NPM1 mutated AML (NPM1mut AML). However, the clinical utility of next generation sequencing (NGS) to detect MRD in AML remains unproven. We analysed the clinical significance of monitoring MRD using ultradeep NGS (NGS-MRD) and flow cytometry (FCM-MRD) in 137 samples obtained from 83 patients of NPM1mut AML at the end of induction (PI) and consolidation (PC). We could monitor 12 different types of NPM1 mutations at a sensitivity of 0.001% using NGS-MRD. We demonstrated a significant correlation between NGS-MRD and real time quantitative PCR (RQ-PCR). Based upon a one log reduction between PI and PC time points we could classify patients as NGS-MRD positive (<1log reduction) or negative (>1log reduction). NGS-MRD, FCM-MRD as well as DNMT3A mutations were predictive of inferior overall survival (OS) and relapse free survival (RFS). On a multivariate analysis NGS-MRD emerged as an independent, most important prognostic factor predictive of inferior OS  and RFS . We establish that DNA based NPM1 NGS MRD is a highly useful test for prediction of relapse and survival in NPM1mut AML.Detection of measurable residual disease (MRD) by mutation specific techniques has prognostic relevance in  RUNX1-RUNX1T1). However, for the majority of AML, immunophenotyping (FCM-MRD) remains as the most accepted method to detect MRD [Recent developments in sequencing cancer genomes have advanced our understanding of the complex molecular landscape of AML. Even after addressing the extensive network of cytogenetic abnormalities, molecular heterogeneity, epigenetic and gene expression profiles in AML, the prognostic stratification in AML mainly relies upon only cytogenetics and a handful of gene mutations. Subsequent to risk stratification and standard treatment, the 5 year survival is less than 60% in adult AML . Therefotect MRD –4.NPM1 is a specific subtype of AML with a recurrent genetic abnormality and favourable outcome [NPM1mut AML is one of the most common subsets of AML in adults and comprises around 35% overall adult AML cases and around 45% of normal karyotype AML [NPM1 gene [NPM1 as reliable marker for MRD [NPM1mut AML, the use of molecular techniques has been more prevalent. Recent studies have shown that serial monitoring of NPM1 mutations from the blood or bone marrow during chemotherapy is highly predictive of relapse. Majority of these studies have been RNA based and have used mutation specific approaches for the detection of MRD [NPM1 mutation must be characterized at diagnosis by sequencing and each type of NPM1 mutation must be validated as an independent MRD assay. This is further complicated by varying performance characteristics of mutation specific primers and fluorescent probe combinations.Acute myeloid leukemia (AML) with mutated  outcome . NPM1muttype AML , 7. MorePM1 gene . These m for MRD . For MRDn of MRD –12. A diNPM1mut AML that can detect NPM1 mutations at a frequency of 0.001%. We have then compared NGS-MRD with FCM-MRD, the gold standard for detection of MRD in AML as well as RQ-PCR for the commonly occurring type A NPM1 mutations. We demonstrate that NGS-MRD for NPM1mut AML is an independent prognostic factor significantly predictive of outcome in NPM1mut AML.DNA based next generation sequencing (NGS) is a scalable solution that has the potential for detection of MRD in AML –15. As aThe mean overall survival (OS) was 40.02 months (95%CI 33.73 to 46.32) for the entire cohort (median not reached). The mean relapse free survival (RFS) was 33.31 months (95%CI 26.85 to 39.77), median RFS was 19.43 months (95%CI 14.27 to 43.03) months . The medNPM1 mutation over post-induction (PI) and first post consolidation (PC) time points and ascertain whether there was any switch in the mutation type in the course of treatment [NPM1 mutations and their frequencies. Type A, B and D were the most common subtypes  and PC (27) time points. Values obtained from RQ-PCR were not significantly different from corresponding NGS variant allelic frequency (VAF)  were positive for DNMT3A mutation with VAF ranging from 3.5% to 50%. The arginine 882 hotspot was the most commonly affected amino acid in our cohort (43.75%). Presence of DNMT3A mutations in NPM1mut AML were predictive of inferior OS (p = 0.027) and possibly inferior RFS  at end of consolidation. The corresponding NPM1 VAF can be also seen in The details of At the end of induction, FCM-MRD was detected in 26 out of 82 31.7%) patients at levels ranging from 0.02 to 6.32% (median: 0.7%). Similarly, at end of consolidation FCM-MRD was detected in 8 out of 40 (20%) patients at levels ranging from 0.01 to 1.8% (median: 0.05%). The presence of FCM-MRD at the end of induction was highly predictive of inferior OS as well as and RFS .A total 82 cases at the PI timepoint and 40 at PC timepoint were analyzed for MRD using FCM and NGS. A comparison of these results can be seen in On a multivariate Cox proportional hazards regression analysis, the presence of NGS-MRD was an independent factor predictive of inferior OS and RFS. In fact, NGS-MRD and PI FCM-MRD were the only significant prognostic factors for relapse and death Table . NGS-MRDNPM1 gene mutations are the largest subset of molecular alterations seen in adult AML. It is therefore imperative that we develop effective strategies for monitoring MRD. Most of the molecular techniques that have been published for MRD detection in NPM1mut MRD are based on quantitative measurement of NPM1-mutant transcripts [NPM1 alleles [NPM1mut AML [NPM1 mutations has been described by a few cohorts [NPM1mut AML. In fact, Ivey and colleagues indicated that real time PCR based NPM1 MRD status was the only important predictor of relapse or death [NPM1 mutations are a reliable marker for monitoring disease progression. There are very few studies that have evaluated the utility of molecular NPM1-MRD in comparison with immunophenotypic MRD. Here, we demonstrate that at the early time-points of therapy (PI) FCM-MRD is important in prediction of relapse. As seen in nscripts , 17–20 o alleles . Recentl1mut AML , 22. The cohorts , 20, 21.or death . They coNPM1 mutation subtypes . Our dataset shows the importance of a generalized approach to the detection of MRD in NPM1mut AML. The fact that we could identify and monitor rare NPM1 mutations using this technique validates our approach. There have been reports of switch of NPM1 mutations at relapse [NPM1 mutation type either at post induction or post consolidation time points, an NGS based approach provides a definitive advantage over mutation specific testing in potentially detecting a NPM1 clonal switch. There have been contradictory observations in the literature regarding the prognosis of uncommon NPM1 mutation as against the common subtypes [DNA based NGS assays overcome many of the obstacles of real time PCR as they do not need patient specific sequence information prior to monitoring. In our dataset, even though majority of the mutations were of type A, B and D, we detected nine uncommon s Figure . These p relapse . Althougsubtypes , 24. In NPM1mut AML patients. As seen in Recent data has shown that NGS based MRD assays are potentially more sensitive than flow cytometry and offer comparable sensitivity as RQ-PCR , 25–28. NPM1mut AML [In our cohort, significant number of patients who were FCM-MRD-negative had detectable MRD levels using deep sequencing. This resulted in a poor correlation between FCM-MRD and NGS-MRD VAF . Unlike 1mut AML , 35. In 1mut AML . These rNPM1 mutations often co-occur with DNMT3A mutations and portend a poor prognosis [DNMT3A mutations in 7 post treatment samples even when there was significant reduction in the NPM1 mutant levels. These are likely to be associated with age related clonal haematopoiesis [FLT3-ITD and high allelic ratio of FLT3-ITD is a poor prognostic factor in NPM1mut AML [We saw agreement with previous studies that rognosis –39. Furtopoiesis , 41. It 1mut AML . HoweverNPM1 mutant VAF in 19 samples . However, we could not confirm the clinical relevance of using such a cut-off in our dataset at either PI or PC time points. The results of Shayegi and colleagues as well as our results need to be confirmed by larger cohorts for incorporation into treatment algorithms.To summarize, this is one of the few papers that has evaluated the clinical relevance of NGS MRD in template . They coNPM1mut AML were accrued into the study after informed consent from October 2012 to May 