Intensive chemotherapy resulted in complete remission (CR) and CR with incomplete count recovery (CRi) rates of 44 and 33%, respectively, with no difference in survival outcome between the two (p = 0.4). Allogeneic hematopoietic stem cell transplant (AHSCT) was documented in 259 patients and provided the best survival rate . After a median follow-up of 13 months, 841 (75%) deaths were recorded. Multivariate analysis identified age >60 years , adverse karyotype , intermediate-risk karyotype , post-myeloproliferative neoplasm AML , and other secondary AML (HR 1.3 (1.1–1.6) as risk factors for shortened survival. These risk factors retained their significance after inclusion of FLT3/NPM1 mutational status in 392 informative cases: FLT3+NPM1− , FLT3+/NPM+ (HR 2.6 (1.3–5.2), and FLT3−NPM1− .Between 2004 and 2017, a total of 1123 adult patients  with newly diagnosed acute myeloid leukemia (AML), not including acute promyelocytic leukemia, were seen at the Mayo Clinic. Treatment included intensive ( From a clinical perspective, AML is subcategorized into primary (de novo) and secondary. The latter includes AML arising from antecedent myeloid malignancies, such as myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN), MDS/MPN overlap, and therapy-related AML.Acute myeloid leukemia (AML) is a rapidly progressing clonal hematopoietic stem cell neoplasm that results in premature death and major comorbidity. The 2016 World Health Organization (WHO) classification system uses blood or bone marrow blast percentage and cytogenetic and molecular information in defining and classifying AMLRUNX1-RUNX1T1), inv(16)(p13.1q22) or t(CBFB-MYH11), and t(PML/RARA), regardless of the blast percentage2. The diagnosis, prognostication, treatment, and monitoring of acute promyelocytic leukemia (APL) is uniquely different from other AML subtypes.Current diagnosis of AML requires the presence of ≥20% myeloid blasts circulating or in the bone marrow; in addition, diagnosis of AML is implied in the presence of certain recurrent cytogenetic abnormalities, such as t or molecular profile to start induction therapy in non-APL AML, even in FLT3-ITD-positive cases where midostaurin is started on day 85. Long-term disease-free survival in AML is possible only with consolidation therapy. This may be in the form of several cycles of chemotherapy or allogeneic hematopoietic stem cell transplant (AHSCT) in the favorable and adverse risk groups, respectively.Treatment of non-APL AML has not changed, in a substantial manner, in the past four decades except the recent addition of some targeted therapies with uncertain long-term benefit. The “standard” involves combination chemotherapy  as induction with the goal of achieving complete remission (CR)In the current study, we share our 14 years’ real-life experience in 1123 consecutive and cytogenetically annotated adult AML patients seen at the Mayo Clinic. Our objectives were (1) to describe clinical and laboratory features and treatment outcome and real-life experience of non-APL AML in a large cohort of unselected adult patients seen at a single institution and (2) to examine the application of previously established risk factors in our study population.NOS, myeloid sarcoma, and AML of ambiguous linage. Patients were subcategorized into primary (de novo) and secondary AML; the latter was further subdivided into AML with MDS-related changes, AML following MPN, chronic myelomonocytic leukemia (CMML) or MDS/MPN, and therapy-related AML. In all instances, bone marrow examination and cytogenetic studies were performed or reviewed at the Mayo Clinic. Cytogenetic analysis and reporting of results was done according to the International System for Human Cytogenetic Nomenclature criteria9. Cytogenetic risk stratification into favorable, intermediate, and adverse groups was based on the 2017 European LeukemiaNet (ELN) criteria2. Whenever available, we have added the molecular profile to this risk stratification. Commonly available molecular information included FLT3-ITD, NPM1, and CEBPA mutational status. Favorable cytogenetic risk category included t, RUNX1-RUNX1T1; inv(16)(p13.1q22) or t, (CBFB-MYH11). Adverse cytogenetic risk category included tDEK-NUP214; t;KMT2A rearranged; t;BCR/ABL1; inv(3)(q21.3q26.2) or t; GATA2,EV1 (or MECOM); −5 or del(5q); −7; −17/abn(17p); and complex karyotype. Intermediate cytogenetic risk included normal karyotype and abnormal karyotype not included the in favorable or adverse groups. Follow-up information was updated as of May 2018 by reviewing charts, telephone calls to patients and their local hematologist, and using social security death index as necessary. For patients with a diagnosis of therapy-related AML, individual patient information was reviewed meticulously about their diagnosis and exposure to chemotherapy and/or radiation.The current study was approved by the Mayo Clinic institutional review board. One thousand one hundred and twenty-three consecutive and cytogenetically annotated patients with the diagnosis of non-APL AML were identified from the Mayo Clinic AML database and the study period spanned from January 1, 2004 through December 31, 2017. Study eligibility criteria included age ≥18 years, diagnosis of AML based on 2016 WHO criteria, and availability of cytogenetic data. The following AML subcategories were included: AML with recurrent cytogenetic abnormalities, AML with MDS-related changes, therapy-related AML, AML following MPN, AML-10. CR was defined as normal bone marrow morphology with <5% blast, absolute neutrophil count >1 × 109/L, and platelet count >100 × 109/L. CR with incomplete blood count recovery (CRi) met all the criteria for CR with the exception of either platelet or neutrophil count recovery; patients not categorized as CR or CRi were classified as “no remission”.Type of therapy included induction (or intensive) chemotherapy, less intensive treatment such as with hypomethylating agents (HMA), and supportive care alone. Information with regard to marrow recovery was collected to evaluate CR according to International Working Group recommendationp value <0.05. Overall survival was calculated from the date of diagnosis to death regardless of cause, and patients who were alive were censored at last follow-up. Patients who underwent AHSCT were censored at the date of transplant. Relapse-free survival was calculated from the date of reported CR to documented peripheral or bone marrow relapse with blast percentage of ≥5%; patients without relapse were censored at the date of last follow-up. Overall and relapse-free survival curves were prepared using Kaplan–Meier method and compared using log-rank test. Multivariate Cox-regression analyses were used on pretreatment variables to identify significant risk factors. The JMP® Pro 13.0.0 software  was used for all calculations.Statistical analysis considered clinical and laboratory variables at the time of diagnosis and as necessary at the time of follow-up or remission. Median (range) and frequencies (percentage) were used for continuous and categorical variables. Those variables were compared based on the time of diagnosis (2004–2010 vs 2011–2017), among the subtypes of AML, and the two main subgroups (primary vs secondary). Categorical variables were compared using chi-square and considered significant for n = 449) of patients were seen from January 2004 to December 2010, and the rest after January 2011 (n = 674); more patients with secondary AML (48%) were diagnosed in the second half of the study period vs the first half (39%), and more patients (17%) received HMA in the second half of the study period vs the first half (7%), which might have been related to the approval and availability of HMAs.A total of 1123 consecutive patients  with AML met the inclusion criteria; the clinical and laboratory characteristics of the study patients are outlined in Table AML was primary in 626 (56%) patients and secondary in 497 (44%); the latter included 388 patients with antecedent myeloid neoplasm and 109 therapy-related AML. Comparison of clinical, laboratory, and outcome measurements between primary and secondary AML are outlined in Table n = 626, median age 62 years, range 18–92) were younger than those with either secondary  or therapy-related AML  (p < 0.01). As expected, more patients with adverse karyotype were seen in secondary (52%) and therapy-related (51%) vs primary (27%) AML , post-CMML/MDS/MPN overlap (n = 30), and post-MPN (n = 89) AML, were 10, 8, and 5 months, respectively , and post-MDS AML  had a better survival (median 22 months) compared to those receiving less intensive chemotherapy  or supportive care alone ; there was no statistically significant difference in survival in patients achieving CR vs CRi  deaths and 241 (44%) relapses were documented. The median overall survival for patients diagnosed from 2004 to 2010 and from 2011 to 2017 was 11 and 15 months, respectively; age-adjusted multivariate analysis including other risk factors confirmed improved survival in patients diagnosed in the more recent period (n = 47) displayed significantly better overall survival (median 148 months) compared to those with either intermediate  or adverse karyotype /NPM1 (+)  and FLT3-ITD (+)/NPM1 (+) cases  compared to those with FLT3-ITD (+)/NPM1 (−)  and a double unmutated profile .FLT3/NPM1 mutational status in 392 informative cases confirmed the adverse prognostic effect of age >60 years , adverse karyotype , post-MPN AML , other secondary AML , and FLT3+NPM1− , FLT3+/NPM+ (HR 2.6 (1.3–5.2), and FLT3−NPM1−  profile. We have published the impact of mutations  in non-favorable karyotype11. Results were unchanged when survival was censored at the time of ASCT. Two hundred and forty-one relapses were documented during the study period. Median survivals of patients with and without relapse were 23 and 154 months, respectively (p < 0.01); median relapse-free survival of patients with and without AHSCT was 59 and 27 months, respectively.Multivariate analysis of pretreatment parameters evaluable in all 1123 patients identified age >60 years (hazard ratio (HR) 2.2, 1.9–2.6), adverse karyotype , intermediate-risk karyotype , post-MPN AML , and other secondary AML (HR 1.3 (1.1–1.6) as risk factors for shortened survival; the inclusion in the model of n = 1123). At diagnosis, the median age was 65 years and with male predominance (3:2) and is comparable to US census data where the median age was reported to be 68 years .We report the largest cytogenetically annotated single-center real-world management experience of patients with non-APL AML  displayed better overall survival than those diagnosed earlier (2004–2010); this may be attributed to continuing improvement in supportive care therapy, including frequent use of prophylactic antibiotics. In addition, more patients (83 vs 78%) received therapy during 2011–2017, including 78% of those who received less intensive therapy. This may be attributed to the wide availability of and physician preference to less intensive therapy.14. In general, patients with primary AML have favorable host factors that enable them to tolerate more intensive chemotherapy. Our observations in this regard are similar to a previously published population-based cohort study13.As expected, patients with primary AML enjoyed better overall survival, compared to those with secondary or therapy-related AML. Multiple factors contribute to this observation, including younger age, better performance status, lesser likelihood of co-morbid conditions, and more favorable cytogenetic and mutation profile, associated with primary vs secondary/therapy-related AML18. Similar to multiple other previously published studies, the current study confirms the value of risk stratification based on age, eligibility