Docking analysis predicts a distinct binding for putrescine and longer polyamines within the pore of the vacuolar TPC1/SV channel, one of the key determinants of the cell ionic homeostasis and signaling under stress conditions, and an additional site for spermine, which overlaps with the cytosolic regulatory Ca2+-binding site. Several unresolved problems are summarized, including the correct estimates of the subcellular levels of polyamines and their catabolites, their unexplored effects on nucleotide-gated and glutamate receptor channels of cell membranes and Ca2+-permeable and K+-selective channels in the membranes of plant mitochondria and chloroplasts, and pleiotropic mechanisms of polyamines’ action on H+ and Ca2+ pumps.This work critically discusses the direct and indirect effects of natural polyamines and their catabolites such as reactive oxygen species and γ-aminobutyric acid on the activity of key plant ion-transporting proteins such as plasma membrane H PAs putignaling . Stress-ignaling . PA effeignaling . Most liper se of these polycations across the plasma membrane, PM (+/H+ antiporter) and SOS2, key elements in response to salinity; within this complex, the activity of PUT3 and SOS1 is synergistically modulated  and fast (FV) vacuolar channels. FV channels are inhibited by micromolar Caeference , 2018. Beference ; Table 1eference , PAs canIn silico analysis predicts two binding sites for Put within the TPC1 pore, one below and one above the filter /H+ antiporter NHX1, which mediates vacuolar K+ uptake, may be counterbalanced by a massive increase in Put under K+ deprivation  and ligand-gated channels  the activity of the PM Hox state . Tonoplalication  and aftelication . Plant mlication .2+ efflux, pinpointing to the Ca2+-ATPase activation  induced eosin-sensitive Cativation . Low miln planta . Spm and+ efflux . The gen+-ATPase . Ca2+-de+-ATPase . PA-induvesicles . Spm butze roots . Contrarterminal . Howeverntration . Whethere ATPase . As Mg2+n Mg-ATP .2O2. It could be converted to a more aggressive hydroxyl radical (OH•) in the presence of the transition metals in the cell wall (2+-permeable) also known as ROS-activated NSCC or ROSIC  per se could also serve as a stress signal  and SKOR . ROS genand SKOR . The souand SKOR . Transmirogenase . In pea ia ROSIC . OH•-inde of Spm , whereasadditive . PAs als+ efflux . This po+ efflux . In cereolerance . However defense . A transs signal .+ efflux via GORK  in plants is produced via decarboxylation of glutamate or by a two-step Put catabolization. Increased DAO expression in response to abiotic stresses  may leadvia GORK . GABA ovolerance . GABA prey roots . Thus, G+-ATPase, inhibition (by direct nitrosylation) of GORK channels, activation of Ca2+ influx across the PM, and Ca2+ release from intracellular stores. The latter could lead to Ca2+-dependent activation of the slow anion and inhibition of inward-rectifying K+ currents, provoking stomata closure . In addition to pore block, long PA Spm likely interferes with cytosolic Ca2+ binding to the EF2 site , controlling mitochondrial Ca2+ homeostasis, metabolism, and cell death (ATP channel, whose activity decreases ΔΨ and damps mitochondrial ROS production under salinity and drought (Polyamines alleviate stress-induced suppression of photosynthesis in different ways, including a stabilization of the thylakoid ultrastructure, control of lipid composition, regulation of the expression, structure and oligomerization of photosynthetic membrane proteins, promotion of the chlorophyll biosynthesis, and improvement of the antioxidant activity . Howeverll death , a mitocll death , and an  drought .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.IP, OD, and SS developed the concept. MO-A performed the docking analysis. IZ-J composed the table. IP and OD wrote the draft. All authors edited the final 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.


Experiments were carried out in isolated VSMCs from mdx (a murine model of DMD) and congenic C57BL/10SnJ Wt mice. We found elevated [Ca2+]i and [Na+]i in VSMCs from mdx mice compared to Wt. Exposure to 1-oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC3 and TRPC6 channel activator, induced a greater elevation of [Ca2+]i and [Na+]i in mdx than Wt VSMCs. The OAG induced increases in [Ca2+]i could be abolished by either removal of extracellular Ca2+ or by SAR7334, a blocker of TRPC3 and TRPC 6 channels in both genotypes. Mdx and Wt VSMCs were susceptible to muscle cell stretch-induced elevations of [Ca2+]i and [Na+]i which was completely inhibited by GsMTx-4, a mechanosensitive ion channel inhibitor. Western blots showed a significant upregulation of TRPC1 -3, -6 proteins in mdx VSMCs compare to age-matched Wt. The lack of dystrophin in mdx VSMCs produced a profound alteration of [Ca2+]i and [Na+]i homeostasis that appears to be mediated by TRPC channels. Moreover, we have been able to demonstrate pharmacologically that the enhanced stretch-induced elevation of intracellular [Ca2+] and concomitant cell damage in mdx VSMCs also appears to be mediated through TRPC1, -3 and -6 channel activation.Duchenne muscular dystrophy (DMD) is an irreversible muscle disease characterized by a progressive loss of muscle function, decreased ambulation, and ultimately death as a result of cardiac or respiratory failure. DMD is caused by the lack of dystrophin, a protein that is important for membrane stability and signaling in excitable cells. Although vascular smooth muscle cells (VSMCs) dysfunction occurs in many pathological conditions, little is known about vascular smooth muscle function in DMD. We have previously shown that striated muscle cells, as well as neurons isolated from dystrophic (mdx) mice have higher intracellular Ca Dystroppatients  and mdx patients , 2014, ipatients  and in cpatients .2+ homeostasis  channels. Furthermore, mechanical stretch elicited a further elevation of [Ca2+]i in VSMCs from mdx compared to Wt, which was inhibited by the removal of extracellular calcium and by TRPC channel blockade.In the current study, we show for the first time that dystrophin deficiency of VSMCs leads to dysfunctional regulation of i or [Na+]i was measured in the stretched VSMCs using ion-selective microelectrodes.VSMCs were seeded on flexible-bottomed culture plates coated with poly-L-lysine . After 48 h to allow for cell attachment and spreading, Wt and mdx VSMCs were bathing with Ringer solution and then subjected to mechanical stretch elongation of 30 cycles/min (0.5 Hz), 20% elongation using a Flexcell FX 5000 tension system for 5 min. After the cyclic stretch, to estimate cell damage, the medium was collected for the determination of lactate dehydrogenase (LDH) activity (released by VSMCs) using the LDH kit from Sigma-Aldrich  according to the manufacturer’s instructions. Parallel series of Flex culture plates not subjected to stretching served as controls. At the time of collection of the medium for LDH determination, i was higher in mdx VSMCs (14 ± 1.2 mM n = 25) than observed in Wt cells (8 ± 0.1 mM n = 25) (p < 0.001) (Striated muscle cells from DMD patients and mdx mice  show ele< 0.001) . Similar< 0.001) .2+]i and [Na+]i in smooth muscle cells, VSMCs were exposed to the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) which activates TRPC3 and TRPC6 channels , and in mdx VSMCs by 73%, from 271 ± 29 nM, n = 25, to 470 ± 55 nM, n = 27 (p < 0.001)  on [Cachannels . Incubat< 0.001) . [Na+]i n in OAG . We examdx VSMCs .2+ ([Ca2+]e) on OAG-induced elevation of [Ca2+]i, Wt and mdx VSMCs were incubated for 5 min in Ca2+-free solution before OAG (100 μM) treatment. Exposure to reduced [Ca2+]e significantly lowered [Ca2+]i in Wt and mdx VSMCs but had a greater effect in mdx  than in Wt  VSMCs  VSMCs . In the dx VSMCs . In addi mM Mg2+ .2+]i elevation upon exposure to OAG, we measured [Ca2+]i in Wt and mdx VSMCs before and after incubation with SAR7334 which is a blocker of TRPC6 and TRPC3 channels  and mdx VSMCs (50%) (2+]i in Wt VSMCs (+]i in Wt (13%) and in mdx VSMCs (35%)  . The FigWt VSMCs , and mdxWt VSMCs . In addiCs (35%)  and prevenotypes .2+]i     . Under these conditions the increase in [Ca2+]i in response to stretch was abolished in both Wt and mdx VSMCs , which is known to inhibit mechanosensitive channels (2+]i in both genotypes (2+]i was mediated via L-type voltage-gated Ca2+ channels by incubating the VSMCs with the Ca2+ channel blocker nifedipine (10 μM). The stretch-induced increase in [Ca2+] was not modified by nifedipine in either genotype (Stretching smooth muscle cells has previously been shown to increase [Ca2+]i . Numerou2+]i  and are < 0.001)  and [Na+< 0.001) . In mdx < 0.001)  and [Na+< 0.001) . To testdx VSMCs . Re-addichannels . GsMTx-4enotypes . Additiogenotype .Resting LDH activity in the supernatant from non-stretched mdx VSMCs (a marker of cell damage) was 35% greater than in the supernatant from Wt VSMCs . Muscle 2+]i and [Na+]i and enhanced response to OAG observed in dystrophic VSMs was associated with changes in TRPC protein in the membrane, the expression of TRPC1, -3 and -6 were measured using Western blot analysis. Analysis of these blots demonstrated that TRPC1, TRPC3, and TRPC6 were significantly upregulated in VSMCs from mdx compared to Wt mice (To determine whether the elevation of [Ca Wt mice .2+]i and [Na+]i dysregulation in mdx VSMCs. The main findings in the present study are the following: (i) quiescent VSMCs isolated from mdx mice have [Ca2+]i and [Na+]i overload; (ii) the increase in [Ca2+]i and [Na+]i induced by exposure to OAG was greater in mdx than Wt VSMCs; (iii) Removal of extracellular Ca2+ or blockade of TRPC3 and -6 channels abolished the increases of [Ca2+]i and [Na+]i elicited by OAG; (iv) Muscle stretch-induced elevation of [Ca2+]i, and [Na+]i was significantly higher in mdx than Wt VSMCs and removal of extracellular Ca2+ or exposure to mechanosensitive channel blockers inhibited the increase in [Ca2+]i linked to mechanical stretch in both mdx and Wt VSMCs; (v) Baseline and stretch-induced LDH leak was significantly higher in mdx than Wt VSMCs; (vi) Expression of TRPC1, -3, and -6 proteins was upregulated in mdx compared to Wt VSMCs.To the best of our knowledge, this is the first comprehensive study of i may exert a myoprotective effect on mdx VSMCs precluding cell death.Intracellular calcium plays an essential role under physiological conditions to regulate many different processes in VSMCs . Quiescef 1.8 mM . The actgradient . Our datmdx mice , 2018. Cll death . Prevent2+]i and [Na+]i in Wt and mdx, however, the increment was more significant in mdx than Wt VSMCs. Western blots showed a significant upregulation of TRPC1 -3, -6 proteins in mdx VSMCs compare to age-matched Wt, which probably contribute to the observed intracellular Ca2+, and Na+ overload and also to the greater responsiveness to OAG found in mdx VSMCs.The TRPC channels are expressed in vascular smooth muscle vessels playing diverse physiological cellular responses . We have2+-free solution inhibited the observed rise in [Ca2+]i induced by OAG and induced a reversible depolarization of cell membrane potential. The effect of removing extracellular Ca2+ on resting membrane potential in smooth muscle has been previously reported by other groups (2+]i in a dose-dependent manner and