2017 (after exclusion of patients with induction deaths and refractory disease). Patients were diagnosed as per current WHO 2016 criteria. NPM1 mutations were detected initially using fragment length analysis [2) and followed up till January 2018. FCM-MRD was assessed from the bone marrow at the end of induction (PI)  and at the end of first consolidation . NGS-MRD was assessed from 82 PI samples  and 55 PC samples, of which 13 samples were sourced from the peripheral blood (PB). In total, there were 54 paired samples between PI and PC time points. ). To prevent contamination from other sample indices, we used a dual indexing strategy with 10 bp sample specific index barcode in both forward and reverse primers. The primer sequences are as listed in We adopted a strategy as described by Salipante et al. with minor modifications as detailed below . BrieflyAssay setup was as follows: For a total reaction volume of 75 μl, 37.5 μl of NEBNext High-Fidelity 2X PCR Master Mix , and 10 uM each forward and reverse primers were used to amplify 600ng of genomic DNA . PCR cycling conditions were as follows: Initial denaturation of 95°C for 15 minutes; then 35 cycles of denaturation at 94°C for 1 minute, annealing at 65°C for 1 minute, and extension at 72°C for 1 minute; followed by extension cycle of 72°C for 45 seconds. PCR products were size selected using Agencourt AMPure XP beads  and quantified using Qubit dsDNA HS assay . Samples were deep sequenced after pooling in equimolar concentration on an Illumina MiSeq  next generation sequencer using a 150 bp paired end V2 chemistry.Runs were demultiplexed using the MiSeq onboard software. Paired-end reads were self-assembled using PANDAseq . Self-asNPM1 mutations at a high sensitivity of 0.001%. A limit of detection experiment demonstrated that the assay could detect the NPM1 mutation at 1:100,000 frequency. To ensure that the assay did not make false NPM1 insertion calls we performed a limit of blank experiment on 30 normal DNA samples. The details pertaining to these experiments can be seen in We increased the analytical sensitivity of the original NGS-MRD assay  to detect To account for systemic drift, we assayed two (high and low level) precision controls  in every run. The results of these experiments can be seen in NPM1 variant allele frequencies obtained after ultradeep sequencing at PI and PC time points were used to calculate log reduction values.  to construct a library that spanned the entire eviously . Sequenceviously , read seeviously , alignmeeviously , pre-proeviously  & GATK veviously . Varianteviously , Mutect eviously , and Plaeviously . Using aeviously  the variNPM1 mutation were subjected to PCR to amplify exon 12 of NPM1. PCR products were purified and cloned into a pJET1.2/blunt cloning vector  following the manufacturer’s instructions. Plasmids carrying mutant and wild type NPM1 were confirmed by sanger sequencing, linearized and used to generate standard curves in further RQ-PCR experiments. MRD detection for type-A NPM1 mutations using DNA as a template was done using a previously published protocol on a LightCycler 96  [DNA extracted from OCI-AML3 cell lines carrying Type-A zerland) .Patients accrued from March 2012 to February 2015 (25 cases) had been acquired on an 8 colour BD FACSCanto II or two 10 colour BC Navios instruments using a three tube 8 colour MRD assay as seen in Overall survival (OS) was defined as time from start of induction therapy to time of last follow up or death. Relapse free survival (RFS) was calculated from date of achievement of remission to the date of relapse, if not, death from any cause. Patient who did not relapse and were alive were censored on the last follow up date . ResultsNPM1 mutation. This assay has a prognostic impact and can be used in the prospective monitoring of NPM1mut AMLs. It has advantages as compared to FCM-MRD in terms of a lower cost of testing, stability of DNA for shipped samples, higher sensitivity and easier interpretation. In addition, it is comparable to established RQ-PCR assay based MRD results with definite advantages. We feel that in view of the high sensitivity of the NGS assay, monitoring of NPM1 MRD from the peripheral blood is feasible.We have evaluated the clinical utility of an ultrasensitive NGS assay for sequential monitoring of


The G Protein-Coupled Estrogen Receptor (GPER) is a novel membrane-bound receptor that mediates non-genomic actions of the primary female sex hormone 17β-estradiol. Studies over the past two decades have elucidated the beneficial actions of this receptor in a number of cardiometabolic diseases. This review will focus specifically on the cardiac actions of GPER, since this receptor is expressed in cardiomyocytes as well as other cells within the heart and most likely contributes to estrogen-induced cardioprotection. Studies outlining the impact of GPER on diastolic function, mitochondrial function, left ventricular stiffness, calcium dynamics, cardiac inflammation, and aortic distensibility are discussed. In addition, recent data using genetic mouse models with global or cardiomyocyte-specific GPER gene deletion are highlighted. Since estrogen loss due to menopause in combination with chronological aging contributes to unique aspects of cardiac dysfunction in women, this receptor may provide novel therapeutic effects. While clinical studies are still required to fully understand the potential for pharmacological targeting of this receptor in postmenopausal women, this review will summarize the evidence gathered thus far on its likely beneficial effects. Among measures of cardiac function, diastolic performance is one of the most comprehensive—integrating myocardial relaxation, mitochondrial bioenergetics, cardiomyocyte/myocardial structure, and left ventricular (LV) ejection with respect to proximal aortic distensibility—and is a potential barometer of cardiac health . LV diasDespite a marked female sex-specific predilection in HFpEF, relatively little is known regarding the mechanisms by which sex hormones, particularly the estrogens, and estrogen receptors (ERs) impact diastolic function. Over the last decade, we and others have explored the roles of the newest estrogen receptor, G protein-coupled estrogen receptor , in the maintenance of cardiac function and structure after estrogen loss. In this review, the effects of pharmacologic activation of GPER by its specific agonist G1 on mitigating the adverse consequences of estrogen loss on relaxation, mitochondrial function, LV stiffness, and aortic distensibility will be presented. The influence of global and cardiomyocyte-specific GPER gene deletion on function and structure at the cardiomyocyte, whole heart, and conduit vessel levels will also be discussed.Diastolic dysfunction denotes a condition whereby the LV cannot fill adequately despite normal filling pressure. Slowing, delayed, and incomplete myocardial relaxation results from alterations in intracellular calcium handling, impairments in energy metabolism, and increases in LV stiffness due to hypertrophic and/or interstitial remodeling. Elevations in LV filling pressure initially compensate, but eventually pulmonary congestion develops as a result of increased left atrial (LA) pressure . While aThe increased prevalence of HFpEF in older women compared with men of the same age appears related to the loss ovarian hormones, and primarily estrogens, that occur during menopause , 26. Epi2) at a similar nanomolar affinity as ERα and ERβ and exerts comparable actions on calcium mobilization and phosphoinositide 3-kinase activation  and β (ERβ) are classical nuclear hormone receptors, which bind estrogen and translocate to the nucleus to regulate target gene expression. However, molecular signaling is also induced by estrogen outside of the nucleus. While some intracellular signaling may be initiated by truncated forms of the steroid ERs , a membrtivation . ERα andtivation  as well tivation . In conttivation  but remativation , 49. Basal women  and in oal women , researcal women  and geneal women . These sal women , direct al women . Howeveral women .d = 11 nM) and highly selective GPER agonist was developed from a library of 10,000 molecules and does not activate the classical estrogen receptors at concentrations up to 10 μM . The rapid signaling events following GPER activation also lead to inhibition of the expression of cell cycle genes, such as cyclin