for AHSCT, karyotype, and mutation profile. In the current study, unfavorable karyotype was more common in older patients and associated with poor outcome, supporting the rationale to wait for cytogenetic and molecular reports in such group of patients before initiating therapy, as previously suggested by another study19. Similar to other studies, NPM1 mutation in the current study was associated with better survival both in the presence and absence of FLT3-ITD and the latter without NPM1 mutation carried the worst prognosis20.Regardless of other factors, patients who received intensive chemotherapy displayed a better overall survival in comparison to those receiving less intensive or no therapy. Furthermore, the current study suggested that the quality of complete remission (CR vs CRi) did not affect overall survival. In this regard, our observation is somewhat different from that of Greef et al. and Walter et al.21. Despite the origin of myeloid blasts, whether primary or secondary, it is the genetic composition of blasts that primarily determines the prognosis and outcome to therapy25. Almost all patients with AML harbor somatic genomic mutations that are often categorized as driver (disease initiation) and second hit or passenger (responsible for disease manifestations and/or progression) mutations. Some of the driver mutations 26 are commonly identified as founding clones29, and may be found in asymptomatic elderly individuals31, rarely cause overt leukemia by themselves but may decrease the threshold for malignant transformation. Some of the passenger mutations may include signal transduction , NPM1, spliceosome , chromatin regulation , or cohesin (STAG2) genes, and others may accelerate the progression to overt leukemia32. Occasionally, passenger mutations, similar to specific cytogenetic abnormalities, may help to separate de novo AML from secondary AML35. Driver mutations may serve as a substrate for relapse after chemotherapy or AHSCT.The prognosis and outcome for non-APL AML depends on both host factors and the biology of the leukemic blasts and their microenvironment. The individual host factors that may affect prognosis and decision with regard to choice of therapy may include age, performance status, and co-morbidityFLT3 and IDH inhibitors) and incorporation of Venetoclax into low-intensity chemotherapy.Despite the limitation of this study because of its retrospective nature, single center, lack of molecular data in the majority, and underrepresentation of newly approved drugs, this real-life experience study could serve as a benchmark against which we may measure outcomes of newly approved therapies. Finally, we were encouraged by the improvement in survival overtime and further improvement is likely, considering the introduction of targeted therapy (e.g., Supplemental Figure 1


In contrast to other cells, they are fully devoid of organelles. Similar to apoptosis of nucleated cells erythrocytes may enter suicidal death, eryptosis, which is characterized by the presentation of membrane phosphatidylserine on the cell surface and cell shrinkage, hallmarks that are also typical of apoptosis. Eryptosis may be triggered by an increase in the cytosolic Ca2+ concentration, which may be due to Ca2+ influx via non-selective cation channels of the TRPC family. Eryptosis is further induced by ceramide, which sensitizes erythrocytes to the eryptotic effect of Ca2+. Signaling regulating eryptosis further involves a variety of kinases including AMPK, PAK2, cGKI, JAK3, CK1α, CDK4, MSK1/2 and casein kinase. Eryptosis-dependent shrinkage is induced by K+ efflux through Ca2+-activated K+ channel KCa3.1, the Gardos channel. Eryptotic cells are phagocytosed and may adhere to endothelial cells. Eryptosis may help prevent hemolysis since defective erythrocytes usually undergo eryptosis followed by rapid clearance from circulating blood. Excessive eryptosis stimulated by various diseases and xenobiotics may result in anemia and/or impaired microcirculation. This review focuses on the significance and mechanisms of eryptosis as well as on the ion fluxes involved. Moreover, a short summary of further ion transport mechanisms of the erythrocyte membrane is provided.Erythrocytes are among the most abundant cells in mammals and are perfectly adapted to their main functions, i.e., the transport of O With a al cells .2+ influx and Ca2+-dependent enzymatic digestion of intracellular proteins as well as breakdown of the phosphatidylserine asymmetry of the cell membrane  . AlthougCells of the mononuclear phagocyte system (MPS) such as macrophages recognize phosphatidylserine on the surface of eryptotic cells by means of specific receptors . This inThe presentation of phosphatidylserine on the surface of eryptotic cells can have two major pathophysiological implications: On the one hand, it initiates phagocytosis of erythrocytes , on the Excessive eryptosis initiating the phagocytosis of many red blood cells may therefore result in an acute loss of erythrocytes, i.e., anemia . In lineThe adherence of eryptotic erythrocytes to vascular endothelium cells also mediated by the phosphatidylserine receptor may impair microcirculation. Hence, stimulators of eryptosis may not only cause anemia, but also cardiovascular complications due to impeded microcirculation .Plasmodium falciparum, an unicellular eukaryote  determine the distribution of phosphatidylserine among the inner and outer membrane leaflet : Flippasramblase . Upon inIn patients with sickle cell disease, phosphatidylserine externalization is found in subsets of reticulocytes, young cells, and partly also in mature erythrocytes . Interes+ ions which is paralleled by efflux of Cl– and osmotically obliged water  encoded by KCNN4  that exhibit a relatively high intracellular Nantration . Such eronophore .2+ concentration is an early event in the orchestration of eryptosis and initiates hallmarks of eryptosis, phosphatidylserine externalization and cell shrinkage (2+ concentration is in the range of 50 nM (2+ concentration (around 1.5 mM) and comparable to the free cytosolic Ca2+ concentration in nucleated cells. Total cytosolic Ca2+ also includes Ca2+ ions bound by Ca2+-binding proteins such as calmodulin and amounts to 5–6 μM  (2 (PGE2) is an important eicosanoid, contributing to pain by sensitizing peripheral nociceptors, to fever and local inflammation (– and hyperosmotic shock induce PGE2 synthesis in erythrocytes which in turn activates Ca2+ influx through the Ca2+-permeable cation channel (2 is a major regulator of eryptosis by inducing Ca2+ influx (Activators of the cation channels include prostaglandin E2 (PGE2) . Phosphoammation . The iso channel . Hence, + influx .2+ contribute to Ca2+ homeostasis in erythrocytes   . Accordihrocytes . This vi by PKCα . PKCα al by PKCα .2+ ATPase (PMCA) is expressed in human erythrocytes , the erythrocyte glutathione level is only 10% of the normal value , members of the CSF family , C-X-C chemokines , FGF growth factors (bFGF), IL-3, IL-5, IFNα2, IFNγ, members of the IL-1 family, LIF, IL-12(p40), IL-12(p70), IL-17, MIF, PDGF-bb, VEGF, TNFα, TNFβ, and TRAIL . WhetherA selection of important mechanisms of eryptosis is summarized in 2+ concentration results in lower O2 affinity of hemoglobin -dependent NO production is stimulated by an increase in the cytosolic Cantration . By the ntration .2+ concentration  is a cysteine protease that is activated by an elevation of the intracellular Cantration . It is entration  and degrntration . Calpainntration .Erythrocytes express the mechanosensitive non-selective cation channel PIEZO1 which is stretch-activated  and open– ions for HCO3– ions which is part of the mechanism of CO2 transport from peripheral tissues and organs to the lung (+ and/or K+ leakage (The anion exchanger 1 (AE1 or SLC4A1) is encoded by the SLC4A1 gene and is also known under the name Band 3 . The twothe lung . In thisthe lung . Several leakage . This ph leakage .P. falciparum endogenous inwardly and outwardly rectifying Cl– channels are activated , presumably VDAC3 . In the survival . In linesurvival .+, K+ as well as Ca2+ conductance. Simultaneously, glucose transport is impaired (Also the gene encoding glucose carrier GLUT1 which accomplishes glucose uptake by erythrocytes can be affected by a mutation that renders the carrier into a cation channel with Naimpaired .3) carrier  is an ammonia , the expression of a low conductance cation channel in erythrocytes was concluded in early studies . It is sIt is yet unknown whether and to which extent the further ion transport mechanisms presented in this section are required for the cellular machinery initiating and executing eryptosis. Clearly, future studies are needed to address this issue.Ion transport in eryptosis is still incompletely understood. Future research should address the molecular identity of the channels and transporters involved. The specific pharmacological targeting of ion transport mechanisms required for the execution of eryptosis may turn out to be favorable in different clinical conditions including anemia, impaired microcirculation, or malaria.MF and FL wrote the review. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Current models assume that cytosolic Ca2+ regulates workload and that mitochondrial Ca2+ uptake precedes activation of matrix dehydrogenases, thereby matching OXPHOS substrate supply to ATP demand. Surprisingly, knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice results in only minimal phenotypic changes and does not alter OXPHOS. This implies that adaptive activation of mitochondrial dehydrogenases by intramitochondrial Ca2+ cannot be the exclusive mechanism for OXPHOS control. We hypothesized that cytosolic Ca2+, but not mitochondrial matrix Ca2+, may adapt OXPHOS to workload by adjusting the rate of pyruvate supply from the cytosol to the mitochondria. Here, we studied the role of malate-aspartate shuttle (MAS)-dependent substrate supply in OXPHOS responses to changing Ca2+ concentrations in isolated brain and heart mitochondria, synaptosomes, fibroblasts, and thymocytes from WT and MCU KO mice and the isolated working rat heart. Our results indicate that extramitochondrial Ca2+ controls up to 85% of maximal pyruvate-driven OXPHOS rates, mediated by the activity of the complete MAS, and that intramitochondrial Ca2+ accounts for the remaining 15%. Of note, the complete MAS, as applied here, included besides its classical NADH oxidation reaction the generation of cytosolic pyruvate. Part of this largely neglected mechanism has previously been described as the “mitochondrial gas pedal.” Its implementation into OXPHOS control models integrates seemingly contradictory results and warrants a critical reappraisal of metabolic control mechanisms in health and disease.Mitochondrial oxidative phosphorylation (OXPHOS) and cellular workload are tightly balanced by the key cellular regulator, calcium (Ca The combination of MCU KO mice with the methodologically refined reconstituted, complete MAS assay allowed us to unequivocally unmask the OXPHOS control mechanisms that so far had escaped accurate assessment. Our data establish the complete MAS as the major provider of pyruvate for OXPHOS regulated by cytosolic, but not mitochondrial matrix, Ca2+ in the different models. This integrates seemingly contradictory results on Ca2+-mediated OXPHOS control and may provide the intellectual basis for novel therapeutic approaches to diseases where cell death is initiated by mitochondrial Ca2+ overload.To overcome the aforementioned limitations and to dissect the Ca2+ from different cellular compartments on the regulation of OXPHOS, we took advantage of an MCU KO mouse model established by Pan et al.   and E–H (heart)). Using RT-PCR analysis, we