also blocked the increases in [Ca2+]i and [Na+]i elicited by OAG in both Wt and mdx VSMCs. Based on SAR7334 pharmacological dose blocking effect on TRPC3 and TRPC6 channels (2+ dyshomeostasis is more significant than TRPC6 channels. The increase of [Ca2+]i induced by OAG was not affected by the Ca2+ channel inhibitor nifedipine, which suggests that the activation of L-type Ca2+ channels is not part of the mechanism by which OAG induced elevation of [Ca2+]i and [Na+]i in VSMCs. Dysregulation of TRPC channels has been associated with diverse vascular pathologies (Car groups . Furtherr groups , reducedchannels , we can hologies  which cohologies .2+]i (2+ influx pathway activated by mechanosensors in VSMCs appears to be mediated by canonical cationic channel, which seems to be more critical in mdx VSMCs than Wt. Extracellular Ca2+ influx mediated by the voltage-dependent L-type Ca2+ channels has been suggested to intervene in VSMC stretch-mediated activation (2+]i elevation does not support this hypothesis.Previous studies have demonstrated that VSCM stretch induces elevation of [Ca2+]i . TRPC ch2+]i . Further2+]i , 2016. G2+]i . Here, wtivation . However2+ elevation after VSMCs stretch was suppressed entirely in both genotypes by removal of extracellular Ca2+ or GsMTx-4, indicating that this event is mediated by Ca2+ influx from the extracellular side which appears to be through a GsMTx-4 sensitive pathway.Basal LDH activity in the extracellular medium was higher in the supernatant from mdx compared to Wt VSMCs, which is consistent with the idea that the absence of dystrophin leads to chronic injury due to a lack of structural support at the sarcolemma. Besides, stretching further increased extracellular LDH activity, an indicator that this muscle stretch yielded some degree of cell injury in both genotypes. However, because the increase was higher in mdx than Wt, these data support the view that the lack of dystrophin makes VSCMs more sensitive to contraction-induced damage . The int2+ observed in VSMCs from mdx mice, and we did not study the functional aspects of TRPC channels. Secondly, we did not carry out experiments in which TRPC1, -3, and -6 channels were individually or collectively downregulated using siRNA. Therefore, we were unable to assess whether decreasing TRPC channel expression rescues or improves intracellular Ca2+ regulation in mdx VSMCs.Despite the novelty of our study, some limitations should be acknowledged. First, we used a pharmacological approach to characterize the involvement of TRPC channels in the dysregulation of intracellular Ca2+ and Na+ in VSMCs from mdx mice. The imbalance of [Ca2+]i and [Na+]i appears to be mediated mostly through TRPC channels since their pharmacological blocking activity markedly protected mdx VSMCs (2+]i in mdx VSMCs, which also appears to be mediated by TRPC channels. The originality of our paper stands in revealing the relevance of TRPC channels in the pathology of VSMCs in mdx mice. TRPC channels could be promising targets to help manage symptoms and slow the progression of this devastating disease.This study provides direct evidence of anomalous regulation of resting intracellular Cadx VSMCs . FurtherThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.All protocols used in the study were performed following the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the IACUC of Mount Sinai Medical.JL and JA contributed to the conception and design of the study. JL, AU, GF, and EE performed the experiments. All authors contributed to the manuscript revision, read 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.


DOT1L and SETD1A. We finally show that selective leukemic cell death is achievable with a small molecule inhibitor of MBNL1. These findings provide the basis for a new therapeutic target in MLL-rearranged leukemia and act as further validation of a burgeoning paradigm in targeted therapy, namely the disruption of cancer-specific splicing programs through the targeting of selectively essential RNA binding proteins.Despite growing awareness of the biologic features underlying MLL-rearranged leukemia, targeted therapies for this leukemia have remained elusive and clinical outcomes remain dismal. MBNL1, a protein involved in alternative splicing, is consistently overexpressed in MLL-rearranged leukemias. We found that MBNL1 loss significantly impairs propagation of murine and human MLL-rearranged leukemia in vitro and in vivo. Through transcriptomic profiling of our experimental systems, we show that in leukemic cells, MBNL1 regulates alternative splicing (predominantly intron exclusion) of several genes including those essential for MLL-rearranged leukemogenesis, such as  The alternative splicing regulator MBNL1 is overexpressed in MLL-rearranged leukaemia. Here, the authors show that MBNL1-mediated alternative splicing leads to a specific splicing profile in MLL-rearranged leukaemia and loss or inhibition of MBNL1 impairs leukaemia development in vitro and in vivo. MLL) gene with one of over 70 fusion partners are commonly found in a subset of aggressive leukemias including infant leukemia, and are associated with poor outcome3. Regardless of the fusion partner and the type of leukemia (acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL)) all MLL-rearranged leukemias share a common gene expression profile that is distinct from that of non−MLL-rearranged leukemias5. Analysis of MLL-rearranged signatures reveals that muscleblind-like 1 (MBNL1) is one of the most consistently overexpressed genes in MLL-rearranged leukemia compared to other leukemias6.Reciprocal chromosomal translocations of the mixed lineage leukemia  mRNA is a main contributor to the pathogenesis of myotonic dystrophy type 1 (DM1), a syndrome characterized by muscle weakness, cardiac abnormalities, intellectual disability, and cataracts. The sequestration of MBNL1 results in missplicing of target mRNAs, leading to the clinical features12. Moreover, MBNL1 and MBNL2 are known to play a central role in regulating the pattern of alternative splicing events that control embryonic cell pluripotency13. Knockdown of Mbnl1 in murine fetal liver cells leads to blockade of terminal erythropoiesis through deregulation of alternative splicing of Ndel1 mRNA14.Muscleblind-like 1 (MBNL1) is an RNA-binding protein that has been shown to regulate RNA alternative splicing, localization, and integrity19. The increased expression of MBNL1 in MLL-rearranged leukemia described above, as well as evidence that the MLL-fusion complex directly binds the MBNL1 promoter20, suggest that MBNL1-mediated RNA splicing may be important to the pathogenesis of MLL-rearranged leukemias. The mechanism of action and extent of essentiality of MBNL1 in MLL-rearranged leukemogenesis, however, remains unknown.Abnormalities in RNA processing, particularly splicing, have been associated with cancer, including AML and ALL)independent of spliceosome mutationsIn this report, through a combination of functional genomic studies, pharmacologic inhibition, and comprehensive analysis of alternative splicing we demonstrate that MBNL1 is required in MLL-rearranged leukemia.MBNL1 expression in MLL-rearranged leukemia, we first compared multiple gene expression studies which identified differentially expressed genes between MLL-rearranged and MLL-wildtype leukemias22. We began by examining the intersection of these gene expression signatures, and found that MBNL1 expression was a common feature of these signatures across both acute myeloid and lymphoblastic leukemias  in human leukemia cell lines. We found MBNL1 expression in all leukemic cell lines tested, with the highest MBNL1 expression in MLL-rearranged cell lines  oncogene demonstrated higher levels of MBNL1 expression compared to normal CD34+ cord blood cells  and H3K79me2 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) datasets from THP-130 and ML-231 cell lines (with MLL-AF9 and MLL-AF6 respectively), as well as from the MV4;1130, RS4;1132, and SEM33 cell lines which bear an MLL-AF4 fusion. We found evidence of MLL-N/fusion-C binding to the MBNL1 promoter and gene body, along with H3K79me2 enrichment across all cell lines studied  despite efficient knockdown  knockdown studies of ure Fig.  and in cure Fig. . Howeverlls Fig.  bearing +). Bulk cells were transplanted into immune-deficient NSGS mice. At the time of visible illness, all mice showed robust human cell engraftment (ranging from 92.4 to 99.4% in the MLL-AF9 group and 7.6–40.2% in the MLL-AF10 group) in the bone marrow. Bone marrow from mice transplanted with shNT-transduced cells contained Venus+ cells , whereas the majority of mice transplanted with shMBNL1-64 had virtually no Venus+ fraction  with shMBNL1-64 or a non-targeting (NT) control. Transduction efficiency ranged from 67 to 85%  and non-MLL-rearranged leukemia cell lines , as well as on normal CD34+ cord blood cells. Cells were incubated with the inhibitor at various concentrations (based on previous experiments). Leukemia cells were sensitive to the inhibitor treatment as evidenced by a decrease in cell viability within 18 h in a variety of leukemia cell lines. Despite the high concentrations necessary to achieve the observed effects, MLL-rearranged leukemia cells demonstrated comparatively higher sensitivity to the compound  KO mice lacking Mbnl1 exon 3 (kindly provided by Dr. Maurice Swanson)38. We transduced Mbnl1−/− and littermate control (Mbnl1+/+) lineage-negative bone marrow cells with an MLL-AF9 GFP-bearing retrovirus34. Four days after transduction, Mbnl1+/+ and Mbnl1−/− cells were plated in semi-solid culture for at least two weekly serial rounds to select transformed cells, which were subsequently transplanted into syngeneic irradiated mice  compared to mice secondarily transplanted with Mbnl1+/+ leukemia cells (49 days)  Fig. . In vitrMbnl1−/− mice. At steady state, Mbnl1−/− mice and Mbnl1+/+ controls demonstrated comparable peripheral blood counts, bone marrow cellularity, and progenitor and stem cell counts. To test the role of Mbnl1 in stress hematopoiesis we injected Mbnl1−/− KO and Mbnl1+/+ wild type (WT) mice with one sublethal dose of 5-Fluorouracil (5-FU). No difference was observed regarding the time to recovery of RBCs, platelets, and WBCs from Mbnl1−/− mice compared to controls  along with CD45.1 competitor bone marrow cells in a 1:1 ratio. Four weeks after transplantation the repopulation ability of transplanted cells was tested by peripheral blood flow cytometry for CD45.1 vs 45.2. Bone marrow analyses showed 73.8% and 30.3% CD45.2 donor chimerism in mice transplanted with Mbnl1+/+ or with Mbnl1−/− bone marrow cells, respectively  HSCs compared to WT mice  AML patients, and non-targeting shRNA controls to MBNL1 knockdown (NT). Similar to other AML datasets, we find that MBNL1 is most highly induced in MLL-rearranged AMLs in RNA-seq derived from the Leucegene cohort and comparable sorted healthy control hematopoietic progenitors  with knockdown of ors Fig. .Fig. 6PaMBNL1 knockdown cell line data46. Breakdown of alternative splicing and promoter events in these datasets by event-type finds that MLL-rearranged leukemias predominantly exhibit exclusion of retained introns when compared to CN-AML   in AS events across different cell lines  among the most concordant  to the