B1 and CDK1, which are involved in cardiac fibroblast and mast cell proliferation and contribute to interstitial remodeling . Moreover, GPER activation by G1 reduces remodeling promoted by hypertrophic regulators, including angiotensin II and endothelin-1, via inhibition of 1/2 ERK signaling and upregulation of PI3K/Akt/mTOR pathways (see section GPER and Anti-hypertrophic Remodeling below).GPER is expressed on the plasma and intracellular membranes of cardiac cells, including cardiomyocytes, cardiac fibroblasts, mast cells, and endothelial cells –59. To cto 10 μM . G15 andto 10 μM . While tto 10 μM . The selSince only a limited number of studies have explored the actions of GPER in diastolic function, future studies are needed to deepen our understanding of its effects in the various cardiac cell populations. Elucidation of these cell-type specific signaling mechanisms will help to clarify the therapeutic potential of cardiac GPER activation in preventing and/or halting the progression of cardiac diseases that involve diastolic dysfunction. Herein, where data exists, we include the signaling pathways of an activated GPER that are linked to the physiologic underpinnings of diastolic function preservation in the context of estrogen deprivation . Initial2+]i within a tightly controlled range is critical for normal systolic and diastolic function. In adult cardiomyocytes, the L-type Ca2+ channel is the main pathway for Ca2+ influx. The Na/Ca2+ exchanger is quantitatively the most important pathway for Ca2+ efflux out of the cardiomyocyte. The sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps and the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors) are pivotal in determining [Ca2+]i and subsequent contraction and relaxation.Maintaining cardiomyocyte calcium concentration [Ca2+ current conducted via L-type Ca2+ channels Cav1.2 . This is the main trigger for Ca2+ release from the sarcoplasmic reticulum, termed Ca2+-induced Ca2+ release (CICR). CICR involves ryanodine receptors (RyR), of which RyR2 is the predominant myocardial isoform. ICa,L and CICR together increase the concentration of intracellular Ca2+ that binds to troponin C on myofilaments to initiate myocardial contraction.At the initiation of myocardial contraction, depolarization of the cardiomyocyte leads to activation of the inward Ca2+ and subsequent dissociation of Ca2+ from troponin is required for myocardial relaxation. This involves multiple components, including Ca2+ reuptake into the sarcoplasmic reticulum via SERCA2a  and activation of the sarcolemmal Na+/Ca2+ exchanger  and, to a lesser extent, plasma membrane Ca2+-ATPase   . ICa,L areviewed .2+ signaling, with a focus on mechanisms that regulate myocardial relaxation and the known role of GPER. A summary of the proteins/messengers examined in this context is provided in We will briefly discuss the effects of estrogen on components of myocardial Ca2+ increase, ICa,L is also a main source for refilling SR Ca2+ in smooth muscle  reduces heart rate and pressure and cAMP production in the isolated perfused heart treated with isoproterenol; these effects are not inhibited by tamoxifen, an ERα/ERβ antagonist, and at the time were attributed to activation of an unknown membrane receptor -dependent phosphorylation of Cav1.2  via beta-adrenergic receptor (β-AR)-mediated, cAMP-dependent mechanisms. Estrogen alters gene expression of β-ARs and calcium-handling proteins -mediated contraction, relaxation, and Ca2+ signals, leading to regression of LV dysfunction in a male mouse model of isoproterenol-induced HFpEF   , 84. By laxation , 85. Saractility . Similar2+ homeostasis and improve diastolic function by increasing either the expression and/or activity of SR Ca2+ regulatory proteins. Past reports showed that estrogen increases the expression of SERCA2a, while its expression is decreased in OVX animal models. We found no evidence for changes in SERCA2, PLB, calmodulin, or RYR2 gene or protein expression in cardiac tissues with chronic G1 treatment (50 μg/kg/day) of OVX mRen2.Lewis rats with systemic hypertension, despite improvements in myocardial relaxation  dysfunction when compared with vehicle-treated counterparts  is one mechanism for LV hypertrophic and interstitial remodeling that contributes to LV stiffness and diastolic dysfunction. Indeed, Ang II is involved in tissue remodeling and the induction of fibrosis . While RP < 0.06), cardiac Ang II content is reduced when compared with OVX vehicle, suggesting a role for mast cell derived-factors and chymase/Ang II in the progression of cardiac aging and diastolic dysfunction after estrogen loss (2 treatments favorably regulate cardiac mast cell number and prevent the adverse effects of OVX on cardiac remodeling and LV function in an Ang II-dependent rodent model of hypertension and LV diastolic dysfunction (With respect to estrogen status (intact vs. OVX) and LV diastolic dysfunction, we demonstrated a positive relationship between cardiac chymase-forming Ang II and echo-derived filling pressures in normotensive Wistar Kyoto female rats  and hypegen loss . Indeed,function  and in mfunction  and volufunction , 157.2-induced inhibition of mast cell proliferation , significantly limits hypertrophic remodeling and improves LV systolic and diastolic function (Using cardiomyocyte-specific GPER KO mice , we founfunction . ConsistWhether G1/GPER-mediated anti-inflammatory responses are related to its effects on mast cells, as discussed previously, is not known. Mast cells are potent innate immune cells that accumulate in chronically inflamed tissues. The IL-1 family of cytokines, and particularly IL-33, activate mast cells and prime them to respond to inflammatory signals . If estrLV ejection with respect to proximal aortic distensibility is another factor that contributes to diastolic function in the female heart. During systole, the long axis of the left ventricle normally shortens by pulling the aortic annulus toward the relatively fixed LV apex , 163. Di2 (Although the exact role of estrogen/GPER in aortic–ventricular interactions with respect to diastolic function is not known, recent preclinical studies suggest that GPER activation limits aortic stiffening and remodeling. GPER is expressed in both endothelial and smooth muscle cells of the aorta , 171 and2 , 172. In2  and the 2 , 171. In2  but is f2 . GPER is2 . While A2 . TherefoThe sex-differential in the prevalence and incidence of human HFpEF is stark. Among women age >/ = 65 years, nearly 90% of new cases of heart failure are HFpEF. In the small number of men who develop HFpEF, the underlying characteristics differ markedly from that seen in women with predominantly ischemic heart disease, mildly dilated LV, and borderline/mild levels of systolic dysfunction. Thus, classic HFpEF is nearly exclusively a disorder of older, post-menopausal women. Despite this overwhelming magnitude of this profound biologic signal, its fundamental basis has not been systematically examined. Doing so could produce major insights into the initiation and progression of human HFpEF. Thus, the emerging data reviewed above has the potential to promote key advances in the understanding of human HFpEF and novel approaches to interrupting the pathways that lead to one of its precursors, diastolic dysfunction. This is greatly needed given the disappointing results of the recent PARAGON trial , and the2+ homeostasis, mitochondrial function, and anti-hypertrophic/interstitial processes (Improvements in preventive medicine and health habits by positively lengthening the human life-span have brought to the forefront the impact of the menopause decline in women's cardioprotection. Estrogen-mediated cardiac health in women, using diastolic function as its monitor, is influenced by non-genomic mechanisms through GPER in the heart, in part by counteracting age and/or estrogen loss-dependent abnormalities in myocardial relaxation, cardiomyocyte Carocesses .LG conceived and designed the manuscript. LG, Q-KT, and SL drafted the manuscript. Q-KT, HW, XS, LG, and SL created the tables and figures. DK provided the translational perspective. DK, CF, and CC were involved in critically reviewing and revising the manuscript for important intellectual content. All authors gave final approval of the manuscript to be published and agreed to be accountable for all aspects of the work.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.