detected residual Mcu transcripts in MCU KO mice , reaching 7.3% in hippocampus and 3.9% in heart tissue as compared with WT . The observed residual transcripts may result from unintended gene-trap splicing  and may account for an observed small, but bioenergetically negligible, extent of Ca2+ uptake by MCU KO brain mitochondria ). Although not followed up specifically, both the residual MCU activity and other MCU-independent Ca2+ uptake pathways may be equally responsible for the minimal matrix Ca2+ content seen in MCU KO mice . A decrease in ADP-stimulated phosphorylating respiration triggered by Ca2+ overload, as seen in WT, was absent in MCU KO mitochondria and/or treatment with RR . We also examined whether the MCU KO-related subtle bioenergetic alterations affect the mouse phenotype in the given genetic background. Visual comparison revealed no abnormalities other than a small but significant decrease in body weight in MCU KO mice , confirming earlier results  and Morris water maze tests ). Generally, WT and MCU KO mice exhibited similar learning performances across all training trials except for a small but significantly decreased swim velocity of MCU KO mice . Taken together, our data confirm that MCU KO effectively impairs mitochondrial Ca2+ uptake while leaving the mouse physiology generally unaffected. This makes the MCU KO mouse strain an excellent model to study mitochondrial OXPHOS in response to free Ca2+ levels in different cellular compartments.To study the effect of Can et al. . We conf KO mice . To test2+-sensitive reaction  and E–H (heart)). To adjust optimal OXPHOS conditions, saturating amounts of ADP  were added, making the OXPHOS activity dependent on the rate of substrate oxidation (state 3). In mitochondria isolated from WT brain and heart tissue, maximal Ca2+-stimulated respiratory rates (Vmax = 100%) were reached at Ca2+ concentrations of 0.4 μm (brain) and 1.5 μm (heart) (red arrow in C and G). Of note, already prior to Ca2+ additions, mitochondrial respiratory rates reached ∼85% of Vmax and remained insensitive to Ca2+ stimulation when the MCU inhibitor RR was present. A similar observation was made previously in heart mitochondria prepared from rats kept on a standard diet . These findings were further validated by the use of isolated brain and heart mitochondria isolated from MCU KO mice and studied in the presence and absence of RR . A caveat of our approach, however, was that all experiments were performed at high pyruvate concentrations. We thus set up pyruvate titration experiments using isolated brain and heart mitochondria under phosphorylating (state 3) conditions at low (∼12 nm), moderately elevated (800 nm) Ca2+ concentrations as well as in the additional presence of RR (A and B (brain) and C and D (heart)). Our experiments revealed a substantial pyruvate dependence of respiratory rates. However, no difference was observed between the shape of the titration curves, indicating similar kinetic constants irrespective of the presence and/or absence of MCU activity. These results argue for a high basal, non-Ca2+-dependent, PDH activity, which makes OXPHOS regulation across broad physiologic ranges of workload through Ca2+ stimulation of mitochondrial pyruvate oxidation an unlikely condition. Conversely, our data indicate that even small changes within the range of physiological pyruvate concentrations of ∼175 μm in the brain (A–D). We conclude that MCU activity, and thus the presence of mitochondrial matrix Ca2+, is largely dispensable for OXPHOS activation when driven by PDH-dependent pyruvate oxidation but is required for stimulation of OXPHOS activity beyond 85% of Vmax.Pyruvate is a chief substrate for OXPHOS in brain mitochondria mainly generated by the complete MAS , 20. Itsreaction , 4, whicrix Ca2+ , C and Ghe brain  can easihe brain , A–D. We2+-independent glutamate carriers  through transamination and oxaloacetate formation facilitated by the malate dehydrogenase (MDH) as well as the exchange of malate against α-OG. Therefore, the Aralar-dependent pathway involves and represents the activity of the mitochondrial part of the MAS. This allowed the set-up of experiments specifically addressing the Ca2+ dependence of the mitochondrial glutamate metabolism without the pyruvate metabolization . A stepwise increase of Ca2+ revealed, in contrast to pyruvate oxidation, a three-phase respiratory response , whereas the mitochondrial membrane potential remained unaffected (E and G) and Ca2+ was not taken up (I and K). In phase 2 at Ca2+ concentrations above 400 nm, we observed an increase of both mitochondrial respiration (A and C) and membrane potential (E and G) as well as mitochondrial Ca2+ uptake (I and K). The detected mitochondrial Ca2+ uptake may account for the increase in respiratory rates (C) through the activation of intramitochondrial dehydrogenases, such as α-oxoglutarate dehydrogenase and isocitrate dehydrogenase. This notion is supported by an observed RR sensitivity. However, our data also indicate that the respiratory activation depends to a great extent on extramitochondrial Ca2+ . In phase 3 at high concentrations of Ca2+ (>1.3 μm), we observed decreased respiratory rates (A and C) and membrane potential (E and G) in WT mitochondria. The latter showed mitochondrial Ca2+ efflux (I and K). This phenomenon is known as mitochondrial Ca2+ overload toxicity and is fully abolished by the addition of RR. This finding is unlikely to be a substrate-specific artifact because similar effects were also seen for other substrate combinations  . Using isolated MCU KO brain mitochondria, we observed a similar Ca2+-dependent stimulation of respiration and membrane potential  with a very small but significant RR-sensitive Ca2+ uptake at concentrations above 1.5 μm (J and L) and DFig. S1). This RR-sensitive Ca2+ uptake affected respiratory rates (B and D) and mitochondrial membrane potential (F and H) and may be attributable to MCU activity based on residual Mcu transcript expression  or other nonspecific side effects of RR.We next tested glutamate-malate as a substrate combination. Of note, there are at least two different types of mitochondrial glutamate transporters , namely carriers , GC1 andd Citrin , 23–25. tate see A throughow in WT A. A stepresponse . In phaspiration , A and Caffected , E and Gtaken up , I and Kpiration , A and Cotential , E and G+ uptake , I and Kry rates C through2+-insensitive glutamate-malate–activated respiration of heart mitochondria was 2-fold larger but decreased significantly in the course of 30 min of preincubation . Ca2+ titration upon preincubation revealed Ca2+ responses comparable with those seen for brain mitochondria (A–L). Taken together, the Ca2+-dependent glutamate-malate–controlled OXPHOS in heart and brain mitochondria with stimulations up to 200–300% represents the minimal version of the MAS . The lack of further MAS substrates and enzymes (restricted complexity), however, limits full MAS capacity. Furthermore, in mitochondria of both origins (brain and heart), a Ca2+-independent glutamate-driven OXPHOS can be measured (brain < heart).Isolated heart mitochondria revealed a very similar behavior, essentially replicating the above experiments in brain mitochondria. In contrast to brain mitochondria, however, the Cachondria , A–L. Ta the MAS , mode 1 y Aralar A. The lain vitro and ex vivo to reach a system of continuous (steady-state) generation of pyruvate from lactate. Together with the use of specific inhibitors and Ca2+ chelators, this allowed us to unambiguously separate the role of cytosolic from mitochondrial matrix Ca2+ (A).To study the true impact of the MAS on pyruvate supply for OXPHOS activation, we reconstituted the MAS in a stepwise manner rix Ca2+ A.B and C). The MAS reconstitution assay was done in EGTA media (to adjust different Ca2+ levels) supplemented with the nonfeeding substrates malate, lactate, and NADH. Expectedly, the addition of 2 mm ADP had only minimal effects on respiratory rates (B). Next, the addition of glutamate, α-oxoglutarate, and aspartate adjusted MAS mode 2 . At this mode, we detected a Ca2+-sensitive OXPHOS stimulation, which was limited by the lack of extramitochondrial pyruvate formation (A–C). In support of this assumption, the addition of MAS enzymes (lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), and MDH, complete reconstituted MAS, 2+-sensitive manner (A–C). To demonstrate that the observed OXPHOS stimulation was due to MAS-mediated extramitochondrial pyruvate generation, we added cinnamate (Cin), a specific inhibitor of the mitochondrial pyruvate carrier (PC) and observed a marked OXPHOS inhibition (B and C). This inhibition was not complete, however, because Cin does not impair MAS activity. Thus, mitochondrial hydride anion uptake  remained unaffected, allowing residual respiration. To confirm this assumption, we next inhibited the transport of hydride anions using the GOT inhibitor aminooxyacetate (AOA), which further decreased OXPHOS activities. The degree of activation of OXPHOS rates controlled by extramitochondrial Ca2+ was comparable in WT and MCU KO mitochondria (B and C). This indicates that the experimental elevation of OXPHOS rates was largely independent from MCU activities. Moreover, and most importantly, we demonstrate that extramitochondrial pyruvate generation and supply to mitochondria in a Ca2+-sensitive manner may be a major task of the MAS (A–C). This puts the MAS at center stage of metabolic homeostasis. Using isolated heart mitochondria, we confirmed the above findings (D and E), albeit revealing an almost double capacity of MAS-controlled pyruvate generation in the heart as compared with brain mitochondria (B–E).In a first set of experiments, we used brain mitochondria isolated from WT and MCU KO mice , B and Cry rates B. Next, 2+ concentrations (A). Chelation of cytosolic Ca2+ using membrane-permeable BAPTA-AM decreased free Ca2+ (A and B) and concomitantly restricted OXPHOS activity (B). Because cytosolic Ca2+ is required for the generation of pyruvate in the cytosol, BAPTA-AM indeed induced a condition of mitochondrial pyruvate starvation (A and C). Pyruvate supplementation partially restored OXPHOS activity in a MAS-independent manner (B) despite the BAPTA-AM–mediated decrease of cytosolic Ca2+. To demonstrate that OXPHOS restoration was indeed based on replenished pyruvate level, we prevented its mitochondrial uptake using Cin (B), which decreased OXPHOS activity to levels seen prior to pyruvate supplementation (B). As an independent control, we repeated the experiments but inhibited MAS activity using AOA (D) to levels previously seen in the presence of BAPTA-AM (B). We hypothesized that in the presence of AOA again a state of pyruvate starvation was induced despite high cytosolic Ca2+ concentrations, and indeed, pyruvate replenishment recovered OXPHOS activity nearly completely in a Cin-sensitive manner (D). This demonstrates that AOA, similar to BAPTA-AM, inhibits the MAS activity but leaves cellular workload unaffected. Specifically, MAS activation by cytosolic Ca2+ is essential to generate a sufficient amount of pyruvate for OXPHOS in intact synaptosomes. This notion was further substantiated by feeding synaptosomes with pyruvate instead of glucose/lactate (E). Under such conditions, decreased cytosolic Ca2+ levels have no effect on mitochondrial pyruvate supply despite a presumed decrease of cellular workload. Indeed, further pyruvate addition failed to reactivate the respiratory rate while Cin inhibited respiration.We next aimed to test whether MAS-stimulated OXPHOS control is equally important within intact cells. This was done in isolated synaptosomes, thymocytes, and fibroblasts from WT and MCU KO mice. We first used intact synaptosomes, which