patient and AML Fig. , top. WeAML Fig. , bottom.ons Fig. . Comparines Fig. . These dwn in K56 cells amnts Fig. .RHOC, RHPN1, DLG4, ARHGEF40, ARHGEF1, DENND4B, ARHGAP17, ARAP1, DNM2), regulators of Notch signaling  and genes mediating membrane trafficking and vesicle-mediated transport   Fig. . FurtherT1L Fig. . Both DOsis Fig. . We alsoIgG Fig. , suggestown Fig.  likely down Fig. . Through.54 Fig. . TogetheMBNL1, suggesting that MBNL1-mediated alternative mRNA splicing patterns contribute to the pathogenesis of MLL-rearranged leukemias.Our findings described above demonstrate that MBNL1 is essential for MLL-rearranged leukemia cell growth, yet only modestly so for normal hematopoiesis. We also demonstrate using novel bioinformatic analyses that MLL-rearranged leukemias exhibit a unique pattern of mRNA isoform utilization that is partially reversed upon knockdown of 11, though an understanding of its role in disease pathogenesis has been largely limited to DM1 and Fuchs corneal endothelial dystrophy55. Recently, MBNL proteins have been reported to control the pluripotency of embryonic stem cells and fetal terminal erythropoiesis as well14, implicating their activity to some extent in the hematopoietic system. In examining results of gene expression profiling of MLL leukemias, we identified MBNL1 as one of the most consistently overexpressed genes in this family of leukemia regardless of lineage, even more so than the HOXA cluster genes. This may be due to the fact that even within MLL-rearranged leukemias HOXA gene expression can be highly variable, especially in pre-B ALL57. Because we sought commonalities in gene overexpression across both myeloblastic and lymphoblastic leukemias, this may explain why the HOXA cluster genes were not consistently overexpressed in every dataset we examined.MBNL1 is well known to regulate RNA alternative splicing, localization, and decayDOT1L and the SET1 histone methyltransferase complex member and cyclin K regulator SETD1A. A frequent splicing outcome following MBNL1 knockdown was intron retention, and MBNL1 knockdown appears generally to reverse intron exclusion. Our findings suggest that MLL-fusion proteins, via MBNL1 overexpression, promote the expression of protein coding genes typically suppressed through intron retention-introduced premature termination codons. The negative impacts of MBNL1 knockdown on cell growth are likely a net effect of multiple transcripts (and the subsequent proteins) being affected, rather than solely arising from alternative splicing of the products of a single gene. Ultimately, it remains challenging to quantify the exact extent to which MBNL1 or other splicing factors mediate specific oncogenic splicing programs in vivo, due to a lack of controlled model systems to quantify such effects. In our analysis, we compared MLL-rearranged patient samples to CN-AML as a control, however even within this comparison there are likely additional splicing differences attributable in part to phenomena such as lineage skewing58, co-occurrence of spliceosome mutations within the patient cohort analyzed above44, or other yet-unidentified MLL fusion-specific transcriptional/splicing programs, hence isolation of a core splicing factor-associated program in vitro will only remain an approximation of molecular programs found in patients. Despite this, we demonstrate a high degree of splicing discordance between MBNL1 loss and MLL fusion-associated patient splicing, indicating a high specificity of our analysis for MBNL1-mediated splicing events.The results of our RNA-seq and splicing analyses suggest that a major function of MBNL1 in MLL-rearranged leukemia is through alternative splicing, and a number of alternatively spliced genes identified using our approach have been implicated in leukemogenesis, including a subset of epigenetic regulators such as the H3K79 methyltransferase Mbnl1−/− mice display splicing defects in heart and skeletal muscle yet do not present clinical features of DM1. Notably, Mbnl2 was found to compensate for Mbnl1 in murine tissues59. We speculate that a similar phenomenon might explain the eventual development of leukemia in our Mbnl1−/− mice despite the growth inhibition in our knockdown studies in human cells. In both Mbnl1+/+ and Mbnl1−/− states Mbnl2 and Mbnl3 are stably expressed in mouse leukemia cells, however neither analogue was meaningfully expressed in the human leukemia lines assayed  may be more deleterious than de novo transformation of an MBNL1-deficient cell, as occurs in the mouse retroviral leukemia model. We hypothesize that transformation of Mbnl1−/− cells could conceivably engage alternate oncogenic pathways which eventually allow leukemia cell survival, though this issue of MBNL1 requirement for MLL leukemia initiation versus propagation requires further study.All three members of the MBNL family regulate alternative splicing during development. + hematopoietic stem/progenitor cells. An alternative therapeutic approach may involve targeting elements of the MBNL1 transcript itself using therapeutic RNA inhibition specifically targeted at hematopoietic or leukemic cells. Specifically, our data suggest that human MLL-rearranged leukemia cells have increased expression of MBNL1 transcripts that exclude exon 5 which influences nuclear vs cytoplasmic localization of MBNL143. We hypothesize that the role of MBNL1 in facilitating productive splicing of the MLL-rearranged oncogenic program may in part be mediated by exon 5 exclusion. Therapeutics which influence exon utilization (such as antisense oligonucleotides) may thus represent an alternate means of disrupting the function of MBNL1. However, the specific role of the exclusion transcript and the associated cellular localization are not fully understood and require further study.Given the poor prognosis of patients with MLL-rearranged acute leukemia, there remains an urgent need for better therapeutic approaches in this subgroup of patients. Our data suggests that targeting MBNL1 may be a valid therapeutic avenue in the treatment of MLL-rearranged leukemia. As a proof of concept, we demonstrate that a small-molecule inhibitor rationally designed against MBNL1 can preferentially kill MLL-rearranged leukemia cells while sparing normal CD34MBNL1 overexpression mediated by the MLL-fusion protein promotes leukemogenesis via an altered splicing profile (primarily changes in intron retention) of MBNL1 targets. We demonstrate that MBNL1 is an important regulator of alternative splicing events preferentially expressed in MLL-rearranged leukemia and which participate in the MLL-rearranged oncogenic program. Our data shows the effect of MBNL1 at the mRNA and protein level, and suggests that MBNL1 plays a key role in the pathogenesis of MLL-rearranged leukemia by stabilizing the transcripts of multiple leukemogenic genes including DOT1L and SETD1A; this in turn supports transcriptional activation of downstream targets of the MLL-fusion protein, including activation of MBNL1, creating a positive feedback loop  or Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin.Fully deidentified primary patient cells were obtained from the Cincinnati Children’s Hospital Medical Center Biorepository. Patient identities have been protected. PDX models were generated using residual diagnostic specimens according to an IRB approved protocol (#2008-0021) following proper informed consent. Cells from patient samples were transplanted into busulfan-conditioned NOD/LtSz-SCID interleukin-2(IL2)RG−/− SGM3 (NSGS)  mice. By the time the mice displayed signs of leukemia, the only human cells that remained in the mice were leukemic cells (MLL-rearrangement was confirmed with FISH). De-identified PDX spleen samples taken from NSGS mice  were used for the ex vivo knockdown experiments described below. Cells were cultured in IMDM supplemented with 20% FBS and 10 ng/ml human cytokines including SCF, FLT3-Ligand, Thrombopoietin, IL-3, and IL-6.35 lentivirus and the MSCV-GFP-IRES-tTA34 retrovirus. After transduction, cells were maintained in IMDM/20% FBS with 10 ng/mL of each cytokine. A pure population of dsRED+GFP+ cells were selected over several weeks of culture. To repress expression of the MLL fusion, cells were treated with 1 µg/mL doxycycline.MLL-AF9 Tet-off human CD34+ cells were a kind gift from Dr. James Mulloy, and were generated as follows. Umbilical cord blood (UCB) was obtained from the Translational Trial Development and Support Laboratory (TTDSL) of CCHMC. UCB units are collected and distributed by the TTDSL under an IRB-approved protocol, and originate from discarded umbilical cords and are thus completely anonymized, rendering them exempt from human subjects research. CD34+ cells were isolated from UCB using the EasySep CD34+ isolation kit (StemCell Technologies). CD34+ cells were pre-stimulated in IMDM/20%FBS containing 100 ng/mL SCF, TPO, Flt3-L, and IL-6 and 20 ng/mL IL-3 for 2 days before transduction. Transduction was carried out using spinoculation onto Retronectin (Takara Bio) coated plates along with 4 µg/mL polybrene. Cells were trasduced with both the pSIN-TREtight-dsRED-MLL/AF9Cell lines were periodically validated by STR genotyping through Genetica Cell Line Testing (LabCorp). Cells were tested and were negative for mycoplasma contamination. None of the cell lines utilized in this study are recognized by the ICLAC as being commonly misidentified.www.vizome.org/aml as indicated.RNA-Seq data for primary patient AML and ALL samples were obtained from the St. Jude Cloud (originating publications/datasets as cited in the “Results” section), and from Mbnl1−/− or Mbnl1+/+ cells. For studies of normal murine hematopoiesis, 1 × 106 CD45.2 Mbnl1−/− or Mbnl1+/+ bone marrow donor cells were transplanted with 1 × 106 CD45.1 competitor cells  or without competitor cells (non-competitive transplantation) into lethally irradiated BoyJ recipient mice (RRID: IMSR_JAX:002014). Four or eight weeks after transplantation, bone marrow of recipients was analyzed for donor chimerism. Mice were assessed for signs of leukemia, and when distressed, were sacrificed and tested for leukocytosis, anemia, thrombocytopenia and splenomegaly. For normal hematopoiesis experiments, peripheral blood of recipients was examined every 4 weeks and bone marrow was tested at the end point of the experiment for the presence of donor cells by flow cytometry. For studies of time-to-leukemia in Mbnl1 knockout mice, BoyJ recipient mice were conditioned by sub-lethal irradiation and i.v. injected with 2 × 105 transduced donor cells. For xenograft experiments with MLL-rearranged cell lines, immunocompromised NOD/LtSz-SCID interleukin-2(IL2)RG−/− (NSG)  recipient mice were treated with busulfan (Sigma) 30 µg/mg intraperitoneally and transplanted with 2 × 105 human cells 24 h later. For xenograft experiments with MLL-rearranged primary patient cells, immunocompromised NOD/LtSz-SCID interleukin-2(IL2)RG−/− SGM3 (NSGS)  recipient mice were conditioned with busulfan and transplanted with 2–7.5 × 105 human cells 24 h later. In xenograft experiments bone marrow samples were collected four weeks after transplantation as well as when signs of leukemia were present; aspirates were analyzed via flow cytometry for the presence of human CD45+ cells and the presence of shRNA-transduced CD45+ and