non-competitive inhibition of mCa2+ uptake by [Mg2+]out and are known to decrease the maximal activity of the uniporter108. To define the mechanism by which Mg2+ regulates mCa2+ uptake, we previously resolved the atomic structure of the conserved N-terminal domain of MCU33. The crystal structure of the N-terminal domain revealed a β-grasp-like fold containing the MCU regulating acidic patch (MRAP)33. MRAP binds Mg2+/Ca2+ with ~mM affinity. The binding of divalent cations to MRAP destabilizes MCU, shifts the self-association equilibrium to monomer, and attenuates MCU-mediated mCa2+ uptake33. We anticipate divalent cations to regulate MCU activity based on our previous study.Mitochondria display two different profiles of Ca2+ threshold for MCU activity111. However, recent seminal work showed that the regulation of MCU activity is set not only by the Ca2+ affinities of the MICU1/2 EF hands but also by matrix [Ca2+] and buffering capacity, allowing for enhanced regulation of mCa2+ homeostasis34. It is known that matrix [Ca2+] regulates MCU channel activity with a biphasic concentration dependence, with potent inhibition at ~100 nM and maximal channel inhibition (~80%) at ~400 nM34. Interestingly, high BAPTA (5 mM) and low matrix [Ca2+] also inhibit MCU activity34. If MCU is regulated by matrix [Ca2+], this result was unpredicted as the matrix Ca2+ regulatory sites should be unoccupied, and MICU1/2 should be fully Ca2+-liganded under the IMCU record conditions, which together would be expected to activate the channel. Nevertheless, channel inhibition was observed.Previously regulation of MCU channel activity was defined by the cooperative activation of MICU1/2 that sets a cytosolic Ca2 in the pipette solution in all their IMCU current measurements. Remarkably removal of Mg2+ from the pipette solution completely abolished MCU channel inhibition observed in high-[BAPTA]/low matrix [Ca2+] and also in 400 nM Ca2+/1.5 mM EGTA. Therefore, Mg2+ binding to the MRAP region might underlie channel inhibition observed in high-[BAPTA]/low [Ca2+]m. Though the possible role of matrix [Mg2+] in regulating MCU activity was proposed in earlier studies34, the molecular mechanism was not explored in detail. To understand the regulation of MCU by matrix [Mg2+], we generated a liver-specific Mrs2 KO mouse model  injury  injury . We anti+ translocation and H+ backflow through the mPTP channel . We also anticipate a fall in the electrochemical proton gradient and membrane potential depolarization in Mrs2 KO hepatocytes during early stages of development. The compensatory pathways induced by the loss of Mrs2 during development possibly reversed the direction of ATP synthase rotation, resulting in ATP hydrolysis. We anticipate the reversal of ATP synthase to extrude protons into the intermembrane space and contribute to restoring the proton gradient and the membrane potential while simultaneously resulting in a net loss of ATP116. We used Oligomycin and BTB treatment to show the reverse activity of ATP synthase . Oligomycin blocks ATP hydrolysis and synthesis, whereas BTB is a specific ATP hydrolysis inhibitor. If the respiratory chain can keep up the membrane potential intact, then oligomycin treatment will hyperpolarize the mitochondria; however, if membrane potential is maintained by ATP hydrolysis, oligomycin/BTB will induce depolarization, a phenomenon termed as “oligomycin null-point”119. ATP hydrolysis can occur in extreme pathological conditions or normal or mildly compromised mitochondria, such as in humans with mitochondrial genetic disease and myopathies120. Our results demonstrate oligomycin null-point in Mrs2 KO mitochondria . We show in Mrs2 KO mitochondrial membrane potential to be maintained by the reversal of ATP synthase, thus depleting cellular ATP and activating mPTP opening121. We anticipate the increased matrix [Ca2+] to replace the available Mg2+ bound to the F1-F0 ATP synthase, inciting a conformational change in the functional protein dimer to the PTP114. Future studies will be necessary to understand the regulation of mPTP by loss of matrix [Mg2+] and the reversal of ATP synthase.Interestingly, our data show Mrs2 KO to have decreased ATP levels despite hyperpolarized mitochondria, suggesting an uncoupling of chemical catalysis from H2+], ~33 – 60% of MRAP will be occupied. Therefore, even at the highest non-physiological matrix [Mg2+], we do not anticipate a complete loss of MCU activity, thus maintaining the physiological MCU activity and energy homeostasis during pathology. Also, we show that Mrs2-mediated