are specifically suited to study cellular energy metabolism . Mode 4 trations A. Chelatree Ca2+ , A and Bactivity B. Becausarvation , A and Ct manner B despitesing Cin B, which entation B. As an sing AOA . Again, sing AOA D to leveBAPTA-AM B. We hype manner D. This d/lactate E. Under C–G). From the data, we concluded that mitochondrial pyruvate oxidation rates are not controlled by mitochondrial matrix Ca2+. Instead, cytosolic Ca2+ levels control pyruvate generation through reversible activation of the MAS.We replicated all of the above experiments using intact primary thymocytes and fibroblasts, essentially giving the same results , C–G. FrA). We found that cardiac power was significantly diminished upon AOA inhibition of the MAS (A and B). The fact that glucose addition did not overcome AOA-mediated inhibition of cardiac power (B) indicates that the underlying mechanism was not based on glucose starvation.To resolve the question whether MAS activity controls OXPHOS also in whole organs, we studied cardiac contractility and metabolism in the perfused isolated working rat heart . In support of this notion, inhibition of mitochondrial pyruvate uptake by Cin impaired cardiac power (A and B). Equally important is the observation that hearts with a pharmacologically impaired MAS and/or pyruvate uptake inhibition showed a reciprocal increase in glycolysis (C). This indicates that cardiac power and metabolism are intimately interrelated with MAS, generating sufficient amounts of pyruvate for OXPHOS (D and E). To test this idea, we measured cardiac power in isolated rat hearts using pyruvate as substrate. With pyruvate as substrate, AOA failed to decrease the baseline power (D), once again excluding a MAS-unrelated toxic effect of AOA under such conditions. Unlike in isolated mitochondria and intact synaptosomes and primary cells, we used in the heart only AOA and not BAPTA-AM to test our hypothesis because the expected slow uptake of BAPTA-AM impedes its use in the working mammalian heart . Current models assume that Ca2+-mediated activation of mitochondrial matrix dehydrogenases controls the substrate supply for OXPHOS with mitochondrial Ca2+ uptake being facilitated by MCU. Although Ca2+-mediated activation of matrix pyruvate dehydrogenase in mitochondria is beyond doubt . We propose an alternative mechanism based on the activity of the MAS, which controls cytosolic pyruvate generation and its mitochondrial supply.In the present study, we addressed a fundamental question of bioenergetics  levels  levels , 35. Con cytosol , are wit neurons . In huma neurons , 37, and neurons . The facc oxidoreductase, for instance described for liver tissue  exist –43. One –2+ handling is essential to understand OXPHOS control mechanisms and metabolic homeostasis. Moreover, our findings may help to develop new models to tackle diseases in which cell death is initiated by mitochondrial Ca2+ overload, a pathophysiological mechanism underlying a number of neurodegenerative diseases and responsible for post-ischemic heart injuries.Taken together, a MAS-mediated pyruvate supply exists in a number of cells and tissues and controls OXPHOS under physiologic conditions to a great extent. The conceptional and pathophysiologic consequences of our findings are of broad importance, considering that a better understanding of mitochondrial CaMcu gene)  and RNA purification using the RNeasy Mini Kit . Complementary DNA was transcribed from template RNA using random primers and the iScript Select cDNA Synthesis Kit .CT values were defined at the inflection points of fitted sigmoid curves (four-parameter Chapman curves) and were compared with mitochondrial processing peptidase (Pmpca).Gene expression was measured on an iQ5 qPCR system (Bio-Rad) using SYBR Green qPCR master mix  or iTaq Universal Probes Supermix . Mcu forward, 5′-GTA CGG CCA CCA AAG AGA GAC CTC-3′; Mcu reverse, 5′-ACA GCA CCA GAG TGG TCC TCT-3′; TaqMan probe, 5′-6-carboxyfluorescein-TAA GCC ATG AAG ATG CAG CGA CGC TGA A-N,N,N′,N′-tetramethyl-6-carboxyrhodamine-3′; Pmpca forward, 5′-GTG ACT GCC AGA CCT CAA GAG ACA C-3′; Pmpca reverse, 5′-GAT CGA TCT TCG CTA TGT TCT CTA CAG-3′.Primers used were as follows: 2) was defined, and the time spent in this area was measured as proxy for absence of anxiety.Spontaneous motor activity was assessed as described . To testThe Morris water maze test was performed as described . Briefly2+ content, heart mitochondria were prepared following the same protocol but in the absence of digitonin.Brain mitochondria were isolated as described , 46. To 2+ uptake was measured using a Cary Eclipse fluorescence spectrophotometer (Agilent) and Calcium GreenTM-5N as fluorescent probe  at 506/532 nm excitation/emission wavelengths, respectively. Measurements were done in EGTA media (I and J) and I and J)) or in EGTA-free media Fig. S1 . Free extramitochondrial Ca2+ concentrations in EGTA-containing buffers were measured using Fura-2  at 340 and 380/510 nm excitation/emission wavelengths, respectively. For these measurements, mitochondria were incubated in EGTA media under conditions used for respirometry. Cytosolic Ca2+ concentrations in fibroblasts, thymocytes, or synaptosomes were measured as described (m) at 340- and 380/510-nm excitation/emission wavelengths, respectively. Free Ca2+ concentrations were estimated by established protocols   as descr2 , mannitol , MOPS , KH2PO4 , KCl , and EGTA  and was used for experiments shown in Figs. 1A and C)Fig. S3  and either glutamate  or pyruvate . RR  was added as indicated, and state 3 was adjusted by ADP . Experiments shown in Fig. S3 were performed using multiple substrate inhibitor protocols as described previously (m), glutamate (10 mm), ADP (2 mm), CaCl2 , pyruvate (10 mm), rotenone , glycerol 3-phosphate , succinate , carboxyatractyloside , antimycin A , ascorbate , TMPD , and azide .Isolated mitochondria  were studied in EGTA-containing or EGTA-free buffers at 30 °C. EGTA buffer (pH 7.4) contained MgCleviously . Substra2+ concentrations of ∼600 nm before any addition of Ca2+ Fig. S3  (m), glutamate (10 mm), pyruvate (10 mm), succinate (10 mm), carboxyatractyloside (2.5 μm), ascorbate (4 mm), TMPD (1 mm), and azide (5 mm). Respiratory rates measured after the addition of succinate (state 3) and after carboxyatractyloside (state 4) were used to calculate the respiratory control index.Experiments using EGTA-free media had free Ca ) . Substra6 cells/ml) was studied in Krebs–Ringer bicarbonate buffer (KRB) at 37 °C. KRB (pH 7.4) contained NaCl , KCl (3.1 mm), KH2PO4 , NaHCO3 , NaTES , MgSO4 (1.2 mm), BSA , FCCP , glucose , lactate , or pyruvate (10 mm). Substrates and inhibitors were as follows: AOA , BAPTA-AM , cinnamate , glucose (10 mm), glutamate (10 mm), lactate (10 mm), malate (2 mm), pyruvate (10 mm), and RR (200 nm).Respiration of synaptosomes (0.4 mg of protein/ml) and thymocytes (10 × 10m) or pyruvate (10 mm) in the presence of 100 nm FCCP and supplemented with pyruvate (10 mm) after the addition of BAPTA-AM (50 μm). All Seahorse assays were analyzed using XF Wave 2.3.0 software, according to the manufacturer's instructions. All oxygen consumption rate measurements were normalized to the protein content quantified using the Bicinchoninic Acid Protein Assay Kit  with BSA as standard.Respiration of intact adherent fibroblasts was monitored using a Seahorse XFp Analyzer (Seahorse Bioscience). Fibroblasts were fed with either glucose (20 mm) as described (m) were taken as zero values, and the difference to the actual signal was calculated as a reciprocal measurement for the mitochondrial membrane potential.Alterations in energization of isolated brain and heart mitochondria were monitored using a Cary Eclipse fluorescence spectrometer (Agilent) and the membrane potential-sensitive probe safranin O  perchloric acid . Samples were centrifuged, and the supernatants were neutralized to pH 7.4 using Tris  and KOH . Samples were used immediately for fluorimetric pyruvate determination as described elsewhere  were measured in the presence and absence of 50 μm), lactate (10 mm), and malate (2 mm) as described (2+ was adjusted to ∼12 nm or 800 nm. The assay started with the addition of ADP (2 mm). Different substrates and inhibitors were sequentially added: (i) glutamate (2 mm), aspartate , and 2-oxoglutarate ; (ii) LDH , MDH , and GOT ; (iii) Cin ; and (iv) aminooxyacetate (2 mm). The assay was terminated by adding carboxyatractyloside.Mitochondria  were incubated in EGTA media additionally containing NADH , MOPS (20 mm), sucrose , EGTA (1 mm), and DTT . Final synaptosome preparations were resuspended in isolation media supplemented with 0.1 mm EGTA.Synaptosomes from mouse brain were isolated using Percoll  gradient centrifugation as described , 52. Theg for 5 min. Cells were washed twice, resuspended in KRB, and kept on ice until use. Cell viability was tested by a trypan blue exclusion assay.For isolation of thymocytes, thymus glands were removed and immediately put in ice-cold KRB. Thymocytes were filtered through a nylon sieve, collected, and pelleted at 1000 × l-glutamine , penicillin-streptomycin , ciprofloxacin , and tylosin . Passages between 3 and 6 were used for experiments.Skin fibroblasts were cultivated as described . Brieflym) was necessary to wash out any blood from the heart and to perform left atrial cannulation. Hearts were then perfused as working hearts at 37 °C with recirculating Krebs–Henseleit buffer (200 ml). Perfusate Ca2+ concentration was 2.5 mm. Hearts were perfused with glucose (5 mm) or pyruvate  as substrates. The perfusate was gassed with 95% O2, 5% CO2 and recirculated. All experiments were carried out with a preload of 15 cm H2O and an afterload of 100 cm H2O. Hearts started to beat spontaneously at a rate of ∼250 beats/min. After a period of stabilization, hearts were perfused, samples were withdrawn, and measurements were performed as indicated. Aortic and coronary flow were measured every 5 min by timing the rise of the fluid meniscus in a calibrated glass tube (2O) was calculated as (systolic + diastolic pressure × 2)/3. Heart rate was measured as beats/min, and cardiac output was measured as ml/min. Cardiac power was determined as described elsewhere . Glucose was added to exclude a decreased availability as limiting factor. Pyruvate was used as malate-aspartate shuttle–independent substrate, and cinnamate  was used to inhibit the mitochondrial PC. At the end of perfusion, hearts were frozen and weighed. The wet to dry ratio was determined, and the dry weight was calculated.The preparation has been described in detail elsewhere . Brieflyass tube . Cardiaclsewhere . The MAS3H2O from [2-3H]glucose  (Samples of the coronary effluent (2 ml) were withdrawn every 5 min for the assessment of glycolysis rates determined as the production of NET331C) .n = 3 independent experiments. All data are shown as the mean. Error bars represent S.E. A p value of <0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism  for comparisons as indicated of at least M. Szibor, W. S. K., and F. N. G. conceptualization; M. Szibor, Z. G., T. G., T. E., G. D.-V., M. K., N. K., K. H., F. S., A. B., M. Schwarzer, V. L., and F. N. G. data curation; M. Szibor, F. S., and F. N. G. visualization; M. Szibor, W. S. K., and F. N. G. writing-original draft; M. Szibor, Z. G., T. G., T. E., G. D.-V., M. K., N. K., K. H., F. S., A. B., M. Schwarzer, T. D., H.-J. H., V. L., S. V., W. S. K., and F. N. G. writing-review and editing; Z. G., T. G., T. E., G. D.-V., M. K., N. K., K. H., A. B., M. Schwarzer, V. L., W. S. K., and F. N. G. formal analysis; T. D., H.-J. H., S. V., and F. N. G. funding acquisition; F. N. G. supervision; F. N. G. validation; F. N. G. investigation; F. N. G. methodology; F. N. G. project administration.