Venus-positive cellsAll animals used for this study were 6–12-weeks old. All animal experiments were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). C57BL/6 mice were used as donors for transplantation experiments using 4 method or FuGENE 6 reagent (Promega) according to the manufacturer’s recommendations. Mouse bone marrow cells were isolated by crushing ischial bones, femurs and tibias. Cells were then lineage-depleted using the Lineage Cell Depletion kit (Miltenyi Biotec) following magnetic separation by using an AutoMACS Pro Separator (Miltenyi Biotec). Lineage-negative cells were then pre-stimulated with 100 ng/ml cytokines  (Peprotech) overnight and transduced with retroviral MLL-AF9 constructs using spinoculation on retronectin-coated plates.Retroviral and lentiviral supernatants were generated by transfection of HEK293T cells using the CaPOThe lentiviral MBNL1 shRNA pLKO.1-Puro plasmids  were purchased from Millipore Sigma. Human cells were incubated with a single dose of lentiviral supernatant overnight. Cells transduced with constructs granting puromycin resistance were selected in 0.5–5 µg/ml of Puromycin for 72 h. Cells transduced with constructs containing a fluorescent marker  were isolated on day 4–5 after transduction by sorting using a MoFlo XDP (Beckman Coulter), FACSAria (BD Biosciences), or a Sony SH800S (Sony Biotechnology.) MSCV MLL-AF9 GFP was a kind gift from Dr. Gang Huang.For murine peripheral blood flow cytometry analysis, peripheral blood was collected by retro-orbital bleeding and incubated with surface antibodies (eBioscience) in staining buffer  supplemented with 0.5% FBS). Peripheral blood cell suspensions were treated with Pharm Lyse Lysing Buffer (BD Biosciences) and washed twice with PBS. For apoptosis assays, cells were incubated with allophycocyanin-conjugated Annexin V (BD Bioscience) for 15 min at RT in 1X Annexin V Binding Buffer (BD Bioscience) following staining with 7-aminoactinomycin (eBioscience). Data were acquired on a FACSCanto I and results were analyzed using FlowJo Version 10 (FlowJo). The list of antibodies used for flow cytometry analysis of mouse and human cells is indexed in Supplementary Data i-U scope was used for images of colonies and automatic counting was performed using Nikon Elements software.Mouse bone marrow cells were plated in methylcellulose-containing media  supplemented with 10 ng/ml of granulocyte-macrophage colony-stimulating factor (Peprotech). After 7–8 days of culture, colonies were counted and re-plated for 3–5 rounds. Human cells were cultured in methylcellulose-containing media . Colonies were scored 14 days after plating. The Nikon Eclipse TTM HS Red Mix (Bioline) according to the manufacturer recommendations. The amplified reaction products were separated on a agarose gel (ranging from 1 to 3.5%). The BioChemi System (UVP Bioimaging system) was used for visualization and analysis (Image Lab 6.0 used for band quantification). For quantitative RT-PCR 5–10 ng of cDNA was analyzed using iTaq Universal SYBR Green Supermix and iTaq Universal Probes Supermix (Bio-Rad) in a StepOnePlus Real-Time PCR machine (Applied Biosystems). Primers for RT-PCR are detailed in Supplementary Data Total RNA was extracted from human Puromycin-selected or sorted Venus-positive cells using the RNeasy Mini kit (Qiagen). RNA was reversed transcribed into cDNA using iScript Advanced cDNA Synthesis kit (Bio-Rad Laboratories). RT-PCR reactions were carried out in an Eppendorf PCR Cycler using MyTaq7 cells were used per immunoprecipitation reaction. Five micrograms of either mouse anti-IgG  or anti-MBNL1  was used. After RNA-immunoprecipitation, RNA was extracted and analyzed via qRT-PCR through the process described in the “RT- and Quantitative RT-PCR” section, with the exception that the qRT-PCR was run using Taqman Probes  instead of SYBR Green.RNA-Immunoprecipitation was performed using the Magna RIP kit (Millipore). 2.0 × 10The primary antibodies used were anti-MLL1 , anti-MBNL1 , anti-DOT1L , anti-SETD1A , anti-Actin , anti-Lamin B1  and anti- β-tubulin . Whole cell lysates were isolated using RIPA buffer (Sigma) while nuclear extracts were obtained using the NE-PER kit (Thermo Scientific) according to manufacturer’s instructions, and the amount of protein was determined by the BCA Protein Assay Kit (Thermo Scientific). Ten µg of protein was separated by SDS-PAGE on a 4–20% gradient gel (Bio-Rad). After transfer to PVDF membranes, blots were blocked with Odyssey® Blocking Buffer TBS (LI-COR) for one hour and incubated with primary antibodies overnight. After washing, blots were treated with appropriate secondary IRDye 680RD goat anti-mouse (LI-COR) and IRDye 800CW goat anti-rabbit (LI-COR) antibodies at a dilution of 1:10,000 for one hour. Images were obtained using the Odyssey CLx Infrared Imaging System (LI-COR).5 cells/ml were plated in a 96-well plate and incubated with the inhibitor at a concentration of 250 µM, 500 µM, or vehicle (DMSO). At various time points after incubation cells were stained with Annexin V and 7-AAD and analyzed by flow cytometry; additionally, viable cells were counted using Trypan Blue.0.5 × 10Blood 2004 Supplementary Table 104, Mullighan et al. Leukemia 2007 Suppl Table 522, Zangrando et al. BMC Med Genomics 2009 Suppl Table 221 and Stam et al. Blood 2010 Suppl Table 56. Probe IDs were reconciled with gene symbols, and intersections were determined and visualized with http://bioinformatics.psb.ugent.be/webtools/Venn/.For Fig. For Fig. All means were compared using two-tailed t-tests unless specifically indicated otherwise. For survival analyses, log-rank tests were used to compare survival curves. The statistics used for the RNA sequencing analyses are described in the section below . Graphpad Prism 8 was used to perform statistical tests and create graphs. Select figures created with BioRender.com.n = 3 MOLM13, n = 3 MV411), sh65  or shNT control  at a depth of >40 million paired-end stranded reads using the Illumina HiSeq 2500 (GSE123441), following library preparation with the TruSeq RNA Library Prep Kit (Illumina), similar to that of the Leucegene RNA-Seq dataset (GSE67040). A longer 12-day MBNL1 knockdown with independent shNT controls (MOLM13) was also performed as biological triplicates, processed with the same protocol on an Illumina NovaSeq platform. AML FASTQ files for MLL (n = 27) and CN AML patients (n = 29) were downloaded from the GEO database as previously described44. All FASTQ files were aligned to the reference human genome (GRCh37/hg19) using the latest version of STAR61. Exon-exon and exon-intron spanning reads were determined from the software AltAnalyze (version 2.1.1) using the Ensembl-72 human database, along with splicing event calculation and annotation with the MultiPath-PSI algorithm . For comparative splicing analyses, >50% of samples for each splicing event were required to have detected PSI value, consistent between the patient dataset and transduced cell line analyses. As statistical thresholds, we used a two-tailed empirical Bayes moderated (eBayes) t-test with p ≤ 0.01 for shRNA analyses and delta PSI > 0.1. Breakdown of splicing event categories is provided through the MultiPath-PSI software and associated heatmap visualization through AltAnalyze. For the MLL patient samples versus CN, an eBayes t-test was also performed (p ≤ 0.01) and delta PSI > 0.1 was used for differential splicing analysis. Only unique junction-cluster ID results were reported. Relative PSI differences were calculated separately for patient dataset and transduced cell line samples for joint visualization. Gene expression analyses were similarly performed in AltAnalyze from the input STAR BAM files, using the same p-value thresholds and 1.5 fold change cutoff, with gene-level RPKM quantification. ENCODE RNA binding protein knockdown and control hematopoetic cell type splicing comparison results were used from a prior study as previously described62. RNA recognition element (RRE) enrichment analysis was performed on alternatively spliced exons and introns (exons with 500nt flanking intron sequence bins) using the software HOMER63 from a large database of RRE motif models in the form of position frequency matrices (PFMs) taken from the CisBP-RNA database64. Evaluation of spilcing concordance was performed using a custom Python script. Pathway enrichment analysis was performed with ToppGene65. GSEAPreranked51 was performed on differentially expressed genes as determined by AltAnalyze.RNA sequencing was performed on total RNA obtained from MOLM13 or MV411 cell lines following transduction with either MBNL1 sh64 (Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary


Stochastic processes leading voltage-gated ion channel dynamics on the nerve cell membrane are a sufficient condition to describe membrane conductance through statistical mechanics of disordered and complex systems.Voltage-gated ion channels in the nerve cell membrane are described by the Ising model. Stochastic circuit elements called “Ising Neural Machines” are introduced. Action potentials are described as quasi-particles of a statistical field theory for the Ising system.The particle description of action potentials is a new point of view and a powerful tool to describe the generation and propagation of nerve impulses, especially when classical electrophysiological models break down.The particle description of action potentials allows us to develop a new generation of devices to study neurodegenerative and demyelinating diseases as Multiple Sclerosis and Alzheimer’s disease, even integrated by connectomes. It is also suitable for the study of complex networks, quantum computing, artificial intelligence, machine and deep learning, cryptography, ultra-fast lines for entanglement experiments and many other applications of medical, physical and engineering interest. In 1952 British physiologists Sir Alan Lloyd Hodgkin 1914–1998) and Sir Andrew Fielding Huxley (1917–2012) at the University of Cambridge demonstrated the existence of selective and voltage-dependent ion channels in the nerve cell membrane with five famous pioneers works published in the Journal of Physiology. They received the Nobel Prize for Medicine in 1963 together with the Australian physiologist Sir John Carew Eccles (1903–1997) .We call this quasi-particle Carrier*Ising in (9) after the trigger of the first spike, because the process is ruled only by the Ising machines.As a first approximation, if we consider the density of ion channels on the axonal membrane of non-myelinated axons almost constant , 31–34, Therefore, we consider the process of generation and transmission of a spike for a time t > τ, where τ is the generation time of each spike by each pair (Na/K) of Ising Machines. If we indicate with s the coordinate along with the axis of the axon , then each section (housing a pair of Ising Machines Na/K) will be traveled in a time t by the coordinate s = s (t) Therefore, the velocity v = ds/dt of the nuon will be given by the limit of the difference quotient ∆s/∆t with ∆t ≠ 0. Our choice of the one-dimensional Ising model is thus clear. Finally, we will havem and thickness dm distributed from the radius a1 of the axon core to the external radius a2, that is to the axon radius  given by the following relations  is a powerful tool that allows us to use the techniques and results of theoretical and general physics , 43. As nectomes . A detainectomes . Furthernectomes ). Other nectomes  to study