The calcium-sensing receptor (CaSR) has been localized and characterized in numerous tissues throughout the body. In the mammalian gastrointestinal tract, the CaSR is known to act as a nutrient sensor and has recently been found to play a role in intestinal fluid and electrolyte balance. This study aims to demonstrate the functionality of the CaSR as a modulator of fluid secretion and absorption along the small intestine.ex vivo intestinal perfusion device that provides independent intraluminal and extraluminal  perfusion. The regions were perfused with 5 and 7 mM of Ca2+, both in the presence and absence of forskolin (FSK), a potent secretagogue. Control experiments were conducted with intraluminal perfusate containing standard Ringer-HEPES buffer with a physiological concentration of Ca2+ (1 mM). A second set of comparison experiments was performed with intraluminal perfusates containing AC-265347, a CaSR activator and agonist, in the presence of FSK. In all experimental conditions, the intraluminal perfusate contained fluorescein isothiocyanate (FITC)-inulin, a nonabsorbable fluorescent marker of secretion and/or absorption. Intraluminal fluorescence signal was utilized as a measure of water movement at the start of the experiment and every 15 min for 90 min.Small intestine regions  were isolated from Sprague Dawley rats and loaded into an 2+ in the luminal perfusate reduced intestinal fluid secretion in all regions. At a Ca2+ concentration of 7 mM, net fluid absorption was observed in all regions. In the presence of FSK, 5 mM Ca2+ significantly decreased fluid secretion and 7 mM Ca2+ abolished FSK-induced fluid secretion. Intraluminal perfusion with 5 mM Ca2+ was as effective as AC-265347, in reducing secretagogue-induced fluid hypersecretion in the proximal and middle regions.Under physiological conditions, increasing the concentration of Ca2+ and/or calcimimetics reduces fluid secretion in a dose-dependent manner, with higher Ca2+ concentrations, or application of a calcimimetic, leading to fluid absorption. We furthermore show that, in the presence of FSK, receptor activation abates FSK secretagogue-induced fluid secretion. This presents a new therapeutic target to address secretory diarrheal illnesses.This study concludes that apical CaSR is active along the small intestine. Its activation by Ca The rats were fasted 12–15 h prior to each experiment.Male Sprague-Dawley rats weighing between 250 and 490 g  after which a midline laparotomy was performed. Cold (4°C) Ringer-HEPES buffer (ex vivo perfusion device previously described by our group (Fasted rats were fully anesthetized with Isoflurane (IsoThesiaS buffer  was usedur group . Each ch2+ (5 and 7 mM) separately. Control regions were perfused with standard Ringer-HEPES buffer containing 1 mM Ca2+, which is within the physiologic concentration of Ca2+ in the small intestine , respectively. Lastly, we compared the effect of 1, 5, and 7 mM intraluminal Ca2+ in the presence of FSK to that of 2 μM of the calcimimetic 1-Benzothiazol-2-yl-1--ethanol , which is an allosteric agonist of the CaSR, which is also known to reduce ionized calcium 2+Ringer-HEPES buffer with 5 mM Ca(2)2+ + 10 μM FSKRinger-HEPES buffer with 5 mM Ca(3)2+Ringer-HEPES buffer with 7 mM Ca(4)2+ + 10 μM FSKRinger-HEPES buffer with 7 mM Ca(5)Standard Ringer-HEPES buffer(6)Standard Ringer-HEPES buffer + 10 μM FSK(7)Standard Ringer-HEPES buffer + 10 μM FSK + 2 μM AC-2653472, thereby keeping chloride concentration constant  was used to measure fluorescence intensity of the intraluminal perfusate over time. The greater the fluorescence of the sampled luminal content, the less water secretion into the lumen was observed, decreases in luminal fluorescence were indicative of a decreased concentration of FITC-inulin. For each experiment, fluorescence measurements were taken at seven-time points over the course of the experiment: 0, 15, 30, 45, 60, 75, and 90 min. Five intraluminal fluid samples of 2 μL each were taken for each experimental time point; the slope and SEM were calculated and graphed. At the end of each experiment, extraluminal perfusate samples were collected to exclude the possibility of leakage due to perforation or damage to the intestinal wall. The absence of any detected FITC-inulin in the basolateral  perfusate ruled out leakage and confirmed preserved integrity of intestinal regions. Presence of FITC-inulin in the extraluminal perfusate warranted discarding of the intestinal region.p value of less than 0.05 (p < 0.05) was considered statically significant.Intraluminal fluorescence values of FITC-inulin concentration were calculated, plotted and analyzed using GraphPad Prism 8.01 (GraphPad Software). Linear regression analysis was performed to determine slopes, which represented average secretion or absorption per minute over 90 min. Statistically significant differences were calculated using mixed-effects analysis and Tukey’s multiple comparisons test. A 2+ . As shown in 2+ completely abolished FSK-induced secretion in all intestinal regions. This effect was statistically significant in comparison to control experiments .Addition of FSK to the intraluminal perfusate dramatically increased fluid secretion in the control intestinal regions perfused with standard Ringer-HEPES . In the 2+, the secretion profiles in all the intestinal regions were compared in the presence of control (1 mM of Ca2+), control with 10 μM FSK, control with 10 μM FSK and 2 μM AC-265347, and 5 mM Ca2+ with 10 μM FSK. There was no statistical significance between control and control with 10 μM FSK and 2 μM AC-265347, a CaSR agonist, in all regions as shown in 2+ in abating secretagogue-induced fluid secretion to that of 2 μM AC-265347 in standard Ringer-HEPES buffer containing 1 mM Ca2+ demonstrated that intraluminal perfusion with 5 mM Ca2+ in the presence of 10 μM FSK was statistically similar to intraluminal perfusion with 2 μM AC-265347 with 1 mM Ca2+ in decreasing FSK-induced fluid secretion in the proximal and middle regions of the intestine , and more effective in the distal region (p = 0.0034). The reduction in secretion observed with 7 mM Ca2+ was larger when compared to the calcimimetic AC-265347 with 1 mM of Ca2+ in the middle and distal regions (p < 0.0001) and had a similar effect in the proximal region (p = 0.0004) as observed in p value for the intestinal regions’ comparison. A detail of each comparison is shown in In order to determine if the CaSR was being activated with Ca2+ or a calcimimetic decreases intraluminal fluid secretion under physiological conditions. Increasing luminal calcium concentrations completely abates the secretory effect of the potent secretagogue FSK along the entire length of the rat small intestine. The receptor has been previously localized along the entire length of the mammalian gastrointestinal tract  with physiological Ca2+ concentration that results in decreased secretagogue induced fluid secretion comparable to a high Ca2+ perfusate. The observed effect of activating the CaSR established a dose-dependence and occurs along the entire length of the small intestine, with net fluid absorption occurring at a Ca2+ concentration of 7 mM in all intestinal regions (2+) increases the activity of the CaSR in modulating luminal fluid secretion, and at elevated levels of Ca2+ (7 mM) induces absorption of fluid.The data obtained in the present study suggests that elevations in intraluminal Ca regions . Accordi2+ would influence secretagogue-induced fluid secretion. FSK is a cyclic nucleotide activator that leads to an elevation of cAMP levels within intestinal epithelial cells and as such mimics the physiology of secretory diarrhea (2+ concentration in the continued presence of FSK significantly decreases the secretory action of FSK in a dose-dependent manner. CaSR activation inhibits cyclic nucleotide activation and increases Na+-dependent absorption, thus inhibiting secretion and promoting fluid absorption. At a Ca2+ concentration of 7 mM, CaSR activation completely abolished FSK-induced fluid secretion along the small intestine. To confirm that the exposure to elevated luminal Ca2+ levels was via activation of the CaSR and not due to a non-targeted effect we exposed small intestinal segments to a physiological luminal Ca2+ concentration in the presence of the calcimimetic AC-265347 (2+ is just as effective as intraluminal AC-265347 in decreasing secretagogue-induced fluid secretion (2+ is significantly better than 2 μM AC-265347 in abrogating fluid secretion (Overstimulation of the secretory capacity of the intestinal tract results in secretory diarrhea, which is characterized by large stool volumes and is a leading cause of mortality amongst children and the elderly worldwide . As suchdiarrhea . As showC-265347  (Figure C-265347 . In the ecretion . In factecretion . These recretion .2+ improves resistance to enterotoxigenic Escherichia coli in young adults and ameliorates infectious diarrhea in immunocompromised children (2+ leads to cessation of cyclic nucleotide-induced hypersecretion, and results in fluid absorption at higher luminal Ca2+ concentrations. These results not only provide a novel approach of Ca2+-based anti-secretory therapeutics, but also provides the possibility of correction of dehydration and nutrient absorption by increasing fluid absorption in secretory diarrheal maladies.The CaSR has recently provided a target for treatment of diarrheal illnesses, with the aim of activating it to induce its anti-secretory, pro-absorptive, anti-motility, and anti-inflammatory effects . Human schildren . Our finMB was involved in conducting the experiments, statistical analysis, and writing of the manuscript. RM was involved in conducting experiments, writing, and editing of the manuscript. TL and MF were involved in conducting the experiments and writing the manuscript. GC was involved in the experimental design. VB was involved in editing of the manuscript. FD and DM were involved in experimental design, manuscript writing, and editing. JG was involved in all aspects of the experimental design, statistical analysis, manuscript writing, and editing.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.