Endosom2+ release from acid stores, for example, Ned-19 and tetrandrine, also severely reduced entry of this virus.2+ signaling studies, it is known that deletion of TPC2 markedly reduced NAADP-elicited Ca2+ release in permeabilized cells, as did an antibody to TPC2 . The endosomal [Ca2+] then declined further to ∼4 μM in late endosomes , acting on IP3 receptors principally located in the endoplasmic reticulum.2+ release,,2+ signaling influences SARS-CoV-2 entry. Taking advantage of the co-existence in pancreatic acinar cells of two different Ca2+ signaling mechanisms, activated by separate receptor pathways, it would seem possible to provide well-defined test and control conditions in such studies.Exocrine pancreatic secretion is principally controlled by the neurotransmitter acetylcholine (ACh) and the hormone cholecystokinin (CCK). Both agents elicit secretion by mobilizing intracellular Ca2+ release from the endo-lysosomal system in both SARS-CoV-2 entry and NAADP-mediated Ca2+ signaling. The pancreatic acinar cells may prove to be a particularly useful system for exploring the relationship between these phenomena.We conclude that there is evidence for the involvement of acidic endosomes and TPC-mediated CaNone declared.Experimental work in the authors’ laboratory was supported by grants from the UK’s Medical Research Council (MR/J002771/1 and G19/22/2).


The present study investigated the modulation of Ca2+ homeostasis in CKD mice. Echocardiography revealed impaired fractional shortening (FS) and stroke volume (SV) in CKD mice. Electrocardiography showed that CKD mice exhibited longer QT interval, corrected QT (QTc) prolongation, faster spontaneous activities, shorter action potential duration (APD) and increased ventricle arrhythmogenesis, and ranolazine (10 µmol/L) blocked these effects. Conventional microelectrodes and the Fluo‐3 fluorometric ratio techniques indicated that CKD ventricular cardiomyocytes exhibited higher Ca2+ decay time, Ca2+ sparks, and Ca2+ leakage but lower [Ca2+]i transients and sarcoplasmic reticulum Ca2+ contents. The CaMKII inhibitor KN93 and ranolazine  reversed the deterioration in Ca2+ handling. Western blots revealed that CKD ventricles exhibited higher phosphorylated RyR2 and CaMKII and reduced phosphorylated SERCA2 and SERCA2 and the ratio of PLB‐Thr17 to PLB. In conclusions, the modulation of CaMKII, PLB and late Na+ current in CKD significantly altered cardiac Ca2+ regulation and electrophysiological characteristics. These findings may apply on future clinical therapies.Cardiovascular complications are leading causes of morbidity and mortality in patients with chronic kidney disease (CKD). CKD significantly affects cardiac calcium (Ca Phosphodiesterase 3 (PDE3) inhibition potentiates this signalling pathway.2+/calmodulin‐dependent protein kinase II (CaMKII) phosphorylates several Ca2+‐handling proteins, including PLB, SERCA, SR Ca2+ release channels  and L‐type Ca2+ channels, which play important roles in the regulation of intracellular Ca2+.2+]i) are the major determinants of cardiac function, but little is known about alterations in [Ca2+]i in CKD‐induced cardiomyopathy.One of the major pathological changes in cardiomyopathy is dysregulation of intracellular Ca22.12 inhalation 6 months later after the sham operation or PNx. Then, we collected blood and the hearts were dissected for analysis. The concentrations of blood urea nitrogen (BUN) and creatinine of serum (sCr) were detected by using the Jaffe method .Chronic kidney disease was induced by way of partial nephrectomy (PNx) in C57BL/6J mice as described formerly.2.2A Philips iE33 ultrasound imaging system with a 7‐15 MHz linear array transducer was used to perform echocardiography . Inhalation of 3% isoflurane was performed to anaesthesia, until animals were sedated and maintained 1% isoflurane during the examination of echocardiography. First of all, it was to obtain 2D left ventricular (LV) short‐axis images. M‐mode was used to measure the thickness of LV wall and dimension of chamber and ejection fraction (EF) at diastole and systole phases. All measurements of echocardiography were averaged for consecutive 5 cardiac cycles.2.3Under isoflurane anaesthesia (5% for induction and 2% for maintenance), electrocardiograms (ECGs) were recorded from standard lead II limb leads via a bio‐amplifier (ADInstruments), were connected to a ML845 Powerlab polygraph recorder (ADInstruments) and were continuously displayed throughout the experiment in sham or CKD mice.2.43, 4 mmol/L of KCl, 2.7 mmol/L of CaCl2, 0.5 mmol/L of NaH2PO4 and 0.5 mmol/L of MgCl2,. The ventricle tissue preparation was dissected first and pinned to the base of a tissue bath. The free end of the ventricle tissue preparation was hooked to a Grass FT03C force transducer (Grass Instrument Co.) with a string. Tissue was perfused at 3 mL/min with normal Tyrode's solution which saturated with 97% O2 and 3% CO2 mixture. The temperature remained constant at 37°C. These preparations were stabilized for 1 hour before the electrophysiological study.Sodium pentobarbital (100 mg/kg) was used to anaesthetize Sham and CKD mice with intraperitoneal injection. The heart and lungs were removed by midline thoracotomy. The ventricle was opened in Tyrode's solution which containing 137 mmol/L of NaCl, 11 mmol/L of dextrose, 15 mmol/L of NaHCO90), 50% (APD50) and 20% (APD20) of the APA were measured accordingly. Data acquisition was performed by using a Digidata 1320 interface (Axon Instruments Inc).Recording Action potentials (APs) of the ventricle were performed with borosilicate glass microelectrodes which contained 3 mol/L KCl. Preparations of tissue were connected to a WPI model Duo 773 electrometer (World Precision Instruments) with tension of 150 mg. A TDS2000C oscilloscope was used to display both electrical and mechanical events simultaneously (Tektronix) and Gould TA11 recorder (US Instrument Services). Electrical signals were recorded by removing DC coupling first and passed through a low‐pass filter with 10‐kHz cut‐off frequency, then feed to a Digidata 1320 acquisition system (Molecular Devices). Electrical stimulation was provided with a S88 stimulator with a SIU5B isolation unit (Grass Instrument). Spontaneous activity was defined as occurrence of spontaneous AP with no electrical stimuli. Early afterdepolarization (EAD) was defined as abnormal depolarization occurred with phase 2 or 3 of the APs. APs were evoked by a 4‐Hz electrical stimulus before and after administration of medicine. The RMP was obtained during the phase between the last repolarization and subsequent onset of the AP. The amplitude of action potential (APA) was obtained from the RMP to the apex of the AP depolarization. The action potential duration (APD) at 90% . The perfusate was replaced with oxygenated Ca2+‐free Tyrode's solution which containing 150 units/ml collagenase  and 0.25 units/ml protease  for 5‐8 min. The ventricle was separated from the atrium and lung and placed in a chamber which containing Ca2+‐free oxygenated Tyrode's solution. Dice the tissue and shaken gently in 5‐10 mL of Ca2+‐free oxygenated Tyrode's solution until single cardiomyocytes were obtained. The Ca2+‐free solution was changed gradually to oxygenated normal Tyrode's solution.Mice used in this study were anaesthetized by using sodium pentobarbital . The heart and lungs were removed quickly after a midline thoracotomy was performed. The heart was perfused in a retrograde manner via a dispensing needle  cannulated through the aorta and left ventricle into the left atrium. The free end of the dispensing needle was connected to a Langendorff perfusion apparatus and perfused with oxygenated normal Tyrode's solution at 37°C  in order to obtain data of intracellular Ca2+ transient [Ca2+]i changes from baseline values (△F/F0), and exclude