These, in turn, produce an inward sodium–calcium exchange current which opposes AP repolarization. Restoration of AP phase 1 repolarization improved Ca2+ release synchrony and reduced late Ca2+ spark rate, suggesting a different approach to reducing the risk of sudden death in heart failure.Sudden cardiac death in heart failure is a major unsolved clinical problem that is linked to the development of a spontaneous arrhythmia. Early afterdepolarizations (EADs) are an arrhythmogenic mechanism, but the cellular trigger for EADs in heart failure is unclear. We show that the reduction in synchronous Ca 2+ measurements were combined with action potential voltage clamp techniques in a physiologically relevant heart failure model. In failing cells, the loss of Ca2+ release synchrony at the start of the action potential leads to an increase in number of microscopic intracellular Ca2+ release events (“late” Ca2+ sparks) during phase 2–3 of the action potential. These late Ca2+ sparks prolong the Ca2+ transient that activates contraction and can trigger propagating microscopic Ca2+ ripples, larger macroscopic Ca2+ waves, and EADs. Modification of the action potential to include steps to different potentials revealed the amount of current generated by these late Ca2+ sparks and their (subsequent) spatiotemporal summation into Ca2+ ripples/waves. Comparison of this current to the net current that causes action potential repolarization shows that late Ca2+ sparks provide a mechanism for EAD initiation. Computer simulations confirmed that this forms the basis of a strong oscillatory positive feedback system that can act in parallel with other purely voltage-dependent ionic mechanisms for EAD initiation. In failing heart cells, restoration of the action potential to a nonfailing phase 1 configuration improved the synchrony of excitation–contraction coupling, increased Ca2+ transient amplitude, and suppressed late Ca2+ sparks. Therapeutic control of late Ca2+ spark activity may provide an additional approach for treating heart failure and reduce the risk for sudden cardiac death.Sudden death in heart failure patients is a major clinical problem worldwide, but it is unclear how arrhythmogenic early afterdepolarizations (EADs) are triggered in failing heart cells. To examine EAD initiation, high-sensitivity intracellular Ca More th+ sparks , which s can occur during the decay of the Ca2+ transient in normal rabbit cardiac myocytes (2+ ripples” are promoted in HF and can generate sufficient NCX current (INCX) to trigger EADs. In addition, normalizing the early AP waveform in HF promotes a faster and larger SR Ca2+ release (which should improve contractility) and at the same time decreases arrhythmogenic LCS activity.We recently showed that “late” Camyocytes . Here, wSI Appendix, Fig. S1), but, to study repolarization mechanisms, an AP similar to human is desirable, and we therefore used an established rabbit model of chronic myocardial infarction that causes HF. When left ventricular ejection fraction had fallen to 44 ± 3% , indicative of moderate to severe HF, ventricular myocytes exhibited t-tubule remodeling  . All of these changes are also seen in human HF  and increased SR release latency  , 19. Alt density C in this density D and bechuman HF , 20. The latency F  in these conditions clearly opposed the normal decline of Ca2+ during the Ca2+ transient.LCS activity depends on SR Ca content  which ge content , 23. A poke EADs . With thoke EADs A. The hinet) and/or repolarization reserve  is directly proportional to Inet because Inet = −Cm·dVm/dt (where Cm is membrane capacitance). Applying this equation allows us to determine how much current is responsible for the repolarization phase of the AP, and net.. In this exemplar, Inet was only ∼0.18 pA/pF during the late AP repolarization phase, and the average Inet during AP repolarization was 0.26 ± 0.08 pA/pF (mean ± SD n/N = 15/4), showing that a small change in membrane currents will affect repolarization trajectory. It follows that any additional inward current of similar amplitude to Inet could stop AP repolarization  and thereby initiate an EAD.In order to generate an EAD, the net cellular repolarizing current  where EADs occur  and evoked inward current (black trace) are shown in Lower. This inward current had a linear relationship to mean Ca2+ with a slope of −1.5 pA/pF/µmol/L , compatible with the expected Ca2+ dependence of INCX  is rather chaotic with no obvious Ca2+ waves being present. However, after image processing, numerous LCS events are seen, some of which form propagating Ca2+ ripples . Vm and Inet can be linked to Ca2+ by two mechanisms: (1) Either Ca2+ activates inward INCX producing an inward shift of Inet and depolarization of Vm or (2) Vm activates L-type Ca2+ channels (LTCCs) which then trigger SR Ca2+ release. As shown by the colored bars in 2+ occurred in synchrony with inward Inet  which also supports the idea that the initiation of EADs can result from the summation of LCS and Ca2+ ripples which activate inward INCX.NCX for EAD generation has also been demonstrated in murine cells by heterozygous NCX knockdown and in failing rabbit ventricle by acute NCX blockade  or a potential cause (by leading the change in Vm). When the Ca load was high, inward INCX clearly preceded the change in Vm  and had2+ transient, and this was associated with an increase in Ca2+ release synchrony. On average, the normal AP increased the rate of rise of the Ca2+ transient from 30 ± 8 nmol/L/ms to 40 ± 9 nmol/L/ms , while the time to peak decreased from 75 ± 9 ms to 65 ± 9 ms . This improvement in excitation–contraction coupling efficiency was also reflected by a decrease in the latency for Ca2+ release  the t-tubule disruption associated with HF would still be present and (2) there is reduced SERCA2a expression in HF which decreases SR Ca2+ uptake rate  Diffusion of Ca2+ from an initiating LCS may trigger additional LCS due to the gain inherent in Ca2+-induced Ca2+ release (CICR). This SR load-dependent process manifests as propagating Ca2+ ripples and, if enough LCS sites are available, more synchronous and larger Ca2+ waves. (2) LCS will increase inward NCX current to depolarize Vm (m couples back onto LCS activity via the Vm dependence of LTCC gating (which may explain the Vm dependence of LCS frequency shown in 2+ load arising from ICa reactivation.Reactivation of Iated ICa . Here weated ICa . This Caarize Vm  while Vmm) and Ca2+ oscillators can couple and synergize  may not be able to initiate cell-wide Ca2+ waves because the effective amplification of Ca2+ release by CICR is insufficient for full Ca2+ wave support. This lack of sufficient amplification could be explained by the stochastic nature of LCS, coupled with local SR refractoriness  Act 1986 and institutional approval by the University of Bristol ethics committee. We used an established coronary artery ligation model that leads to heart failure in adult New Zealand White rabbits (3–3.5 kg) which were daily monitored for health status. The target endpoint for HF was an ejection fraction of 40% . Some rabbits did not quite reach this endpoint but presented other indicators of heart failure including dilated left ventricle and lung congestion . This model, due to repolarizing current behavior, can be used to gain insight into repolarization reserve with human relevance  and euthanasia. Enzymatic dissociation was carried out using 1 mg/mL collagenase I (Worthington), 0.05 mg/mL protease (type XIV Sigma), and 0.1 mmol/L Caeviously . Guinea eviously , 43, 44,2PO4, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 glucose, 1.8 CaCl2, 1 MgCl2, pH 7.4 with NaOH) at 36 ± 1 °C. Patch pipettes were pulled from borosilicate glass using a P80 micropipette puller (Sutter Instruments). Pipettes were filled with an intracellular solution containing, in mmol/L: 120 aspartic acid, 20 KCl, 10 HEPES, 10 NaCl, 5 glucose, 5 Mg.ATP, 0.05 Fluo-4 pentapotassium salt, with KOH added to adjust to pH 7.2. Tip resistance was typically 1.6–2.0 MΩ when filled with this solution. Membrane potential and currents were recorded using an Axopatch 1D amplifier (Molecular Devices), Power1401 digitizer (Cambridge Electronic Design), and Signal data acquisition software . Cell membrane capacitance was measured by step depolarizations to −75 mV from a holding potential of −80 mV for 25 ms. Series resistance was compensated by ∼70%. Liquid junction potential (10 mV) was subtracted from recordings.Electrophysiology experiments were performed in a modified Tyrode’s solution  added directly to the cell-recording chamber  for 2–3 min. Excitation was at 488 nm, and emission was collected at >600 nm.Cacan mode  from then individual independent cell experiments from N animals are given in the text as n/N. Data were tested for normality using the Shapiro-Wilk test ; in any cases where data were skewed, the test was reapplied to log-transformed data. Paired or unpaired t tests were performed on normally and log-normally distributed data. Results are presented as mean ± SEM. The limit of statistical confidence was P < 0.05.Statistical analyses were performed at the level of the cell, and statistics on replicates of All data and computer codes are available upon request from the authors.Supplementary File


The electromechanical function of myocardial tissue depends on the intercellular communication between cardiomyocytes (CMs) as well as their crosstalk with other cell types. Cell injury, and subsequent death trigger inflammation as in myocardial infarction (MI) resulting in myocardial remodeling. Although mechanisms underlying myocardial cell death have been studied so far, the signaling events following single cell death and spontaneous response of connected cells in the myocardial tissue is still barely understood.2+) concentrations and transport in myocardial cell clusters in vitro. Spatial and temporal changes in intracellular Ca2+ concentrations [Ca2+]i were studied using a fluorescent calcium indicator, Fluo-4AM. Spontaneous signaling events following cell death were studied in rat embryonic cardiomyocytes and non-myocytes using separate cell culture systems.Here, we investigated the effect of laser-induced single cell death on Calcium (Ca2+ levels ([Ca2+]i) of surrounding cells. The spread of the observed propagating Ca2+ signal was slow and sustained in myocytes while it was rapid and transient in fibroblasts (Fbs). Further, sustained high Ca2+ levels temporarily impaired the contractility in CMs. The cell-type specific effect of ablation was confirmed using separate cultures of CMs and Fbs. Comparing Ca2+ propagation speed in myocytes and fibroblasts, we argue for a diffusion-driven Ca2+ propagation in myocytes, but not in fibroblasts. Radial and sequential Ca2+ diffusion across the CMs through cell–cell contacts and presence of Cx43-based intercellular junctions indicated a gap junction flow of Ca2+.Cell death triggered spontaneous increase in intracellular Ca2+-mediated functional interplay in myocardial cell clusters upon mechanical injury and, further, the difference in Ca2+ signaling in cardiomyocytes and fibroblasts.These findings