mitochondrial Mg2+ uptake is a critical regulator to prevent MCUmediated Ca2+ overload during I/R injury. However, future studies must study how MCU-mediated Ca2+ overload occurs during I/R injury, despite Mrs2-dependent MCU regulation in reality. We anticipate the loss of Mrs2 (either decreased channel density or decreased activity) to relieve MCU from Mg2+-dependent negative feedback regulation and increase MCU-mediated Ca2+ overload during disease pathology.Our results show expression of gain-of-function Mrs2 mutant to protect cells from mPTP opening . From ou2+] is pivotal in preserving mitochondrial function and facilitating adaptation to increased workload and disease states. Thus, changes in the free matrix [Mg2+] due to mitochondrial Mg2+ entry or changes in matrix phosphate, ATP, or ADP levels, the MRAP domain of MCU will sense these differences and regulate MCU channel activity.In conclusion, our results show that regulating MCU by matrix out) clearance as an indicator of mCa2+ uptake was achieved by loading the permeabilized cells with JC-1 (800 nM) and Fura2-FF (0.5 μM), respectively. Fluorescence was monitored in a multi-wavelength excitation dual-wavelength emission fluorimeter . [Ca2+]out is represented as the excitation ratio (340 nm/380 nm) of Fura2-FF/FA fluorescence, and Δψm as the ratio of the fluorescence of J-aggregate (570 nm excitation/595 nm emission) and monomer (490 nm excitation/535 nm emission) forms of JC-1. A single or series of Ca2+ bolus  and mitochondrial uncoupler, CCCP (2 μM), were added at the indicated time points. All the experiments were performed at 37°C with constant stirring128.An equal number of Mrs2To measure mMg2+ uptake, we performed a similar experiment using the ratiometric dye, Mag-Fura-2 tetra potassium salt. Mag-Fura-2 was calibrated, and the [mMg2+] was determined by MCU) were performed at 30°C, as reported earlier128. In brief, freshly prepared mitoplasts from HeLa, HEK293 cells, or hepatocytes of Mrs2fl/fl and Mrs2Δhep mice were plated on the Cell-Tak–coated coverslips and mounted on the microscope. The mitoplasts were bathed in a solution containing 150 mM sodium gluconate, 5.4 mM KCl, 5 mM CaCl2, and 10 mM HEPES (pH 7.2). The pipette solution contained 150 mM sodium gluconate, 5 mM NaCl, 135 mM sucrose, 10 mM HEPES, and 1.5 mM EGTA (pH 7.2). After the formation of GΩ seals (20 to 35 MΩ), the mitoplasts were ruptured with a 200 to 400 mV pulse for 2 to 6 ms. After capacitance (2.5 to 3.0 pF) compensation, mitoplasts were held at 0 mV, and IMCU was recorded with a voltage ramp . The external/bath solution (5 mM Ca2+) was chosen based on our previous measurements128. Samples were discarded if the break-in took longer than 5 s after adding 5 mM Ca2+. IMCU currents were recorded using an Axon200B patch-clamp amplifier with a Digidata 1320A acquisition board . For measuring the effect of matrix Mg2+ on IMCU, varying concentrations of Mg2+ were present in the pipette solution.Mitoplast patch-clamp recordings for MCU , HEPES (10 mM) (pH 7.4 with N’-Methyl-Dglucamine (NMDG)), NaCl (5 mM), sucrose (135 mM), and 5 mM MgCl2. In some measurements, 200 nM ruthenium red (Ru360) or 1 μM hexamine cobalt(III)chloride was added to the bath. IMrs2 was recorded as described above.Mitoplasts were prepared from Mrs22+ during recordings on the cis side. α-L-phosphatidylcholine (Sigma) was used for forming the bilayer membrane. ePatch amplifier (Elements) was used for lipid bilayer recordings. Purified Mrs2  was used for single-channel recordings, and Co2+  was added into the bath during the recordings without perfusion. pCLAMP-10 software was used for electrophysiology data acquisition and analysis (Molecular devices). All current measurements were adjusted for the holding voltage assuming a linear current-voltage relationship: The resulting conductances are expressed in pS according to the equation G = I/V where G is conductance in pS, V is the membrane holding voltage in mV, and I is the peak membrane current in pA after subtraction of the baseline electrode leak current. Group data were quantified in terms of conductance. All population data were expressed as mean ± SEM.The construct for Mrs2 was expressed in HEK 293 cells and purified using the EZ view Red ANTI-FLAG M2 Affinity Gel (Sigma), according to the manufacturer’s protocol. A silver-stained gel assessed the purity of isolated Mrs2 protein. Planar lipid bilayer recordings were performed in 150 mM NMDG and 10 mM HEPES solution buffered at (pH 7.4); supplemented with 20–104 mM Mgfl/fl/Mrs2Δhep /HEK293 NegShRNA/Mrs2KD