fluctuations in the fluorescence intensity with different volumes of injected dye. Calcium transients at peak systolic and diastolic activities were measured by 1‐Hz field stimulation with 10‐ms twice‐threshold strength square‐wave pulses. The Ca2+ content of SR was measured by adding of 20 mmol/L of caffeine with pacing at 1 Hz for at least 30 seconds. All measurements were performed at a temperature of 35 ± 1°C. Background subtraction was also performed for fluorescence measurement.Isolated cardiomyocytes were loaded with 10 μmol/L of fluo‐3/AM for 30 min at room temperature (RT). The bath solution was changed to remove excess dye at 35 ± 1°C for 30 minutes. A 488‐nm argon ion laser was used to excite Fluo‐3. The emission fluorescence was recorded at >515 nm. Cells were scanned repetitively at 3‐ms intervals for a total duration of 6 seconds. Fluorescence imaging was performed using a confocal microscope (Leica TCS SP5). Signals were corrected by dye concentration via the normalization of the fluorescence (F) against the baseline fluorescence  for 5 minutes to reducing sample buffer and then transferred to 10% SDS‐PAGE. Sample proteins were transferred to nitrocellulose membranes which were blocked at 4°C in 5% milk in PBS with 0.01% Tween‐20 (PBST) overnight. These nitrocellulose membranes were incubated with diluted primary antibodies for 1 hour at RT. Then, washing them in PBST for 15 minutes and repeat 3 times. Membranes were incubated in diluted (1:10 000) secondary horseradish peroxidase (HRP)‐conjugated antibodies for 1 hour, then washed thoroughly in PBST for 15 minutes (repeat for 3 times). ECL Plus Western blotting detection reagents (Amersham Biosciences) and X‐ray film (Eastman Kodak) were used to visualize bands. The following primary antibodies were diluted in BSA: rabbit anti‐RyR , mouse anti‐SERCA2 (Santa Cruz Biotechnology), rabbit anti‐phosphorylated RyR , rabbit anti‐phosphorylated SERCA2 , PLB phosphorylated at Thr17 (PLB‐Thr17) (Badrilla), phosphorylated CaMKII at Thr 286 (pCaMKII), total PLB (Thermo), CaMKII and NCX (Swant). A GAPDH antibody was used to normalize protein bands in each blot. ImageJ software was used to quantify relative protein level.Lysis buffer  containing 10 μL/mL of a protease inhibitor (Sigma) was used to homogenized tissue for 15 minutes on ice, followed by 14 000 2.9t test or one‐way analysis of variance (ANOVA) with Tukey's test for multiple comparisons, as appropriate. A value of P < .05 was considered significant.All quantitative data are expressed as the means ± the standard error of the mean (SEM). Statistical significance between different groups was determined by an unpaired 33.1P < .05) and creatinine levels  in CKD mice confirmed the successful induction of experimental renal failure. The heart rate and left ventricular mass in the sham and CKD mice were not significantly different. CKD mice exhibited a greater left atrium diameter to aortic root diameter ratio (LA/AO), left ventricular internal diameter at end‐diastole (LVIDd), left ventricular internal diameter at end‐systole (LVIDs), end‐diastolic volume (EDV) and end‐systolic volume (ESV) compared with the sham mice. The fractional shortening  and stroke volume  were decreased in CKD mice compared to sham mice. The ECG data showed that CKD mice exhibited longer QT intervals and corrected QT (QTc) prolongation than the sham group  is defined as interruption of phase 2 or 3 of action potentials prior to full repolarization, and EADs are related to a shortening of action potential durations (APDs).3.3CKD ventricles exhibited higher phosphorylation of RyR2 and CaMKII than the sham groups Figure . The pro3.42+ transients in the CKD group but little effect in the sham group  and RAN (late sodium current inhibitor) to CKD and sham cardiomyocytes produced a significant decrease in Cap Figure A,4. RAN 4We successfully created an animal model of CKD‐inducing heart failure in mice and investigated the role of inflammation on heart failure progression.4.12+ is a major ionic modulator of the heart, and it plays an important role in the excitation‐contraction coupling process. Ca2+‐induced Ca2+ release (CICR) describes a biological process whereby Ca2+ activates the release of further ionic Ca2+ from SR stores via the RyR to increase [Ca2+]i and aid in the binding of Ca2+ to myofilaments in the initiation of cardiac contraction.2+ transients, prolonged transient decay, increased Ca2+ leak and decreased SR Ca2+ content, which decreased fractional shortening and was arrhythmogenic with more EADs.Ca2+ leakage also hyperphosphorylates RyR, which leads to early afterdepolarizations.2+ sparks represent a major release of Ca2+ in cardiomyocytes during excitation‐contraction coupling.2+ sparks are stochastic activations of a cluster of RyR2s that are organized into a Ca2+‐release unit.2+ sparks in cardiomyocytes,2+ handling and myocardial dysfunction. We also found an increased frequency and incidence of Ca2+ sparks in CKD mice. Thus, the increased Ca2+ leakage from the SR may produce a Ca2+ deficiency, which leads to myocardial dysfunction in CKD cardiomyopathy. These findings suggest that increased CaMKII in CKD ventricles was arrhythmogenic, and the increased SR Ca2+ leak was a crucial mechanism, which is consistent with our findings.CaMKII plays multiple roles in cellular ionic regulation. CaMKII inhibition diminishes cardiac arrhythmias in vitro and in vivo, and transgenic CaMKII‐overexpressing mice exhibit an increased incidence of cardiac arrhythmogenesis.2+ modulation to cause heart failure, which was associated with increased ROS.2+ transient, decay time and AP, which suggests that a deterioration of Na+ regulation also modulated intracellular Ca2+ handling in CKD.A review from Harvath and Bers found that the increased late sodium current also contributed to Ca4.290 but prolonged QT and QTc intervals. We only recorded APDs from right ventricle, but not from left ventricle. It is possible that the prolongation of QT and QTc intervals may be contributed mostly by prolonged APDs from left ventricle. Therefore, the discrepant effects of CKD on APDs between right and left ventricle may increase interventricular dispersion of APDs, which could increase the genesis of micro‐reentry circuits.CKD patients exhibit a high incidence of cardiovascular complications that are characterized by complex alterations in the mechanical and electrical properties of the heart.4.32+ contents and calcium transient amplitudes, which may be due to the enhanced phosphorylation of PLB or RyR2 and SERCA2a activity.2+ contents and Ca2+ transient amplitudes than sham mice. RyR2 phosphorylation levels increased, but SERCA2 and PLB phosphorylation decreased. These distinct findings suggest a different remodelling of Ca2+‐regulating proteins at different sites in cardiomyocytes, which produces arrhythmogenic or contractile dysfunction and heart failure.The CKD pulmonary vein (PV) cardiomyocytes of rabbits exhibit higher SR Ca52+ regulation and electrophysiological characteristics (Figure + current. The antagonists KN93 and RAN may eliminate the observed effects.Our findings suggest that CKD induces uraemic cardiomyopathy, which exhibits alterations in cardiac Cas Figure . These aAll authors declare that they have no competing interests.HYK, LHC and CYL devised the project, the main conceptual ideas. CST and CYL performed the numerical calculations for the experiment and revised the manuscript. LHC, FZL, YHC and HYK performed the measurements, SMH and YCC were involved in supervised the work, HYK and YCC processed the experimental data and performed the analysis. HYK drafted the manuscript and designed the figures. YLC and LHC performed the excel calculations. CST, SMH, YCC and CYL aided in interpreting the results. All authors discussed the results and commented on the manuscript.