illustrate the spontaneous CaVideo Abstract Myocardial tissue function is orchestrated by a network of cardiomyocytes (CMs), electrically and mechanically connected to each other to effect coordinated heart muscle contraction. The electrical connectivity in CMs is established through the conduction of depolarization current  from pacemaker cells  while thThough cardiomyocytes make up about two-third of the heart by volume, non-myocytes such as fibroblasts (Fbs) and vascular cells outnumber them  owing to2+ has been used in functional studies as it regulates vital cellular functions in mammalian cells i in cultured myocardial cells upon single cell injury. Based on the spatial and temporal [Ca2+]i changes, we describe the functional connectivity and Ca2+ signal propagation in both myocytes and fibroblasts.In our present study we demonstrate the effect of single-cell death induced by controlled laser-ablation of cultured myocardial cells (CMs and Fbs). This technique has been used successfully before both in vitro and in vivo to describe wounding pattern and stress response in cells and tissues –25. Ca2+an cells . Especiatraction , (2) Ca2poptosis , and (3)y in CMs . Moreove2 at 37 °C. Cultured cells contracted synchronously in clusters from 48–72 h and were used for subsequent experiments.Primary cardiac cell culture was obtained from embryonic rat (18–19 days post fertilization) hearts as described in Hersch et al.   from Avanti Polar Lipids, Inc.,  and 1,10-dioctadecyl-3,3,30,30-tetramethylindotricarbocyanine iodide  were prepared in the molar ratio 1/1/0.1/0.05. For fusion, 1–2 × 106 freshly isolated cells were incubated with 20 µl biotin-FLs in 1 ml Dulbecco’s modified eagle medium  for 2 min at room temperature. After biotinylation, cells were centrifuged and incubated at 4 °C for 20 min with 20 µl anti-biotin magnetic microbeads  diluted 1:5 in cell culture medium. The resulting suspension was further diluted with culture medium, centrifuged, and the pellet was resuspended in culture medium. The cell suspension was introduced into the magnetic separation column . Due to high fusion efficiency for non-myocyte cells and low efficiency for myocytes, the flow-through was enriched with myocytes while all other cells were retained in the magnetic column. Removing the magnetic field freed the attached cells and enabled cell collection. The cells were seeded separately to culture dishes and cultured as described above.Myocytes were separated from remaining myocardial cells using fusion based biotin-labelling of cells for subsequent cell-separation using magnetic anti-biotin microbeads as described in Hersch et al.. FusogenCultured cells were washed with pre-warmed culture medium and calcium indicator Fluo-4AM  was added to cells at a final concentration of 5 µM in culture medium. The cells were incubated for 20 min at 37 °C. Extracellular fluorophore was removed by washing with warm medium. For confocal microscopy  the fluorophore was excited at 488 nm and detected with a 490–550 nm bandpass filter. Continuous imaging at 160 ms per frame was used to record calcium waves using a 20 × Plan-Apochromat objective (NA 0.8) and a pixel size of 1.66 µm.2+ sparks were chosen. A pulsed UV-laser, λ = 355 nm  was used for targeted killing of single chosen cells in a cluster. Before ablation the laser position was calibrated in x, y and z directions. The effective laser power, beam width, and number of iterations were chosen. Single cells of a cluster were irradiated with laser intensities between 2 and 5% of total laser output. Cell death was observed from membrane disruption and cell retraction in phase contrast imaging and from spontaneous loss of Ca2+ fluorescence in these cells.Clusters of contracting cardiomyocytes exhibiting rhythmic Ca2; 5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA); 5 mM glucose; 10 mM 2-(4-morpholino)ethanesulfonic acid (MES); pH 6.1 i after ablation recovered to normal levels with time. This effect went along with renewed detection of spontaneous Ca2+ oscillations and contractility.To investigate the effect of single-cell death, the coculture systems described above were used. Cultured myocardial cells contracted rhythmically and synchronously in clusters from 48 to 72 h. Periodic Cangs Fig. b. Ca2+ f2+]i increase was higher in rings closer to the ablated cell and decreased with distance  for each ring, we found a time delay for maximum intensity between one ring and the next ring, suggesting that Ca2+ propagated along the successive rings starting from the point of ablation. The radial Ca2+ propagation speed across each ring was calculated with speeds in the order of µm/s, ranging from 7 µm/s (s.e.m. 2 µm/s) to 4 µm/s (s.e.m. 0.7 µm/s)  Fig. c for N =2+ oscillations, for most experiments no complete recovery was observed in many of the inner analysis rings  and 4 µm/s (s.e.m. 0.9 µm/s) for N = 6 (n = 11), as shown in Fig. In order to study the impact of single-cell death on a homogeneous cell population, we performed the same experimental procedure in myocytes-enriched cultures. Cell clusters with predominantly contracting myocytes were chosen for ablation. Laser-induced death of CM in these clusters resulted in increased [Caore Fig. a. [Ca2+]ime Fig. b. Ca2+ p2+]i increase in beating cardiomyocytes, we observed upon ablation a single, transient Ca2+ spike in non-myocytes that was propagating like a solitary wave front through the surrounding non-myocytes . Such values were at least three-fold higher and significantly different when compared to the propagation speed in myocytes  in cocultures and observed the effect particularly pronounced for surrounding non-myocytes. Cell clusters were chosen such that the central target cells were surrounded by numerous non-myocytes on all sides. In contrast to the briefly sustained Ca+i increa2+ spike propagating like a solitary wave front through the cells in all directions . As before, also this speed was significantly higher when compared to myocytes  than in non-myocytes   and non-myocytes  showed significantly faster recovery in non-myocytes over the complete distance of 100 µm from the ablated cell as seen in Fig. 2+]i fold-increase as well as recovery time were largely independent on distance to the ablated cell for non-myocyte cultures. In contrast, for myocytes a clear decreasing effect was present with distance. Thereby, the clear difference between the above detailed outcomes of ablation argues for a different mechanism underlying Ca2+ propagation and regulation in cardiomyocytes and fibroblasts.Since fibroblasts constitute a major proportion of the non-myocyte population, we next investigated if the effects of ablation in non-myocytes were also present in myocyte-depleted cultures. Although some samples of myocytes-depleted cultures still contained numerous myocytes, several clusters of non-myocytes could be always found. Thereby, cell clusters with predominantly non-myocytes were chosen in these samples. Upon single cell ablation we observed the same single, transient Caons Fig. : movie 3tes Fig. c. [Ca2+]tes N = 1, n = 26 2+ propagation in cardiomyocytes in more detail, clusters of three to four contracting cardiomyocytes were chosen from an enriched myocytes culture. Ablation of a single cardiomyocyte confirmed the increase in [Ca2+] in connected cells over time. Following the Ca2+ changes in cells with high temporal resolution, we observed the flow of Ca2+ from one cell to another in a sequential manner  in cocultures as well as enriched cultures. Immunocytochemical analysis confirmed the localization of Cx43 in cardiomyocytes at the cellular periphery where intercellular junctions are formed while being absent in CM-Fb and Fb-Fb cell–cell contacts  and sarcoplasmic reticulum Ca2+ ATPase (SERCA) pumps [2+]i of the same cells with time must depend on an adaptation mechanism that either activates Ca2+ efflux rates of underlying transport proteins in a time range of seconds or regulates Ca2+ flow through gap junctions. Especially for the latter, gating mechanisms have been identified that can cause uncoupling of GJs at high [Ca2+]i [2+]i additionally allowed recovery of contractility as indicated by regained synchronized Ca2+ spikes. Only for some cells [Ca2+]i remained high to result in retraction of membranes after ablation  as well as mechanical  manifestations in the heart. Moreover, increased [Ca2+]i triggers signaling cascades leading to calpain-mediated proteolysis, hypertrophy and mitochondria-mediated necrotic death of CMs i . Reducedh of CMs –43.2+ propagation speeds between myocytes were comparable in coculture and cardiomyocytes-enriched systems and similar to values reported in previous studies i the contractility reappeared. The difference in Ca2+ propagation in CMs and Fbs indicated a difference in Ca2+ handling in these cell types. Since dysregulation of [Ca2+]i have been found underlying several heart pathologies, understanding these mechanisms could provide valuable knowledge on cell death/survival signaling pathways in myocardial cells.Laser-induced death of single myocardial cells resulted in CaAdditional file 1: Movie 1. Ablation-induced Ca2+ propagation in coculture myocytes: Time series depicting Ca2+ propagating along the myocytes upon ablation of a single myocyte. The ablated myocyte is indicated by red circle. An increase in Ca2+ can be seen in connected myocytes after ablation (t = 0 s). Distant cells are little or not affected by ablation, as indicated by continuing Ca2+ waves even after ablation. Ca2+ intensity values of closely connected myocytes return to values identical to intensity values before ablation.Additional file 2: Movie 2. Ablation-induced Ca2+ propagation in non-myocytes in cocultures: Time-series depicting Ca2+ propagating along the non-myocytes upon ablation of single-central myocyte. The ablated myocyte is indicated by red circle. A spike in Ca2+ can be seen after ablation (t = 0 s), after which the Ca2+ intensity drops to values values identical to intensity before ablation.Additional file 3: Movie 3. Ablation-induced Ca2+ propagation in non-myocytes in myocytes-depleted cultures: Time-series depicting Ca2+ propagating along non-myocytes upon ablation of a single non-myocyte in the cluster. The ablated cell is indicated by a red circle. A spike in Ca2+ can be seen after ablation (t = 0 s), after which the Ca2+ intensity drops to values that are identical to the intensity before ablation.Additional file 4: Movie 4. Time-series showing sequential diffusion of Ca2+ across connected cardiomyocytes: Ablation-induced diffusion of Ca2+ across Fluo-4 labelled cardiomyocytes through cell-cell contacts. Ablated region is indicated by red circle and the time point of ablation is set to t = 0 s. Ca2+ sequentially diffuses across the three connected cells in about 2 s from the time point of ablation. The closely connected myocyte exhibits rapid intracellular Ca2+ oscillations with time.Additional file 5: Movie 5. Ablation-induced changes in surrounding cardiomyocytes in cluster: [Ca2+]i-increase in connected cells after ablation is accompained by membrane retraction in connected cells of the cluster. The ablated region is indicated by red circle and the retraction of cells in the cluster is indicated by arrows. The time point of ablation is set as t = 0 s.