DMPK transcripts. Abnormally expanded (CUG)n repeats in these transcripts form hairpin-like structures that cause the RNA to accumulate in the cell nucleus by sequestering isoforms of the Muscleblind (MBNL) family, tissue-specific regulators of developmentally programmed, post-transcriptional processes in RNA metabolism. Through this mechanism, the function of MBNL in RNA processing becomes dominantly perturbed, which eventually leads to aberrant alternative splicing and the expression of foetal splice variants of a wide variety of proteins, including the MBNL isoforms themselves. Here, we employ a patient-derived muscle cell model for DM1 to examine in detail the expression of MBNL RNA and protein variants during myogenic differentiation. This DM1 model consists of a panel of isogenic myoblast cell lines that either contain a pathogenic DMPK allele with a congenital mutation of 2600 triplets, or lack this expanded repeat through CRISPR/Cas9-mediated gene editing. We found that the temporal expression levels of MBNL1, MBNL2 and MBNL3 RNAs are not influenced by presence of the (CTG)2600 repeat during myogenesis in vitro. However, throughout myoblast proliferation and differentiation to myotubes a disproportionate inclusion of MBNL1 exon 5 and MBNL2 exons 5 and 8 occurs in cells with the (CTG)2600 repeat. As a consequence, a reduced quantity and imbalanced collection of splice variants of MBNL1 and MBNL2 accumulates in both the cytoplasm and the nucleus of DM1 myoblasts and myotubes. We thus propose that both the quantitative and qualitative changes in the intracellular partitioning of MBNL proteins are a pivotal cause of skeletal muscle problems in DM1, starting already in muscle progenitor cells.Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder caused by the expression of trinucleotide repeat-containing  MBNL isoforms, MBNL1, MBNL2 and MBNL3 are expressed. MBNL1 and MBNL2 are found ubiquitously, with MBNL1 being more prominent in skeletal muscle and MBNL2 relatively abundant in brain . Th. ThMBNL  alleles . FollowiMBNL expression, we performed RNA-seq analysis on proliferating myoblasts. Transcript levels of MBNL1, MBNL2 and MBNL3 in (CTG)2600 repeat-containing cells did not differ from those in cells from which the repeat expansion was removed  is an accepted hallmark for the foetal splice pattern characteristic for the disease [MBNL2 in DM1 has not been studied in detail yet, but it has been reported that also for this isoform the conserved exon 5 (54 nts) is overrepresented in mature transcripts [Alternative splicing of is paper ,9,12,14. disease ,9,12. Abnscripts ,10.MBNL1 and MBNL2 splicing in proliferating myoblasts using RNA-seq and focussed on the main alternatively spliced regions, encompassing MBNL1 exons 5–9 and MBNL2 exons 5–8  (MBNL1 exon 7 (64% vs 61%) nor exon 8 (94% vs 91%). For MBNL2, we measured increased inclusion of exon 5  and exon 8  in myoblasts with a (CTG)2600 repeat (When comparing myoblasts carrying the (CTG)2600 repeat with cells from which the repeat had been recently removed, we found that  0.0001) . No sign0 repeat .MBNL exons 5 to 9/8 on RNA isolated from proliferating and differentiating myoblasts to verify the RNA-seq data and to investigate the combinatorial use of multiple alternative splice events during myogenesis. We could confirm that in proliferating myoblasts containing the (CTG)2600 expansion the majority of MBNL1 transcripts included exon 5, unlike in myoblasts without the expanded repeat . When we looked more specifically at combinatorial use of exons 5 and 7, we found that the +ex5+ex7 and +ex5-ex7 MBNL1 variants, encoding MBNL143 and MBNL142 proteins respectively, were around nine-fold higher expressed in cells carrying an expanded repeat than in cells without repeat. In contrast, the -ex5+ex7 and -ex5-ex7 variants, corresponding to the smaller MBNL141 and MBNL140 proteins respectively, were four-fold less abundant in cells with repeat. Of note, despite the fact that alternative splicing and myogenesis are intrinsically related processes, the MBNL1 splice pattern did not undergo an overt change upon the onset of differentiation or the next five days of further myoblast-to-myotube differentiation in vitro, irrespective of repeat presence.We subsequently performed semi-quantitative RT-PCR across MBNL2 splice pattern confirmed that proliferating myoblasts containing the (CTG)2600 expansion showed a higher degree of exon 5 and exon 8 inclusion than those without  2600 containing myoblasts. The +ex5-ex8 mRNA variant, encoding MBNL241 protein, could barely be detected at all. Remarkably, MBNL2 exon 5 and exon 8 inclusion increased significantly during myogenic differentiation, but only in cells with the (CTG)2600 repeat. This phenomenon seems a distinct feature of MBNL2 expression, as it was not observed for MBNL1.RT-PCR analysis of the ctively) . We obseMBNL splicing is noticeably perturbed during the proliferative phase of DM1 myoblast cell formation and involves increased inclusion of MBNL1 exon 5 and MBNL2 exons 5 and 8. Upon induction of myogenic differentiation, this situation is maintained during the subsequent phase of quiescence and myoblast fusion into myotubes, whereby a specific transition occurred between MBNL2 +ex5+ex8 (MBNL240) and -ex5+ex8 (MBNL238) variants.All data combined, we conclude that alternative MBNL1 and MBNL2 mRNAs would significantly alter MBNL protein expression in cells with a CTG(2600) repeat compared to cells lacking the repeat. To this end, whole-cell protein lysates of proliferating myoblast cultures and of cultures harvested at different time points after initiation of differentiation were prepared and used in western blotting. The MBNL1 antibody MB1a that we used for detection was directed against an epitope encoded by exon 3 [We next examined whether the abnormally spliced, yet similar total levels of y exon 3 , present43 (+ex5+ex7) cosegregating with MNL142 (+ex5-ex7), while in the bottom signal MBNL141 (-ex5+ex7) and MBNL140 (-ex5-ex7) migrated together. Total MBNL1 levels were three to four-fold lower in proliferating myoblasts with a (CTG)2600 repeat than in cells without the repeat (40/41 (-ex5±ex7) variants , the dominant proteins in cells lacking the repeat . More specifically, during five days of differentiation, the total MBNL1 level, again mainly the MBNL140/41 variants, decreased around two-fold, particularly in cultures with a (CTG)2600 repeat.From the inferred amino acid (aa) sequences, we predicted that exon 5 or exon 7 inclusion would contribute an extra 2.0 kDa (18 aa) or 1.2 kDa 12 aa), respectively to the total molecular weight of the MBNL1 variants. The blots showed a complex collection of MBNL1 signals with migration behaviour around the 40 kDa marker protein, as expected . Two ban aa, respe repeat . This di40 (+ex5+ex8), cosegregating with MBNL241 from the barely expressed +ex5-ex8 splice variant. We assumed that the bottom signal represented a mix of MBNL239 (-ex5-ex8) and MBNL238 (-ex5+ex8). Total MBNL2 levels were not significantly lower in cells with a (CTG)2600 repeat than in cells without the repeat (40/41 (+ex5±ex8) variants were significantly more abundant under differentiation conditions; in fact, these MBNL2 variants were essentially absent in cells from which the repeat expansion had been removed. In contrast to MBNL1, total MBNL2 level remained relatively constant during myogenic differentiation.Also for MBNL2, multiple signals were detected, using antibody MB2A directed at the N-terminus shared by all MBNL2 variants  Fig 3B)Fig 3B). e repeat . HoweverIn sum, the total level and protein composition of both MBNL1 and MBNL2 is altered in the presence of the expanded (CTG)2600 repeat. During myogenic differentiation, MBNL1, but not MBNL2, levels decreased slightly, particularly in cells with the repeat.MBNL1 exon 5 is implicated in the nuclear localization of MBNL1 [MBNL2. We employed immunofluorescent microscopy using the same antibodies as for western blotting, and examined the subcellular distribution of MBNL1 and MBNL2 in myoblasts with and without (CTG)2600 repeat.We wondered about the effects of lower protein expression and altered protein composition on MBNL localization in the cell. It has been reported that inclusion of of MBNL1 ,11,13. MThe typical DM1 foci in the nuclei of cells with the expanded (CTG)n repeat contained MBNL1 as well as MBNL2 . Notablyin situ using antibodies MB1a and MB2a did not allow for discrimination between MBNL variants, we performed subcellular fractionation of proliferating myoblasts followed by western blotting to identify the MBNL1 and MBNL2 variants in the cytoplasmic and nuclear-enriched fractions. As expected, the MBNL142/43 and MBNL240/41 variants, corresponding to exon 5 inclusion, were enriched in the nuclear fractions, particularly in cells with the expanded (CTG)2600 repeat 2600 repeat 2600 repeat-containing cells, we observed a diffuse granular staining in the nucleus and in the cytoplasm . Excisio0 repeat .MBNL family members must be spatiotemporally expressed with high accuracy during development. Functional loss or change in the composition of the cellular MBNL isoform population is thought to be, at