Use of mitochondria-targeted genetically encoded Ca2+ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca2+ dynamics, which are assumed to be determined by distinct thresholds of Ca2+ increases at each subcellular mitochondrial domain. The balance between Ca2+ influx through the mitochondrial calcium uniporter and extrusion by cation exchangers across the inner mitochondrial membrane may define the threshold; however, the precise mechanisms remain to be further explored. We here report the new red fluorescent genetically encoded Ca2+ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca2+ affinities are engineered to match the intramitochondrial Ca2+ concentrations. They enable visualization of mitochondrial Ca2+ dynamics with high spatiotemporal resolution in parallel with the use of green fluorescent probes and optogenetic tools. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca2+ dynamics and their functions.Mitochondrial Ca Ca2+ concentrations in the mitochondrial matrix are regulated by the balance between the influx of Ca2+ through the mitochondrial Ca2+ uniporter (MCU) and the efflux of Ca2+ by Na+/Ca2+ or H+/Ca2+ exchangers3. Recent studies have elucidated the molecular identity of the channel and regulatory components of MCU11 as well as Na+-Ca2+-Li+ exchanger in the mitochondrial inner membrane13. Furthermore, the advent of genetically encoded Ca2+indicators (GECIs) that are targeted to the mitochondrial matrix has enabled monitoring mitochondrial Ca2+ dynamics with high spatiotemporal resolution, revealing both the subcellular and intercellular heterogeneity of mitochondrial Ca2+ responses upon agonist-induced increases in the cytosolic Ca2+ concentration16. These imaging results suggest that the threshold of the net Ca2+ flux into the mitochondrial matrix is differentially determined in individual cells or even in each subcellular mitochondrial domain. However, the mechanism of these heterogeneous mitochondrial Ca2+ dynamics and their functional significance remains to be clarified. Thus, further analyses combined with high-resolution mitochondrial Ca2+ imaging are required.Mitochondrial Ca2+ indicator protein family of Calcium-measuring organelle-Entrapped Protein IndicAtors (CEPIA) to visualize Ca2+ signals in both the endoplasmic reticulum (ER) and mitochondria15. ER-targeted CEPIAs have Kd values for Ca2+ ranging between 558 and 672 µM, which are higher than those of other ER-targeted GECIs such as D1ER17, GCaMPer18, ER-GCaMPs19, and ER-LAR-GECOs20. Mitochondrial Ca2+ imaging analyses using green fluorescent protein (GFP)-based CEPIA variants with lower Ca2+ affinities (Kd = 14.5 or 90.2 µM) than cytosolic Ca2+ indicators suggested that mitochondrial Ca2+ concentrations can increase beyond 50 µM in a small fraction of HeLa cells. While a red fluorescent protein (RFP)-based low-affinity GECI, mito-LAR-GECO1.2 (Kd = 12 µM)20, has been developed, lower-affinity mitochondrial RFP-based CEPIAs have not been developed, yet. Moreover, GFP-based GECIs cannot be used simultaneously with other green fluorescent imaging tools, including synthetic Ca2+ indicators, nor are they simultaneously used with optogenetic tools that are activated by blue light, such as channelrhodopsin-2 and OptoXRs22. These limitations can be circumvented by GECIs with longer excitation and emission wave lengths. Therefore, CEPIA variants with red fluorescence may allow simultaneous use of other optical tools to increase the utility of mitochondria-targeted GECIs.We have previously developed a Camt variants, R-CEPIA3mt and RCEPIA4mt, of which the Ca2+-affinity was optimized to measure mitochondrial Ca2+ concentrations. These variants allow visualization of mitochondrial Ca2+ signals with high spatiotemporal resolution that enables the detection of mitochondrial Ca2+ dynamics at subcellular local domains. Furthermore, simultaneous use of green fluorescent Ca2+ indicators and optogenetic tools is possible. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a valuable tool for obtaining deeper insight into the cellular functions of mitochondrial Ca2+ dynamics.To this end, we generated the red-fluorescent CEPIA2+ affinity variants of CEPIA15, we generated R-CEPIA3mt and R-CEPIA4mt by modifying one (E31D) and three  amino acids, respectively, in the calmodulin domain of R-GECO1mt  and high dynamic ranges  demonstrated that only a fraction of the cells  showed a transient mitochondrial Ca2+ increase in response to cytosolic Ca2+ elevations induced by the inositol trisphosphate-producing agonist, histamine 15. Additionally, the cells were loaded with fura-2 for simultaneous detection of cytosolic Ca2+ dynamics. As shown in Fig. 2+ signals were detected either by both CEPIA2mt and one of the R-CEPIAmt variants  compared with the GFP-based CEPIAs  of the phospholipase C (PLC) activator, Opto-α1AR-YFP22, we performed mitochondrial Ca2+ imaging during optogenetic activation. In response to 448-nm laser irradiation, several mitochondrial domains 25. The effect of such pH changes would be less in R-CEPIA3mt and R-CEPIA4mt than in GFP-based CEPIAmts.We have previously shown that there is no significant pH change within the mitochondrial matrix in histamine-stimulated HeLa cells2+ dynamics is of great interest. Recent studies have identified molecules involved in Ca2+ influx through the MCU complex, including MCU, MCUb, EMRE and MICU1–311. Furthermore, the Na+-Ca2+-Li+ transporter has been identified as a Ca2+ extrusion mechanism on the mitochondrial inner membrane13. Therefore, it is important to study whether there is intercellular and subcellular heterogeneity in the expression levels or activities of these Ca2+-handling proteins, and whether protein heterogeneity corresponds to the heterogeneity in mitochondrial Ca2+ dynamics. Moreover, it will be informative to study whether the heterogeneity in mitochondrial Ca2+ dynamics produces intercellular heterogeneity in cell death as well as subcellular heterogeneity in mitochondrial motility, ATP production, opening of permeability transition pores, and mitochondrial membrane potentials. Various optical tools for tagging, probing and controlling these cellular processes have been developed. Simultaneous use of these optical tools and the R-CEPIAmt variants is expected to make a great contribution to the field.The mechanism underlying the intercellular and subcellular heterogeneity in mitochondrial Ca2+ indicators that cover a broad range of mitochondrial Ca2+ concentrations. Further imaging analyses using these potent and useful tools may facilitate uncovering the mode of action of mitochondrial Ca2+ dynamics and their functions in health and disease.In summary, we added new members to the library of green and red emission mitochondrial Ca15 using primers 1–6 (Supplementary Table mt15) by restriction enzyme digestion.For engineering R-CEPIA3 and R-CEPIA4, we introduced point mutations into pET19b R-GECO1–1). After harvesting the bacteria by centrifugation, the cells were resuspended in KCl/MOPS buffer  and lysed by French press  at 20,000 psi. The recombinant proteins were purified using TALON metal affinity resin  and eluted with KCl/MOPS buffer containing 250 mM imidazole.BL21-CodonPlus(DE3)-RIL bacteria  were transformed with the pET19b plasmids having R-CEPIA3, R-CEPIA4, R-GECO1 or CEPIA2. The bacteria were incubated for 16–24 h at 37 °C in 2 × YT medium containing ampicillin and chloramphenicol  in KCl/MOPS buffer containing 1 mM EGTA or 1 mM CaCl2+ titration curves were obtained by adding small aliquots of CaCl2 to the recombinant indicators in KCl/MOPS or KCl/HEPES  buffer. The indicators’ concentration was 25–75 nM. The Ca2+ concentrations were clamped with any of the following Ca2+ buffers: 1 mM EGTA, 1 mM BAPTA, 1 mM Br2BAPTA  and 1 mM Nitrilotriacetic acid (NTA). Free Ca2+ concentration was calculated by Maxchelator (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/). Fluorescence intensity was measured with F-4500 FL spectrofluorometer  at 562 ± 5/584 ± 5 nm (excitation/emission) wavelength for R-CEPIA3, R-CEPIA4, and R-GECO1, and 492 ± 10/514 ± 10 nm for CEPIA2. The obtained relationship between the Ca2+ concentration and the fluorescence intensity was fitted by the following single Hill plot equation with the KaleidaGraph software .K′d represents apparent dissociation constant or the Ca2+ concentration at which half of the indicator molecules bind to Ca2+. n represents Hill coefficient. The fluorescence intensity at various Ca2+ concentrations was normalized by (F − Fmin)/(Fmax − Fmin). The dynamic range of the indicator was calculated as the ratio of Fmax to Fmin.Ca2+. pKa was obtained by fitting the obtained fluorescence intensity with a single Hill equation.pH titration curves for each indicator were obtained by measuring fluorescence intensity in the solutions containing 130 mM KCl and 50 mM pH buffer  with 1 mM EGTA or 1 mM Ca–1) and streptomycin (100 U•ml–1). For Ca2+ imaging, the cells were plated on collagen type-I-coated glass-bottom dishes  and cultured for 16 h before imaging.HeLa cells were cultured on collagen-coated plastic dishes  in DMEM supplemented with 10% fetal bovine serum, penicillin  or 5 µM fura-2 AM  at room temperature (22–24 °C) for 30 min in culture medium. Before imaging, the loading solution was washed three times and replaced with physiological salt solution (PSS) containing (in mM) 150 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 5.6 glucose and 25 HEPES (pH 7.4).Cultured HeLa cells were transfected using Lipofectamine 3000 (Thermo Fisher) 2 or 3 days before imaging. The plasmids used in the current study were: pCMV R-CEPIA3mt and R-CEPIA4mt  were captured using an inverted IX81 microscope  equipped with a ×20 UPlanSApo oil immersion objective , an electron-multiplying cooled-coupled device (EM-CCD) ImagEM camera , a Lambda 10–3 filter wheel , an ebx75 xenon lamp  and an EL6000 metal halide lamp (Leica) at a rate of one frame per 1 or 2 s with the following set of excitation and emission filters, respectively: 472 ± 15 nm and 520 ± 17.5 nm for Cal-520; 562 ± 20 nm and 641 ± 37.5 nm for R-CEPIA3/4mt.Time-lapse images of Cal-520, R-CEPIA32+ imaging in subcellular mitochondrial domains, time lapse or snapshot images of R-CEPIA3/4mt, CEPIA2mt, fura-2 and Opto-α1AR-YFP were captured using a TCS SP8 confocal microscope (Leica) equipped with a × 63 HC PL APO oil immersion objective  at a rate of one frame per 1–3 s with the following wavelengths : R-CEPIA3/4mt , CEPIA2mt , fura-2  and Opto-α1AR-YFP [for confirmation of transfection by YFP fluorescence, shown in Fig. For Camt-expressing cells were stained by a 30-min incubation in culture medium containing 500 nM MitoTracker Green (Thermo Fisher) at 37 °C. Images were captured with a TCS SP8 confocal microscope using a ×63 HC PL APO oil immersion objective at excitation: 488 nm and emission: 500–540 nm for MitoTracker Green and excitation: 552 nm and emission: 560–750 nm for R-CEPIA3/4mt.For imaging of subcellular localization of CEPIA, the mitochondria in R-CEPIA3/4t-tests were performed to determine the statistical significance using Origin 7 . The calculations, processing and analysis of obtained images were performed with ImageJ and ImageJ Fiji . Graphs and time course traces were produced with Origin 7 and arraigned with Illustrator CC , respectively.Two-tailed Student’s Sepplementary Information.