cells using CellTiter-Glo® luminescent assay as per manufacturer’s instructions128.Total ATP abundance was assessed in Mrs2fl/fl/Mrs2Δhep /HEK293 NegShRNA/Mrs2KD cells were subjected to oxygen consumption rate (OCR) measurement at 37°C in an XFe24 extracellular flux analyzer (Seahorse Bioscience). Cells (2.5 × 105) were sequentially exposed to 2 μM oligomycin, 0.5 μM FCCP, and 0.5 μM rotenone plus antimycin A at indicated time points to measure basal and maximal respiration, ATP production, proton leak, spare respiratory capacity, and non-mitochondrial respiration as described previously128.Intact Mrs26 HeLa cells grown on 0.2% gelatin-coated glass coverslips were co-transfected with Cox8-mRFP and Mrs2-GFP. Forty-eight hours post-transfection, confocal images (Leica SP8) were obtained at 561 and 488 nm excitation using a ×63 oil objective. Overlap of RFP and GFP signals was quantified using Leica LAS-X software. (ii) Mitochondrial localization of Mrs2: SIM super resolution microscopy. 0.5×106 HeLa cells grown on 0.2% gelatin-coated glass coverslips were co-transfected with Cox8-mRFP and Mrs2-GFP. Forty-eight hours post-transfection, cells were fixed with 4% paraformaldehyde and stored at 4°C until imaging. The slides were imaged under a Nikon N-SIM/STORM SR microscope in SIM mode with an SR Apo TIRF100x, NA1.49 objective, and the images were captured with an Andor DU 897x EMCCD camera. The GFP channel was excited by a 488 nm laser to collect an emission peak at 522 nm, while the RFP was excited by a 561 nm laser to obtain an emission peak at 605 nm. Images taken in 3D SIM mode were processed and reconstructed with Nikon Elements. The reconstructed images were analyzed for colocalization in Nikon Elements and ImageJ. (iii) TMRM and Calcein fluorescence (Time-series/intact cell) measurements: 0.5×106 hepatocytes isolated from Mrs2fl/fl and Mrs2Δhep mice grown on 0.1% collagen-coated glass coverslips were stained with the Δψm indicator TMRM or PTP opening indicator calceinAM. Coverslips were mounted in an open perfusion micro incubator  and imaged at 561 or 488 excitations using a ×63 oil objective. After baseline measurements, ionomycin (5 μM) was added, and changes in TMRM or calcein fluorescence were recorded every 3 s (Leica SP8) for 10 mins. The loading and imaging buffer contained cobalt chloride  for calcein measurement. The presence of CoCl2 quenched the calcein fluorescence in the cytosol and nucleus, leaving the mitochondrial fluorescence intact. As the PTP opens, CoCl2 enters the mitochondrial matrix and quenches calcein fluorescence. The decrease in calcein fluorescence after ionomycin treatment was measured using the Leica LAS-X software. (iv) Confocal imaging of mitochondrial Δψm: 0.5×106 hepatocytes isolated from Mrs2fl/fl and Mrs2Δhep mice grown on 0.1% collagen-coated glass coverslips were stained with the Δψm indicator TMRM and dihydrorhodamine 123 (DHR123). Coverslips were mounted in an open perfusion micro incubator  and imaged at 561 or 488 excitations using a ×63 oil objective. Images were analyzed, and the mean TMRM fluorescence was quantified using Image J software (NIH). (v) Measurement of cytosolic Mg2+ dynamics. 0.5×106 hepatocytes isolated from Mrs2fl/fl and Mrs2Δhep mice grown on 0.1% collagen-coated glass coverslips were stained with MagGree-AM and MitoTracker Red. Coverslips were mounted in an open perfusion micro incubator  and imaged at 488 and 561 excitations using a ×63 oil objective. After baseline measurements, glucagon (10 μM) and 2 mM Mg2+ were added at the indicated time. MagGreen fluorescence changes  were recorded every 3 s (Leica SP8) for 10 mins. The rate of cytosolic MagGreen decay and the mitochondrial peak fluorescence was measured using Leica LAS-X software.(i) Mitochondrial localization of Mrs2, confocal imaging: 0.5×10fl/fl and Mrs2Δhep were homogenized in ice-cold mitochondrial isolation buffer  containing 1 mg/ml fatty acid-free bovine serum albumin. The homogenate was centrifuged for 10 min at 1000 x g, and the supernatant was centrifuged again at 14,000 x g for 10 min. The mitochondrial pellets were washed twice and centrifuged at 11,200 xg. The isolated mitochondria (1 mg protein) were added to 0.2 ml of mitochondrial swelling buffer . Mitochondrial swelling was measured by the decrease in absorbance at 540 nm after adding Ca2+ (250 μM)27.Hepatocytes isolated from Mrs2fl/fl and Mrs2Δhep were treated with ionomycin (25 μM) for 6 hr. For necrotic cell death measurement, cells were stained with propidium iodide. The cells were analyzed with the BD LSR Fortessa (BD Biosciences). Relative PI staining was plotted on a logarithmic scale using Flow-Jo software38.Hepatocytes isolated from Mrs2125.Gel filtration was performed by fast protein liquid chromatography , and the Superdex 200 10/300 column was equilibrated with PBS. Column calibration was carried out with a gel filtration protein standards kit (Bio-Rad). Cleared lysates from HEK293 NegShRNA and Mrs2KD cells stably expressing MCU-FLAG were directly loaded onto a Superdex 200 FPLC column at a 0.5 ml/min flow rate. Forty fractions were collected, concentrated, and fractionation of MCU complex was assayed by Western blot analysisPlasma aspartate (AST) and alanine (ALT) aminotransferase activities were measured using a commercially available kit (Cayman) as per the manufacturer’s instruction. We used Mouse Cytokine Array Panel A  to determine the relative levels of 40 mouse cytokines.The median and left liver lobes were harvested and fixed in 4% formalin. Fixed tissues were subsequently sectioned, ten μm thick, collected on (+) charge slides, and stained with hematoxylin and eosin. Examination and scoring of each lobe were carried out by a pathologist who was blinded to the experimental group.Data from multiple experiments (≥3) were quantified and expressed as Mean ± SE, and differences between groups were analyzed using the two-tailed paired Student’s t-test or, when not normally distributed, a nonparametric Mann–Whitney U-test (Wilcoxon Rank-Sum Test) for two groups. The data were computed with GraphPad Prism version 9.0 or SigmaPlot 11.0 software. Differences in means among multiple datasets were analyzed using one-way ANOVA with Tukey correction performed. A P ≤ 0.05 was considered significant in all analyses.


The micronutrient manganese (Mn) plays essential roles in diverse biological processes, including photosynthesis, antioxidant defense and cell wall biosynthesis (Marschner 2+) is a critical signaling transducer in all eukaryotic organisms. In plants, Ca2+ signals are decoded by two signaling networks, Ca2+-dependent protein kinases (CPKs) and the CBL-CIPK network composed of calcineurin-B-like Ca2+ sensor proteins (CBLs) and CBL-interacting protein kinases (CIPKs)  of Arabidopsis roots exhibited oscillatory Ca2+ signals after exposure to low Mn. These Ca2+ oscillations initiated 126 ± 4 min after exposure to low Mn and displayed a frequency of 31 ± 6 min and amplitude of 0.14 ± 0.04 ΔR/R0 on average. They also observed a remarkable differentiation of Ca2+ oscillation formation in regard to its longitudinal and transversal dimensions. Together, their results revealed an interesting finding that Mn deficiency induces long-lasting multicellular Ca2+ oscillations in the EZ of Arabidopsis roots, although whether these Ca2+ oscillations are triggered directly by Mn deficiency or indirectly by Mn deficiency-induced other changes such as growth inhibition remains further clarification.Previously, a group of researchers from Northwest A&F University  have used aequorin reporter to detect Ca2+ signals in response to Mn deficiency, Fu et al. (cpk mutants and mutant combinations and then examined which mutants showed impaired plant growth under Mn deficiency. Based on this phenotypic analysis, they revealed that CPK21/23 but not CPK4/5/6/11 are redundantly involved in the regulation of low Mn tolerance. They therefore further carried out protein interaction assays to confirm that CPK21 and CPK23 interact directly with NRAMP1 both in vitro and in vivo.To identify components potentially decoding the oscillating Cau et al.  performenramp1 complementation assays and persuasively demonstrated that NRAMP1T498 phosphorylation is required for plant high-affinity Mn uptake and low Mn tolerance.Next, by in vitro kinase assays, Fu et al.  identifiT498 phosphorylation in plants, which relies on the presence of CPK21/23, and overexpression of CPK23 increases plant tolerance to low Mn via the regulation of NRAMP1. Overall, their results revealed a key Ca2+ signaling mechanism for plant tolerance to Mn deficiency. In this mechanism, Mn deficiency triggers Ca2+ oscillations in the root EZ, which activate CPK21/23 kinases that promote NRAMP1 phosphorylation at Thr498 site, and the NRAMP1T498 phosphorylation increases its transport activity and consequently enhances plant Mn uptake and tolerance to Mn deficiency. Thus, the study by Fu et al. (2+ signaling modulates plant Mn uptake but also provides a new tool to enhance plant tolerance to Mn deficiency by engineering NRAMP1 phosphorylation.Finally, Fu et al.  demonstru et al.  not only