least in part, responsible for the spliceopathy typically seen in DM1 [MBNL expression, with a focus on MBNL1 and MBNL2, in a well-controlled human cell model for DM1 with normal expression of an expanded repeat. Our unique isogenic muscle cell panel, comprising several independent cell lines with and without a congenital DM-type mutation, enabled us to study repeat effects without possible confounding influences of genetic background or other forms of interpatient variability. Using independent methods for the identification and quantification of RNA and protein, we investigated MBNL expression in detail in proliferating myoblasts and in differentiating myotubes. Earlier, we and others had already shown that (CTG)n repeat excision in DM1 cells restores the normal cellular phenotype [MBNL expression as early as in myoblasts, the muscle progenitor cells.As important cell type-specific regulators of RNA processing, n in DM1 . In the henotype ,35,36. WMBNL isoforms in our cell model. This observation matches findings from a comparative RT-qPCR study on muscle biopsies from a cohort of DM1 patients and unaffected individuals [MBNL1 expression, but not MBNL2 and MBNL3, in tibialis muscle from DM1 patients compared to controls, indicating that expression levels can vary in different muscle tissues [MBNL RNA levels did not overtly change during myoblast alignment and myotube formation during five days in vitro. All in all, these RNA quantifications indicate that changes in MBNL protein expression in early myogenesis in DM1 must be predominantly the result of (CTG)n repeat-mediated effects at the posttranscriptional level.RNA-seq and RT-PCR analyses demonstrated that removal of the (CTG)2600 repeat did not alter RNA expression levels of any of the three ividuals  and a reividuals . Interes tissues . We alsoMBNL is alternative splicing. Alternative splicing of MBNL1 and, to a limited extent, MBNL2 has been studied by a number of laboratories, but only rarely under well-controlled conditions of normal expression of an expanded DM1 repeat [MBNL gene structure, but using the nomenclature summarized in MBNL1 exons 3 (204 nts), 5 (54 nts), 7 (36 nts) and 8 (95 nts) in muscle cells. Of these, we basically only found a significant increase of exon 5 inclusion in DM1 cells. We did not detect increased inclusion of exon 7, which was reported in DM1 brain [MBNL2 exons, exon 5 (54 nts) and exon 8 (95 nts), were both preferentially included in DM1 cells. It is important to note that we mainly detected a shift in splice variant composition of MBNL1 and MBNL2 and did not identify significant expression of uncommon or novel splice variants.Probably the main level of posttranscriptional regulation of 1 repeat –14. The s) and 8 5 nts in MBNL mRNA and protein splice variant composition, indicating additional regulatory mechanisms after the MBNL mRNA has been made. The most likely explanation for the difference in the regulation of MBNL mRNA and protein abundance are variations in mRNA translation efficiency and protein stability [When comparing the data on RNA and protein expression from either cells with or without (CTG)2600 repeat, we did not observe a quantitative one-to-one correlation between alterations in tability ,40. The 41 (-ex5+ex7) was significantly reduced in myoblasts with repeat and even stronger in myotubes. As a result, MBNL143 (+ex5+ex7) was overrepresented in these cells. Total MBNL2 protein level did not significantly change upon repeat expansion, but MBNL240 (+ex5+ex8) increased at the expense of MBNL239 (-ex5-ex8), particularly in myotubes. We therefore conclude that the dysfunction of MBNL1 and MBNL2 in DM1 consists of a moderate (two to five-fold) reduction in protein expression, combined with a modified splice variant content, i.e. overrepresentation of predominantly +ex5 variants. Effects of MBNL protein loss in DM1 have been studied in detail in vivo in Mbnl knockout mice, which showed that Mbnl1 and Mbnl2 have distinct functions, predominantly in muscle and brain respectively, but also that the two isoforms can partially compensate for each other’s absence [We found that MBNL1 protein, in particular MBNL1 absence ,22. EffeDMPK, MBNL1 and MBNL2 [Alternative splicing determines MBNL1 and MBNL2 localization in the cell, next to effects on RNA and protein binding, dimerization behaviour and splicing activity ,11,13–16nd MBNL2 . Recentlnd MBNL2 , but we DMPK (CUG)n transcripts trigger the prominent changes in the posttranscriptional regulation of MBNL. Presumably, expanded (CUG)n RNA-MBNL binding and subsequent formation of foci is the initiating event of the toxic cascade. After all, the use of antisense oligonucleotides and small molecules that specifically block this interaction has demonstrated that repeat RNA toxicity effects are reversible and can be neutralized [MBNL splicing. Whether the number of MBNL1 and MBNL2 molecules that can be sequestered in foci is sufficient to significantly reduce the active MBNL concentration in the cell is questionable. However, MBNL1 is known to stimulate its own exon 5 skipping [MBNL1 splicing and protein variant composition. Alternatively, there might be specific signalling mechanisms that are activated following (CUG)n RNA-MBNL complex formation and that feed into splicing regulation, but these pathways have not been identified thus far.An important question that remains is how pathogenic tralized ,45. We askipping , so an iMBNL1 and MBNL2 in DM1 is independent of differentiation cues, already occurring in myoblasts, the in vitro equivalent of muscle progenitor cells to the satellite cells in vivo. Disrupted MBNL expression pertains to protein level and splice variant composition throughout the entire cell. To obtain further insight in the exact sequence of events during muscle development, it is important to study MBNL expression even earlier during embryogenesis, right at the first appearance of toxic expanded (CUG)n RNA.In conclusion, we have demonstrated that dysregulation of S1 Fig42/43 and MBNL240/41 variants (the top two bands in both panels), corresponding to exon 5 inclusion, were enriched in the nuclear fractions, particularly from cells with the expanded (CTG)2600 repeat.Western blot analysis of  MBNL1 and  MBNL2 in cytoplasmic (cyto) and nuclear-enriched (nucl) fractions of  proliferating myoblasts and  five-day old myotubes with and without (CTG)n repeat expansion. Lamin A/C and tubulin served as nuclear and cytoplasmic marker, respectively, to demonstrate enrichment of both fractions. Molecular weights are indicated in kDa. Note that the blots generally show four distinct signals for both MBNL1 and MBNL2, instead of the two usually detected, which represent comigrating protein variants e.g. . MBNL142(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


However, the subcellular determinants of these SCaEs remain incompletely understood. Structural differences between atrial and ventricular cardiomyocytes, e.g., regarding the density of T-tubular membrane invaginations, may influence cardiomyocyte Ca2+-handling and the distribution of cardiac ryanodine receptors (RyR2) has recently been shown to undergo remodeling in atrial fibrillation. These data suggest that the subcellular distribution of Ca2+-handling proteins influences proarrhythmic Ca2+-handling abnormalities. Here, we employ computational modeling to provide an in-depth analysis of the impact of variations in subcellular RyR2 and L-type Ca2+-channel distributions on Ca2+-transient properties and SCaEs in a human atrial cardiomyocyte model. We incorporate experimentally observed RyR2 expression patterns and various configurations of axial tubules in a previously published model of the human atrial cardiomyocyte. We identify an increased SCaE incidence for larger heterogeneity in RyR2 expression, in which SCaEs preferentially arise from regions of high local RyR2 expression. Furthermore, we show that the propagation of Ca2+ waves is modulated by the distance between RyR2 bands, as well as the presence of experimentally observed RyR2 clusters between bands near the lateral membranes. We also show that incorporation of axial tubules in various amounts and locations reduces Ca2+-transient time to peak. Furthermore, selective hyperphosphorylation of RyR2 around axial tubules increases the number of spontaneous waves. Finally, we present a novel model of the human atrial cardiomyocyte with physiological RyR2 and L-type Ca2+-channel distributions that reproduces experimentally observed Ca2+-handling properties. Taken together, these results significantly enhance our understanding of the structure-function relationship in cardiomyocytes, identifying that RyR2 and L-type Ca2+-channel distributions have a major impact on systolic Ca2+ transients and SCaEs.Spontaneous Ca Although potential proarrhythmic effects of changes in RyR2 expression and phosphorylation have been extensively discussed  at the sarcolemma to RyR2 in the cell center cellular structure and functional Ca2+-handling in human atrial cardiomyocytes, as the former usually requires fixation of the cardiomyocyte for antibody staining. The perfect control and observability provided by computational