Malassezia spp. have rarely been reported in rodents and lagomorphs. In 2011, Malassezia cuniculi was described in two rabbits. Further microscopic studies showed M. cuniculi-like yeasts in more than 50% of samples from rabbits’ ear canals, but no isolation was made. The present study details the presence of Malassezia spp. and tries to typify it from ear canals of healthy rabbits. Seventy-eight half-breed rabbits from rural farms and 98 companion dwarf rabbits from northern Italy were considered. A first attempt to screen ear swabs was performed by microscopic and cultural examination on Sabouraud Glucose Agar (SGA), modified Dixon Agar (mDA) and Leeming and Notman Agar (LNA). Additionally, ear swabs from eight further microscopically positive rabbits for M. cuniculi-like cells, were used for both isolation on LNA medium and nine of its variants and for DNA extraction, PCR and sequencing. The microscopic observation of the swabs of the screened 168 rabbits highlighted the presence of yeasts in one or both of the external ear canals of 98 rabbits (58.3%). Rabbits used for meat production were more frequently diagnosed positive than pet rabbits (P = 0.001), and young ones were more often positive compared to rabbits older than 3 months (P = 0.027). No yeast growth was observed in culture. From the eight selected rabbits, Malassezia isolation failed both on LNA and on the modified mediums. Sequences of ~300 bp fragments of 18s rDNA, obtained by PCR from swabs, showed 99.9% identity with Malassezia phylotype 131 described from human ear canals. As Malassezia-like yeasts have been observed in more than half of the examined population, its colonization of ear meatus can be considered as physiological in rabbits. The results outline how much remains to be discovered on Malassezia as a component of the skin mycobiota of rabbits and that the use of the culture examination alone is not the best choice to detect Malassezia-like yeasts in rabbits. Malassezia, belonging to the class Malasseziomycetes of Ustilaginomycotina  [Malassezia spp. is rarely reported in rabbits. In 1985, Dufait [Malassezia pachydermatis from the hair of several rabbits. Later, in 1994, Guillot et al. [Malassezia in the auditory conduit of these animals could be related to the small amount of cerumen, an essential source of lipids for these yeasts. After that, Malassezia spp. has been considered to not affect lagomorphs. Only one case of dermatitis is associated with Malassezia spp. growing on Sabouraud dextrose agar, therefore, most likely M. pachydermatis, was recently described in Peru [Malassezia spp. morphologically different from M. pachydermatis in histological sections of rabbits’ skin with sarcoptic mange. Cabañes et al. [Malassezia-like yeast with round budding cells from the external ear canal and inguinal skin of two subjects, growing only on Leeming and Notman Agar (LNA) but neither on modified Dixon Agar (mDA) nor on Sabouraud Glucose Agar (SGA). After morphological, physiological and molecular characterization, the authors described this isolate as a new species named Malassezia cuniculi. Subsequently, Quinton et al. [Malassezia yeast in 54% of the samples, speculating that it was probably M. cuniculi without performing cultural or molecular tests.The genus , Fungi) , include, Dufait  isolatedt et al.  did not s et al. , in 2011n et al. , in a cyMalassezia spp. in ear canals of healthy rabbits through cytological and cultural examination, assessing correlation with gender, age, breeding, housing and feeding systems. Additionally, further cultural and molecular assays were performed to typify the involved yeasts.This study aims to survey the presence of The study was performed on 176 rabbits, either for meat production or for companionship, from northern Italy. No invasive procedures were used—only ear swabs in agreement with the owners— and no personal data were collected. Included animals were in a good state of nutrition, showed neither cutaneous alterations nor signs of otitis.A first screening was performed on 168 rabbits, 70 mixed breed from rural farms and 98 companion dwarf rabbits. Samples were collected from both the external ear canals of each animal using swabs soaked in phosphate buffered saline (PBS) containing 0.1% Tween 80 [Malassezia cuniculi-like yeasts in both external ear canals were selected from a rural farm. From each animal, a further five swabs were used to collect yeasts: One was soaked in Tris-EDTA (TE) buffer and used for molecular analysis, four were soaked in PBS containing 0.1% Tween 80, and used for cultures. The culture medias used were LNA and nine of its variants, empirically created modifying LNA mainly by adding various lipid sources  according to manufacturer’s instruction for yeast cells, slightly modified as follows: Each swab was put in 500 µl Zymolase Buffer with 15 U Lyticase  and incubated at 37 °C for one hour, shaking periodically, then centrifuged at 3000 × g for 10 min at room temperature to pellet the spheroplast. As a positive control, one ear swab from a dog positive to M. pachydermatis was used, whereas a sterile swab without any skin contact submitted to the same procedures was used as negative one.DNA extraction from swabs was performed using PureLinkMalassezia [Malassezia spp., included into the D1/D2 region.The primers Mal-F 5′ GTC CGC GTT GTA ATC TCG AGA CGT and Mal-R 5′ TGG CTG ACT TCA AGC GCT TCC CTT, highly specific for lassezia , were usMalassezia spp. sequences, retrieved from GenBank, were constructed using BioEdit 7.2.5 [For sequencing, bands were excised, purified by NucleoSpin Gel and PCR clean-up , sequenced with an ABI 3730 DNA analyzer at StarSEQ GmbH  and assembled with Vector NTI Advance 11 software . Blast analysis was performed and multiple sequence alignments with it 7.2.5 . The seqMalassezia spp. Results were considered significant when P ≤ 0.05. Stata Statistical Software  was utilized for the descriptive statistical analysis.Pearson’s chi-square test was used for the comparison of gender, age, breeding, housing and feeding systems as potentially affecting the presence of P = 0.967) and by involved category of rabbits (P = 0.419), on the contrary age was dependent on such categories (P < 0.001). Malassezia-like yeasts were cytologically observed from at least one ear conduct of 98 (58.3%) rabbits . The yeasts, usually observed in cluster not uniformly distributed, were rounded, 2–4 µm in diameter, with buds formed in a monopolar pattern on narrow bases  compared to pet rabbits (48%) . Furthermore, Malassezia was more often bilaterally detected in meat production systems compared to companion animals, 76.5% and 56.5%, respectively . As for sex, 67.1% of the females and 49.4% of the males harbored yeasts . Determination of the Pearson χ2-test showed age patterns, where rabbits 3-months-old or younger were more often cytologically diagnosed positive for Malassezia-like yeasts compared to rabbits older than 3 months, 68.1% and 51%, respectively . Additionally, differences among housing methods were found , since Malassezia-like yeasts were diagnosed in 12 (92.3%) rabbits from cages in a rural environment, in 39 (56%) rabbits permanently fenced in and finally in 47 (54.6%) out of the rabbits free to roam in-home. Considering feeding methods, rabbits fed only with pellet were more frequently diagnosed positive for yeasts (83.8%) compared to mixed feeding rabbits (51.1%), this difference showed a statistical significance to the Pearson χ2-test . Age class and feeding methods were strictly dependent on housing and breeding methods (P < 0.001). Results are summarized in Considering the two categories, meat production and companion intent, Malassezia phylotype 131, isolated from human external auditory canal, not growing on LNA medium [Malassezia species, as already reported [Concerning the culture test performed on the eight selected rabbits cytologically positive, no ear swab yielded colonies on LNA, and none of the modified medium used in the present study allowed the yeast growth. All the eight swabs from these rabbits and the one from dog, used as a positive control, tested positive with PCR, giving a band at around 300 bp. The sequences obtained from all rabbits were identical to each other and showed a BLAST identity of 99.9% with uncultured Basidiomycota clone 131 (AB663497), later on named A medium . Our spereported .M. pachydermatis, considered a single non-lipid dependent species, new lipid-dependent species have been described over time, not only in humans but also in animals. The most recently described M. brasiliensis, M. psittaci [M. arunalokei [M. vespertilionis [With the exception of psittaci , M. arununalokei  and M. vtilionis , led to Malassezia spp. was rarely described in rabbits [Malassezia spp. growing on SDA, therefore most likely M. pachydermatis, was described in the literature [M. cuniculi, characterized by growing only on LNA medium, was isolated and described in Spain from only two rabbits by Cabañes et al. [Malassezia in rabbits. In the present study, we have shown that M. cuniculi-like yeasts are frequently detected in healthy rabbit ear swabs (58.3%) in agreement with Quinton et al. [Malassezia-like yeast detection. Unlike other mammals, in the past  rabbits ,3 and, tterature . M. cunis et al. , who wern et al. , who repn et al.  observedMalassezia cuniculi-like yeasts in cytological examination on healthy rabbits, suggests that these yeasts are common commensal in these animals. However, there are few studies describing the presence of Malassezia spp. and its role in rabbits. Malassezia is not usually considered among the causes of dermatitis in rabbits [M. pachydermatis due to its ability to grow on SDA, has been described [M.cuniculi. The frequent finding of  rabbits . Only onescribed . A retroescribed  highlighMalassezia-like yeasts in rabbits may be explained by several reasons: i) Malassezia had not been considered in rabbits, ii) low number of animals checked, iii) the diagnostic methodologies applied. Although, in mycology, culture methods are usually more sensitive than microscopic examination, the use of fungal cultures may only have produced an underestimation of Malassezia presence in rabbits, associated or not to dermatitis. These rare diagnoses of M. cuniculi, we have not observed any growth using this medium, as well as using mDA or SDA. This supports the hypothesis that some variants could be present, likely related with geographical origin [M. pachydermatis, which shows many variants with different morphological, biochemical and molecular characteristics [Although the LNA has been described as the suitable medium for the growth l origin , having eristics ,18,19,20Malassezia species in culture are yet to be fully determined and this hinders the study of these yeasts. Our findings reveal that the sensitivity of the culture system for the detection of Malassezia-like yeasts can be equal to zero, even though several mediums are used.For this reason, in this study, in order to obtain isolates from the ear of cytologically positive rabbits, nine LNA variants, empirically created modifying LNA mainly by adding various lipid sources , were usMalassezia directly from biological samples has been described in the literature, basically in the human field [M. cuniculi in a patient, in addition to other more frequent species such as M. restricta and M. globosa (predominant), M. slooffiae, M. sympodialis, M. dermatis and M. pachydermatis.In order to overcome this problem, DNA extraction directly from the swab could be a useful tool. To our knowledge, molecular detection of an field ,22,23,24Malassezia using universal primers. Recently, Puig et al. [M. pachydermatis. In the animal field, the abundant fungal flora contaminant or resident over the skin, can complicate the direct amplification of g et al.  developeMalassezia spp. directly from the ear swabs. The amplified fragment, even though consisting of only ~300 bp, is included into the D1/D2 domain of the 26S rRNA. This domain could be used for the identification of most basidiomycetous yeast species, although the internal transcribed spacer region is required to distinguish closely related species [M. cuniculi ones and showed 99.9% identity with Malassezia phylotype 131, a strain not growing on LNA and detected only by PCR by Zhang et al. [The primers used in our study  are highg et al.  from theMalassezia yeasts. Nevertheless, the cluster in which our sequences are included is well supported (98%). Differences with other Malassezia spp. can support the hypothesis of distinct variants having different metabolic requirements and would explain troubles in obtaining isolates by culture media. The short sequences obtained are not suitable for inferring accurate phylogenetic relationships if used as the only marker, therefore the tree obtained herein was only aimed at showing the relationship with the other Malassezia-like yeasts have been observed in more than half of the healthy examined population, its colonization of ear meatus can be considered as physiological in rabbits, being part of the normal skin microbiota. In the future, the control for Malassezia based on the rabbits’ physiological status, and the presence of ear or skin disease, could be useful to clarify if Malassezia is able to play a pathogenic role in this species. Additionally, it appears clear that the use of culture examination alone is not the best choice to detect Malassezia-like yeasts in rabbits. In conclusion, since Malassezia as a component of the rabbits mycobiota. Thus, we strongly believe that our results might be useful for further future studies. The strain here reported certainly cannot be considered a new species, due to the lack of isolation and further genetic information, however, the results obtained outline how much remains to be discovered on