Therefore, we develop an analytical pipeline for automatic assessment of Ca2+ transient abnormality, by employing advanced machine learning methods together with an Analytical Algorithm. First, we adapt an existing Analytical Algorithm to identify Ca2+ transient peaks and determine peak abnormality based on quantified peak characteristics. Second, we train a peak-level Support Vector Machine (SVM) classifier by using human-expert assessment of peak abnormality as outcome and profiled peak variables as predictive features. Third, we train another cell-level SVM classifier by using human-expert assessment of cell abnormality as outcome and quantified cell-level variables as predictive features. This cell-level SVM classifier can be used to assess additional Ca2+ transient signals. By applying this pipeline to our Ca2+ transient data, we trained a cell-level SVM classifier using 200 cells as training data, then tested its accuracy in an independent dataset of 54 cells. As a result, we obtained 88% training accuracy and 87% test accuracy. Further, we provide a free R package to implement our pipeline for high-throughput CM Ca2+ analysis.Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an excellent platform for potential clinical and research applications. Identifying abnormal Ca Although current hiPSC-CMs display fetal-like phenotypes in terms of their structural and electrophysiological properties2, they have increasingly been used to study normal cardiac functionality5 and human cardiovascular diseases such as long QT syndrome, catecholaminergic polymorphic ventricular tachycardia and viral myocarditis as well as for high-throughput cardiotoxicity screening9. Furthermore, hiPSC-CMs are under active investigation for use as a cell source for possible clinical usage11. For these applications, extensive functional characterization of hiPSC-CMs is required.Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) are highly desired for drug discovery and modeling human development and disease, as alternative models such as human primary CMs are hard to obtain2+ transients are a fundamental characteristic of cardiomyocyte functionality, although cardiac action potentials and contractility are also commonly used to study cardiomyocyte functionality by methods such as patch clamp, multielectrode array, microscopic video analysis, and fluorescence imaging12. Coordinated movement of Ca2+ at single cell level plays a key role to control contraction of the heart by the conversion of electric excitation into mechanical contraction. Specifically, each action potential induces Ca2+ influx, which triggers a much greater Ca2+ release from the sarcoplasmic reticulum (SR). The increased cytosolic Ca2+ binds to and activates the Ca2+-sensing protein of the contractile apparatus and initiates CM contraction. Then Ca2+ is removed from the cytosol through reuptake into the SR or extrusion into the extracellular space, which leads to CM relaxation. Thus, the rapid release and reuptake of Ca2+ between the SR and the cytosol create a Ca2+ transient inside the CM13. Abnormal Ca2+ signals are indicative of various cardiac pathologies, such as arrhythmia8.Ca2+ transient analysis is an important component of hiPSC-CM phenotype analysis. Ca2+ transient characteristics are commonly captured with Ca2+-specific fluorescent dye, and fluorescence imaging is the most optimal for high-throughput application. Human experts’ assessment of Ca2+ transient signals is often based on Ca2+ transient morphology characterized by rapid upstroke and decay kinetics. Although manual identification of abnormal Ca2+ transients by human experts is often taken as gold standards, such visual assessment is labor-intensive, time-consuming, and subjective to the assessor’s expertise. Moreover, manual identification of abnormal Ca2+ transients by human experts becomes a bottleneck hindering its application to high-throughput analysis. Thus, a user-friendly computational tool is in pressing need to mitigate the bottleneck of manual analysis and to enable automatic assessment of Ca2+ transient abnormality.An accurate Ca. proposed an Analytical Algorithm to detect cycling Ca2+ transient peaks, quantify peak variables, and assess the abnormality of transient peaks and signals14. This analytical algorithm identifies signal abnormality based on whether the assessed cell signal contains at least one abnormal transient peak based solely on characteristics of a single peak. The assessment did not leverage shared characteristics of normal and abnormal Ca2+ transient peaks and signals across all samples, which are expected to provide valuable input to improve the accuracy of signal abnormality assessment. Further, the analytical algorithm fails to account for the valuable manual assessment results about existing data.Previously, Juhola et al15 is used for abnormality assessments of peaks and signals, which leverages shared data characteristics and experts’ manual analysis results of training data.To overcome these limitations, we develop an improved automatic pipeline that is composed of peak detection, peak variable quantification, peak abnormality assessment, signal variable extraction, and signal abnormality assessment. We adapt the existing Analytical Algorithm for peak detection, peak variable quantification, and peak abnormality assessment. Additionally, the advanced machine learning method of Support Vector Machine (SVM)2+ transient data as well as functions for peak detection, peak variable quantification, training peak-level SVM classifier, cell variable quantification, training signal-level SVM classifier, and predicting signal abnormality. Our R library is freely available through GitHub and is expected to serve as a convenient tool for people in need of a Ca2+ transient analysis software with high speed and accuracy.Further, we provide an R library to implement this pipeline, which includes SVM classifiers trained using our Ca14 to better characterize signal abnormality by including additional peak variables such as nearby peak distance, varying peak amplitude, and peak asymmetry, as well as considering irregular peak phases for peak abnormality assessment.A flowchart is provided in Fig. 2+ signals as the training data, our pipeline first trains a peak-level SVM classifier by taking peak assessments by human experts as responses  and 14 peak variables , no oscillations of the diastolic Ca2+ signal, and no obvious spontaneous Ca2+ release between transients  is considered as the beginning  timeframe point of the current peak. Starting from the peak left, first derivative values should be positive before peak maximum point while negative after peak maximum. Thus, the first timeframe point after the peak left point with a negative derivative value is taken as the maximum timeframe point of the current peak . The first timeframe point after peak maximum with a positive derivative value whose absolute value is greater than a pre-defined threshold rtup  is taken as the end of the current peak . The default value for tup is set as used by Juhola et al.14, and the default value for rtup is determined based on our experiments. In particular, taking rtup = 0 is equivalent as taking the first timeframe point after peak maximum with a positive first derivative value as peak end.To detect the peaks of CaTo avoid the identification of a partial or noisy first peak within a signal, we exclude the first peak that is asymmetric with left amplitude less than 50% of the right amplitude and intensity value < 5. We also exclude noisy peaks with peak amplitudes less than 15% of the maximum amplitude within the signal. To ensure that our detected peaks are valid with minimal noise or partial peaks, signals with no peak or a single peak are excluded from our analyses.Fourteen peak variables are quantified after peak detection Fig.  and thenThe 14 peak variables are as follows: peak left amplitude (A_l), peak right amplitude (A_r), amplitude difference between A_l and A_r (A_d), duration from peak left to peak max (D_l), duration from peak max to peak right (D_r), maximum first derivative value from peak left to peak max (Dy_max), absolute minimum first derivative value from peak max to peak right (Dy_min) maximum second derivative value from peak left to peak max (D2y_max), absolute minimum second derivative value from peak max to peak right (D2y_min), peak area under the intensity curve from peak left to peak right (R), duration from the previous peak max to current peak max , duration from peak left to Dy_max (delta_l2Dymax), duration from peak max to Dy_min (delta_m2Dymin), and median of delta values within a signal (Peak_distance_median). These quantified peak variables are used for training peak-level SVM classifier and subsequent cell-level SVM classifier.14, our improved Analytical Algorithm also considers irregular phase to assess peak normality based on peak distances (delta) within one signal.Here, peak max amplitude and min amplitude respectively refer to the maximum and minimum of A_l and A_r. In addition to peak amplitudes and asymmetry as considered by previous methodWe first assess peak normality with respect to peak amplitudes. That is, the first peak will be labeled as abnormal if the peak max amplitude is less than 50% of the average peak max amplitude within the same signal. Peaks other than the first one will be labeled as abnormal if the preceding peak is abnormal and the peak max amplitude is less than 50% of the average peak max amplitude within the same signal, or if the peak amplitude is less than 50% of the preceding normal peak. Second, a peak with normal amplitude characteristics will be labeled as abnormal when the peak min amplitude is less than 85% of the peak max amplitude . Last, irregular phase assessment will be considered. A symmetric peak with normal amplitude but distance from previous peak to current peak  greater than 90% of the median delta within the same signal  will be labeled as abnormal. All thresholds are chosen based on our experimental training data and can be adjusted according to new data characteristics.17 to fit peak-level SVM classifiers and make predictions for all samples in the training dataset. In particular, peaks within a signal are taken as test data and a corresponding peak-level SVM classifier is trained using peaks from all other samples, which is iterated for all signals to obtain predictions of all peaks. The peak normality predictions by the LOOCV approach will then be used to train the follow-up cell-level SVM classifier.To employ expert peak assessments and peak characteristics of training data, we train a peak-level SVM classifier to predict peak normality status, taking expert peak assessments as outcome and these 14 peak variables as described in previous subsection as predictive features. To avoid the issue of overfitting for accuracy assessment with training data, we take the LOOCV approachThe cell normality labels based on peak normality assessments obtained by our improved Analytical Algorithm and SVM-LOOCV approach are considered as cell variables. We consider additional cell variables as follows: proportion of abnormal peaks per signal , variance of peak amplitude per signal (var_A), variance of peak distances per signal (var_delta), and variance of peak areas per signal (var_R). These cell variables are centered and standardized and then used as predictive features to train a cell-level SVM classifier to predict cell abnormality, where outcomes are taken as human-expert assessments about cell normality. This trained cell-level SVM classifier can then be used to predict cell normality for additional independent signals.2+ transient data, we applied the pipeline to study the Ca2+ transient data of 254 cells generated in our lab. In particular, we took 200 cell signals (containing 1893 peaks) as our training data and 54 cell signals (454 peaks) as our test data. We first manually assessed the normality of all of these signals and peaks that were considered as gold standards and taken as outcome variables for training SVM classifiers. Second, by applying our improved Analytical Methods to assess peak normality, we obtained 93.3% accuracy, 91.1% sensitivity, and 95.8% specificity 18.In addition, we constructed a receiver-operating curve (ROC)2+ transient signals, an otherwise labor-intensive and time-consuming phenotypic analysis for CMs. Specifically, we improve the existing Analytical Algorithm14 by accounting for irregular phases within signals and employ the advanced machine learning SVM method for peak and cell abnormality prediction. We also validate our approach of using advanced machine learning SVM method in this pipeline by using training and test hiPSC-CM Ca2+ transient signals generated by our lab. With independent test data, we demonstrate that our SVM approach obtained 87.0% accuracy .In this study, we develop an automatic pipeline for assessing the normality of hiPSC-CM CaOur results show the advantages of learning normal and abnormal characteristics across multiple peaks and cells as well as employing the valuable human-expert assessments of training data. Although our improved Analytical Algorithm yielded excellent peak assessment accuracy of 93.3% with our training data, its cell-level assessment accuracy was 87.5% with our training data and 83.3% with our test data. The decent peak abnormality assessment accuracy by Analytical Algorithm is probably because the Analytical Algorithm is developed to mimic human-expert assessment. In contrast, our cell-level SVM classifier obtained accuracy 89.9% with our training data by the LOOCV approach and accuracy 87.0% with our test data. The relatively lower accuracy for signal abnormality assessment is likely because the Analytical Algorithm fails to account for abnormality due to abnormal characteristics of