modeling may help to overcome this challenge , is projected to increase, placing a significant burden on modern healthcare systems . All animal handling conformed with directive 2010/63/EU and experimental protocols were approved by the local ethical committee DEC2014-112). New Zealand white rabbits (2.5–3.5 kg) were anesthetized and hearts were rapidly removed, excised, washed, perfused and cut into smaller pieces, as previously described . The resulting RyR2 distribution matrix was implemented in the computational model and employed for simulation.Image processing was employed to enable simulation with the experimental data .Simulations were based on a previously published model of the human atrial cardiomyocyte in which local Capost-hoc test for multiple comparisons, or independent t-test, depending on the number of groups and the type of the data. Statistical analyses were performed using GraphPad Prism 7 .Due to the stochastic nature of the simulations, each condition was simulated at least 6 times and data were expressed as mean ± standard deviation. Statistical differences between conditions were evaluated using one- or two-way ANOVA with Tukey 2+ waves and corresponding DADs during follow-up after pacing at 0.5 Hz, in line with experimental data  and the average size of a Ca2+ wave as fraction of cardiomyocyte volume was 5x smaller than with σ = 0.0 . Increasing RyR2 heterogeneity also reduced the longitudinal  and transversal  velocity of Ca2+ waves. We compared the magnitude of the effect of altered RyR2 distribution to a 25% change in total RyR2 expression. In line with previous results  produced a few large Ca Figures . LikewisNext, we investigated the origins of SCaEs in simulations with heterogeneous RyR2 distributions . Although an increased RyR2 inter-band distance indeed slowed down the longitudinal velocity of SCaEs, it surprisingly did not impair propagation of SCaEs and resulted in an increased SCaE incidence Figure . Subsequ−1 in the normalized RyR2 expression group vs. 0.191 ± 0.018 s−1 in the group without inter-band clusters (σ = 0.0), p > 0.05 and 0.470 ± 0.138 s−1 for normalized RyR2 expression vs. 0.565 ± 0.105 s−1 without inter-band clusters (σ = 0.1), p > 0.05; Figure Similar to the results described by Macquaide et al. , our con2+-handling is not fully understood. Axial tubules contain LTCCs, modulating Ca2+-induced Ca2+ release and centripetal Ca2+-wave propagation. We incorporated various configurations of axial tubules in the 100-segment model and investigated their impact on depolarization-induced, LTCC-triggered Ca2+ transients and SCaEs. Addition of an axial tubule to the model reduced time-to-peak of the regional LTCC-triggered Ca2+ transient  and slightly increased the Ca2+-transient amplitude , including proarrhythmic triggered activity cellular structure and functional Ca2+-handling in the same cardiomyocyte. Here, we extended a computational model of the human atrial cardiomyocyte with a more physiological subcellular structure, including heterogeneous RyR2 distributions and axial tubules with LTCC. Our computational analyses showed that increasing RyR2 heterogeneity resulted in more, smaller SCaEs arising from regions with high local RyR2 expression. LTCC located in axial tubules produced a faster, more synchronous CICR, which was modulated by the location and extent of the axial tubular network. Moreover, hyperphosphorylation of RyR2 surrounding axial tubules increased the incidence of SCaEs and DADs. Finally, we developed and validated a novel human atrial cardiomyocyte model with physiological RyR2 distribution and axial tubules with LTCC based on experimental observations, which can serve as a tool for future studies.Recent studies have identified a major role for cardiomyocyte Ca2+ load leading to store-overload-induced SR Ca2+ release   to study the molecular determinants of SR Ca2+ release. For example, Hake et al.  but have not simulated whole-cell Ca2+-handling abnormalities, which would be relevant to study arrhythmogenesis.Several computational models have been developed to study cardiomyocyte Cae et al.  developer et al.  developeahradnik  construc2+-handling: Walker et al.   was insufficient to simulate local Ca2+ dynamics around a single Ca2+-release unit sparks). Detailed Ca2+-release unit models have been developed . The use of super-resolution microscopy would be beneficial to obtain a more detailed overview of the 3-dimensional RyR2 distribution. In this case, dual staining of RyR2 and axial tubules should be performed to obtain information on both distributions in a single cardiomyocyte, which was not performed in the current study. Instead, a representative experimental axial tubule geometry from previously published work  of both LTCC and RyR2, highlighting the need for detailed immunocytochemistry and functional studies to explain differences in whole-cell Ca2+-handling properties between conditions.We employed the perfect control and observability provided by computer models to overcome experimental challenges in the analysis of the subcellular determinants of cardiomyocyte CaHS, BvS, and JH conceived the study. PS, BvS, JH, MvZ, and GA performed and supervised the experiments. HS and JH performed the computational simulations and data analysis. HS and JH drafted the manuscript. All authors critically revised the manuscript and approved 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.


The rationale is to develop a tool that will enable a cross-talk between modeling and experiments, using modeling results to guide targeted experimental efforts. The model is built based on biophysical events and molecular cascades known to unfold as part of neurons' Ca2+ homeostasis mechanism, as well as on Ca2+ signaling events. The state of ion channels is described by their probability of being activated or inactivated, and the remaining molecular states are based on biochemically defined kinetic equations or known biochemical motifs. We estimate the parameters of the model using experimental data of hyperosmotic stimulus-evoked Ca2+ transients detected with a FRET sensor in young and aged worms, unstressed and exposed to oxidative stress. We use a hybrid optimization method composed of a multi-objective genetic algorithm and nonlinear least-squares to estimate the model parameters. We first obtain the model parameters for young unstressed worms. Next, we use these values of the parameters as a starting point to identify the model parameters for stressed and aged worms. We show that the model, in combination with experimental data, corroborates literature results. In addition, we demonstrate that our model can be used to predict ASH response to complex combinations of stimulation pulses. The proposed model includes for the first time the ASH Ca2+ dynamics observed during both "on" and "off" responses. This mathematical and computational effort is the first to propose a dynamic model of the Ca2+ transients' mechanism in C. elegans neurons, based on biochemical pathways of the cell's Ca2+ homeostasis machinery. We believe that the proposed model can be used to further elucidate the Ca2+ dynamics of a key C. elegans neuron, to guide future experiments on C. elegans neurobiology, and to pave the way for the development of more mathematical models for neuronal Ca2+ dynamics.We propose a mathematical and computational model that captures the stimulus-generated Ca C. el neurons –14, in f neurons ,15 as we neurons ,17 or ad neurons –22. The  neurons ,19,22,23esponse) ,25, in a2+ transients, neuronal behavior [2+ transients in the ASH neuron and C. elegans behavior has been the object of several studies that suggest an interesting correlation not only between the "on" response and specific behaviors [2+ dynamics in the ASH neuron participate in fine-tuning the worm's rich behavioral repertoire.The “on” and “off” responses that ensue in the ASH neuron upon its stimulation are the object of several studies, which explore the connection between Cabehavior ,24–26 anbehavior ,19,27. Iehaviors –30, but 2+ transients [2+ dynamics has been performed in a variety of organisms and cells, but the literature is sparse and far from complete for mathematical modeling of Ca2+ response in C. elegans neurons [2+ dynamics using ordinary differential equations. However, that work does not include the ASH "off" response, and the model does not account for the dynamics of the molecular players involved.Caansients  have beeansients ,23, oxidansients ,22, foodansients  and oxygansients . Extendeansients ,24,26,32 neurons ,33,34. W neurons  has focu2+ dynamics observed during both the "on" and "off" responses in the C. elegans ASH neuron. The approach integrates biophysical models that describe several aspects of the overall Ca2+ signaling mechanism and it merges them into an inclusive model forged by novel phenomenological adjustments and known biochemical motifs. Thus, the model succeeds in capturing the Ca2+ dynamics in ASH neuron with excellent fidelity. Moreover, we show how the model can be used to suggest potential changes in molecular components that can explain modifications in Ca2+ dynamics due to aging and oxidative stress to guide