multiple peaks and signals such as signals with irregular phases.2+ transients can overcome limitation of traditional manual assessment. Manual signal abnormality assessment is difficult since recordings are often short, contain small number of peaks, with varying morphologies of signals and peaks within them. Abnormal cell signals are often difficult to be identified consistently by multiple human-experts. This may be due to the fact that (1) there are peaks of small amplitude that are borderline noise, (2) the nature of the cell signal morphology renders it difficult to exactly characterize, for instance due to continuously decreasing fluorescence intensity, among other reasons.Automatic identification of Ca2+ transient experts, we expect our model to be better modified for more nuanced prediction of novel Ca2+ transient data. Various other types of Ca2+ transient signals can be assessed by a human expert to further modify the prediction model based on any potential need of any user.Our signal classification results are already applicable for current use. Its potential is even larger as more data can be fed into the training set with ease. By incorporating more data collected and analyzed by different Ca2+ transient signals. This could render the machine learning model biased toward certain signal patterns. As we accumulate more data, we expect to see further improvements in the overall accuracy and efficiency of our proposed machine learning method. In addition, training the model with various other hiPSC-CMs derived from different cell lines including disease cell lines  at multiple differentiation stages will significantly improve the generalizability of our machine learning method in such a way that will allow us to capture the underlying essence of seemingly different patterns of Ca2+ transient signals among CMs of different sources. This will, in turn, enhance its ability to be used as a scalable tool for analyzing high-throughput CM data for various purposes, such as drug screening. As our machine learning model incorporates more diverse sets of data, we anticipate its usage to evolve as well: from an aide for a busy human-expert to eventually fully replicating the decision-making of a human-expert on all patterns of Ca2+ transient signal, regardless of its origin and experimental procedures employed.Currently, our machine learning SVM classifiers have been trained using hiPSC-CMs derived from two different strains of stem cells—SCVI-273 and IMR-90—which produce very similar Ca2+ transient analysis software that implements our automatic pipeline with machine learning SVM method is available in the form of R package for everyone in need of Ca2+ transient analysis tool.Our Ca9 and IMR90 hiPSCs (WiCell Research Institute)20 were fed daily on Matrigel-coated plates with mTeSR1 defined medium  and passaged using Versene  when compact colonies reached 90–100% confluence. For CM differentiation, hiPSCs were induced using a growth factor-guided differentiation protocol22. At the day of induction (day 0), medium was replaced with RPMI 1640 medium supplemented with 2% B27 minus insulin  and 100 ng/ml activin A . After 24 h (day 1), RPMI supplemented with 2% B27 minus insulin was used for 24 h. After 24 h (day 1), activin A was replaced with 10 ng/ml BMP4 , and cells were cultured without any medium change for the next 3 days. From day 4, the growth factor-containing medium was replaced with RPMI supplemented with 2% regular B27  and the medium was changed every other day. hiPSC-CMs were further enriched by the metabolic selection method using RPMI without glucose  supplemented with 2% B27 and 5 mM lactate from day 11 to 1423. Alternatively, enriched hiPSC-CMs were generated by microscale generation of cardiospheres at day 624. Cells were observed under a microscope daily for beating cells, which typically appeared by day 8–10. At day 14, a parallel culture of cells were harvested to determine CM purity before subsequent assessments.Undifferentiated SCVI-273 hiPSCs (Stanford Cardiovascular Institute)2+ transient was performed using Fluo-4 AM . At differentiation day 18, cells were seeded in a 96-well plate at a low density to acquire single-cell Ca2+ transients. At differentiation days 20 to 22, cells were treated with or without arrhythmogenic drugs including TNF-α, ethanol, and melphalan for 3 to 5 days. At differentiation days 23 to 25, cells were acquired for Ca2+ transient signals. Beating hiPSC-CMs were incubated with 10 µM Fluo-4 AM for 25 min at 37℃ followed by a 5 min wash with warm 1 × Normal Tyrode solution . Fluorescence images were acquired in 1 × Normal Tyrode’s solution immediately after the wash using ImageXpress Micro XLS System (Molecular Devices) with excitation at 488 nm and emission at 515–600 nm at a frequency of 5 frames/sec and 20× magnification for 12 or 32 s. Fluorescence intensity plots from spontaneously beating cells were obtained using MetaXpress software (Molecular Devices) by region of interest measurements.Live cell imaging of intracellular Ca2+ transients from SCVI-273-derived CMs with and without TNF-a treatment and IMR90-derived CMs with and without ethanol treatment were used as training datasets for machine learning algorithm. Ca2+ transients from SCVI-273-derived CMs with or without melphalan treatment were used as test datasets for machine learning algorithm.Ca2+ transients  with patterns similar to those observed in patient-derived hiPSC-CMs . We also note that the patterns of Ca2+ transients at days 23–25 observed in this study were similar to those from cells of 30 ± 2 days old8.We note that treatment conditions we tested  caused abnormal Ca2+ transient analysis described in this study, called SVMCaT, is available on the Github website (https://github.com/hyunmhwang/SVMCaT).An R-package for the CaSupplementary Information.


The molecular regulation mechanism of GRB7 in bladder cancer was investigated by treatment with AKT inhibitor. GRB7 mRNA was upregulated in bladder cancer samples compared with that in normal tissue samples. Overexpressing GRB7 significantly promoted the proliferation and tumorigenesis of bladder cancer. However, silencing GRB7 played the retarding part. GRB7 promoted G1/S transition by activating the AKT pathway. Our results indicate that GRB7 plays an important role in promoting proliferation and tumorigenesis of bladder cancer.Growth factor receptor-bound protein 7 (GRB7) has been found closely related to the occurrence and development of various tumors, but its function in bladder cancer has not yet been elucidated. The study is aiming at investigating the expression and function of GRB7 in bladder cancer. The Cancer Genome Atlas (TCGA) database was selected to analyze mRNA levels of GRB7 in bladder cancer. RT-qPCR and Western blot were conducted to detect the expression of GRB7 in normal bladder epithelial cells, seven bladder cancer cell lines and eight pairs of malignant/nonmalignant bladder tissues. The role of GRB7 in tumor proliferation and tumorigenesis was explored by establishing stable cells,  Bladder cancer is a malignant tumor that occurs in the bladder mucosa and is the most common malignant tumor of the urinary system Growth factor receptor-binding protein 7 (GRB7) is a member of the GRB7 signal transduction protein family including GRB7, GRB10 and GRB14 In the present study, we find that GRB7 mRNA and protein expression are upregulated in bladder cancer. Overexpressing GRB7 promotes, while silencing GRB7 inhibits the G1/S transition in the cell cycle as well as tumorigenesis through phospho-AKT pathway. These results suggest that GRB7 may be a diagnostic marker and a valuable therapeutic target for bladder cancer.http://www.broadinstitute.org/gsea) was adopted to analyze them. Besides, chi-square test was used to judge whether there were differences in data between groups. In this study, a double tailed p value less than 0.05 was considered statistically significant.The RNA detection values of 408 BC tissues and 19 adjacent normal bladder tissues were downloaded from the Cancer Genome Atlas (TCGA). Excel and MeV 4.9 software were used to sort out these data and Gene Set Enrichment Analysis  were cultured in Dulbecco's Modified Eagle's Medium , supplemented with 10% fetal bovine serum . Primary normal bladder epithelial cells  and eight paired BC tissues  were kindly collected and presented by Dr. Yang for this study. As previously described GAPDH . Considering about the variability in expression level, all the expression data were normalized to the geometric mean of the housekeeping gene GAPDH. The main antibodies adopted in the present study were as followed: anti-GRB7 , anti-p-AKTSer473 , anti-p-AKTThr308 , anti-AKT , anti-cyclin D1 , anti-CDK6 , anti-CDK4 , anti-p21Cip1 , anti-p27Kip1 , anti-pRB , anti-RB, anti-p-GSK3β , anti-p-GSK3β . Anti-β-actin monoclonal antibody  was taken as a loading control.Real-time Quantitative PCR (RT-qPCR) and Western blot analysis were performed following standard methods as mentioned pSUPER.retro.puro plasmid . As described before, retroviral production and infection were performed The full-length human GRB7 was subcloned into the pMSCV-retro-puro vector . The forward PCR primer was 5'-AGATCTATGGAGCTGGATCTGTCTCCAC-3' and reverse PCR primer was 5'-GAATTCTCATCAGAGGGCCACCCGC-3'. Besides, under the help of RNA interference (RNAi) method, the GRB7 knock down bladder cancer cell lines was also established. To endogenously downregulate GRB7, two hairpin GRB7 siRNA oligonucleotides  were inserted into the subcloned 3 cells/well. The cell proliferative activity was tested by 3--2,5-diphenyl-2-H-tetrazolium bromide  assay. After cultured for 24 h, 48 h, 72 h, 96 h, 120 h or 144 h, each well was added with 10 µl of MTT solution (5 mg/ml). After incubation with the MTT reagent at 37 °C for 4 h, cells were added with 150 µl dimethyl sulfoxide (DMSO) and then the absorbance was measured at 490 nm on a microplate reader . All tests have been repeated at least three times.In cell counting part, the established cells were seeded in 96-well plates with a density of 2×10As for colony formation assays, 1000 cells per well were plated in six-well plates in triplicates and cultured for ten days before staining viable colonies by nitro blue tetrazolium (Sigma). All experiments were conducted in triplicate for each cell line.2 at 37 °C, replacing the medium every 2 days. 12 days later, the cells were counted by the help of an inverted microscope, and during this observation, any opaque spot bigger than 0.1 mm was regarded as one colony. Images were photographed and stored for later analysis.500 cells were lightly blown and suspended evenly in 1 mL complete DMEM supplementary with 0.7% agarose. Then the suspension was all lightly plated into 6-well plates container which have been pre-covered with 1.5% agarose. Each group had 3 parallel samples. The cells were then incubated with 5% CO5 cells/well were seeded in 100-mm dishes and normal cultured for 48 hours. Then, cells were harvested and fixed with ice-cold 70% ethanol before being kept overnight at 4 °C. Next, they were washed with PBS, collected, and centrifuged at 1500 rpm for five minutes, and after that the cells were incubated by the use of bovine pancreatic RNase  at 37 °C for 30 min and stained with propidium iodide . Cells were further subjected to cell cycle analysis by FACSCanto II flow cytometer , and the data were analyzed with the help of the FLOWJO software . All experiments were conducted repeatedly three times.5×10Cells in the logarithmic growth stage were seeded at the initial density of 2000 cells per well in 24-well plates with coverslips  placed inside the wells, and then the cells were allowed to attach for 72 h. BrdU (working concentration was 10 µM) was then added into each well and allowed to be incubated for 1h. Then the cells were washed twice in PBS, fixed in 100% methanol (chilled at -20 °C) for five min, permeabilized with 0.2% TritonX-100 for ten min followed with the addition of immunostained with an anti-BrdU antibody . A laser scanning microscope  was taken for counting and taking typical pictures.in vivo experiments were performed as described before 3), where A was the largest diameter (cm) and B is the smallest diameter (cm). On the day of 24, the animals were euthanized, and the tumors were excised and weighed.The P < 0.001). To exclude individual differences between patients, we further classified the 427 samples and obtained 18 pairs of bladder cancer tissues and adjacent normal tissues from the same patient. Among the 18 pairs of specimens, GRB7 mRNA was significantly higher in bladder tumors than in adjacent normal tissues .The mRNA expression of GRB7 in 427 bladder cancer tissues in the TCGA database was analyzed. A point on the graph of Figure For the experimental verification, seven bladder cell lines and five pairs of clinical bladder specimens were taken further detection. Compared with normal bladder epithelial cells, the mRNA levels of GRB7 in 7 cultured bladder cell lines were significantly increased Figure C. The GRP < 0.05). For experimental verification, 5637 and RT4 cells were stably transfected with the GRB7 plasmid  and GRB7 low expression group (GRB7-L). Shown as Figure in vitro experiments. By observing the growth rate of the transplanted tumor and weighing the final transplanted tumor, it was found that the tumorigenic ability of 5637 cells was significantly enhanced when GRB7 was highly expressed, but was greatly attenuated when GRB7 was silenced  via its PH domain In general, this study shows that GRB7 is upregulated in human bladder cancer and plays an important role in promoting proliferation and tumorigenesis via phosphorylated-AKT pathway, which suggests a potential role of GRB7 as a diagnostic marker and valuable therapeutic target in bladder cancer.