future experiments. Lastly, we demonstrate how the proposed model can be used to predict Ca2+ transients in ASH neuron when delivering arrays of complex stimuli.We propose a mathematical and computational model that is based on biochemical pathways and for the first time encompasses the Ca2+ transients and their connection with the rest of the model components is given in the Discussion.The pathways and molecules included in the proposed model are portrayed in 2+ transients which occur when the ASH neuron of young unstressed worms is stimulated by a hyperosmotic solution  the original case, in which after the knockout component is removed the values for all parameters remain the same as in the model that generates the output shown in One way to explore if a component of the model is necessary is to knock out that component and look for parameter sets that can still capture the complete response of the cell. To this end, we removed different components of the model, one at a time, to investigate their contribution to the predicted Cadynamics . For eacin silico knock out approach. We find that the model completely diverges without the PMCA . The times each parameter is present in a plausible parameter set for young stressed , aged unstressed  and stressed  worms is shown in The overall sensitivity of the model parameters can be visualized by a sensitivity plot . For eac2+ transients for such in silico experiments. In all panels, in addition to the %FRET ratio change, the Ca2+ concentration in the ER is also presented to show the long-lasting effects of the stimulus on the system. Compared to the changing Ca2+ concentration in the cytoplasm (represented by %FRET ratio change), the Ca2+ concentration in the ER has a slower dynamic, which affects the system’s response especially during sequential stimuli. At rest, the Ca2+ concentration in the ER is equal to its equilibrium value. When the stimulus is delivered, a small increase in the ER Ca2+ concentration is observed due to influx of Ca2+ through TRPVs and VGCCs. This is followed by a rapid large decrease due to Ca2+ release from the ER through IPRs. Then, the Ca2+ concentration in the ER remains relatively constant while the stimulus is sustained, since the influx and efflux of Ca2+ balance each other. Finally, when the stimulus is withdrawn, a second rapid large decrease in the ER Ca2+ concentration is observed during the “off” response. Then, the Ca2+ concentration in the ER starts returning to its equilibrium. However, if a stimulus is delivered before the Ca2+ concentration in the ER reaches its equilibrium  pulses . When weLastly, we applied ramp pulses, where the stimulus intensity (magnitude) changes gradually over time  to 6G. W2+ that flows in leads to IPRs opening via Ca2+-induced Ca2+ release, and to a voltage change that activates the VGCCs. The important role of TRPVs in the model is verified when they are in silico knocked out . Th. Th2+ re2+ response is then similar to the normal transient , like the ones activated by hyperosmotic stimulus in the ASH neuron ,61, can ed worms . TherefoC. elegans [kO and 2+ transients.Although TRP channels are involved in lifespan regulation in  elegans , changes elegans ,69. Our ed worms  as well 0, kP0−) . Thus, tkO and C. elegans and other systems.Oxidative stress has a significant impact on TRPV channels, mainly through oxidation of cysteine residues in channels' subunits ,71, resu2+ influx rises in both cases [2+ transients in the neuronal body, where our efforts are focused. Moreover, VGCCs are modeled coarsely in the present work, not allowing us to draw solid conclusions.VGCCs are known to be affected by oxidative stress  via oxidth cases ,78. Inte3 receptors [2+ into the cytoplasm [2+-induced Ca2+ release [GIPR), and to the rates for IPRs , which changes in stressed worms (GIPR) and to the rates for IPRs  . Hence, 2+ through PMCAs occurs also in aged neurons [C. elegans. This could be related to changed affinity of PMCAs and SERCAs for Ca2+ (KPMCA and KSERCA) in aged worms (2+ of SERCAs (KSERCA) in young stressed worms (GPMCA), as is suggested in the model . However, the decreasing stimulus through stepped pulses results in different “off” responses, as has been reported experimentally (33). This suggests that the proposed model can encapsulate Ca2+ dynamics in the ASH neuron in alignment with the experimental data.A step-increasing pulse, instead of a continuous gradient,  does notThe ability of ASH to respond reliably to a sequence of identical stimuli has been shown experimentally , and intKc parameter in Eq . Theref2+ transients, to flickering stimuli of 1 sec long pulses  and does not depend solely on the actual duration or magnitude of each pulse  suggests how molecular players that are known to be affected by aging and/or oxidative stress may be involved in generating the altered Ca2+ transients features (e.g. IP3 receptors), ii) suggests a possible effect of aging and/or oxidative stress on molecular players not yet documented to play a role toward aging- or stress-driven modifications of the Ca2+ transients features (e.g. activation/inactivation of TRPV channels), and iii) can be used to propose and guide future experimental work, targeting specific molecular players involved in Ca2+ dynamics. Finally, the model can be used to predict Ca2+ transients in the ASH neuron in the case of complex stimuli. We envision the proposed model as a tool used to further elucidate the Ca2+ dynamics of C. elegans neurons and to pave the way for the development of more mathematical models for neuronal Ca2+ dynamics.We propose a model for the Ca2+ transients. Numerous molecules and pathways are involved in this process [C. elegans neurons [The model is based on intracellular molecular events responsible for the generation of Ca process ,93. We f process ,77,79–81 neurons . We mode2+, the concentration of free Ca2+ in the cytoplasm and endoplasmic reticulum (ER) can be written as the following system of ordinary differential equations [c and cER are the concentrations of free Ca2+ in the cytoplasm and in the ER. JPMCA represents the Ca2+ efflux through plasma membrane Ca2+ ATPase (PMCA) pumps to the extracellular space (ES), and JSERCA is the Ca2+ flux from the cytoplasm into ER through ER ATPase pumps. JTRPV and JVGCC denote the influxes of Ca2+ into cytoplasm from ES through transient receptor potential-vallinoid (TRPV) channels and through voltage gated Ca2+ channels (VGCC). JLeak and JLeak,ER represent constant influxes of Ca2+ into the cytoplasm from ES and ER through other mechanisms. γ denotes the ratio of the cytoplasmic volume to the ER volume, which can also account for fast linear Ca2+ buffers in the ER [Assuming a cell with well-mixed free Caquations :dcdt=JIn the ER .GPMCA and GSERCA are the maximum fluxes through PMCA and SERCA pumps. KPMCA and KSERCA model the affinity of PMCA and SERCA for Ca2+. Ca2+ in the cytoplasm and the ER is always at equilibrium before applying the stimulus. To that end, the leak parameters across the cell membrane and the ER need to be adjusted based on the parameter values for PMCA and SERCA, and on the initial concentration of Ca2+ in the cell and the ER. The necessary leak values for equilibrium can be found by setting the time derivatives in Eqs in silico, the leak across the membrane will be found equal to zero).The PMCA pumps are modeled following  as:JPMCC. elegans neurons, this behavior is not included in the model. The Ca2+ influx through TRPV channels is modeled as:GTRPV is the maximum influx of Ca2+ through TRPV channels, and cES denotes the Ca2+ concentration in the ES, which is assumed to be constant and cES = 2000 μM. O and I are the probabilities of TRPV channels to be activated and inactivated, and are governed by:kO, kI are forward and O and I states. P1 and P2 represent two molecular players that control activation and inactivation of TRPV channels with the following kinetics:P1 and P2, and P2 for an equivalent player P0 that coarsely represents the dynamic effects of the cascade of molecular players that are activated by the external stimulus. We model the dynamics of P0 as:S represents the strength of the stimulus, P0 for S, and P0.Even though TRP channels show weak voltage dependence , due to P0 coarsely accounts for the pathway that is triggered upon delivery of the stimulus, including the G-proteins which are coupled with the receptors [P1 coarsely represents the pathway downstream of EGL-30, which leads to activation of TRPV channels [P2 represents the cascade of events in which fatty acids participate, modulating TRPV channels [Player eceptors ,98  to observe a stronger Ca2+ influx when a stronger stimulus is applied, as dictated by the underlying assumption . Therefore, P2 in our model saturates with P0  show that a stronger stimulus leads to a stronger Ca show thaallenged . Therefoh P0 see  to allow2+ influx through IP3 receptors (IP3Rs) is modeled as:GIPR is the maximum Ca2+ flux through IP3 receptors, and OIPR is the probability of IP3 receptors (IPRs) to be open, which is modeled using a reduced form [p is the concentration of IP3, and y is the faction of inhibited IPRs. The faction of inhibited IPRs is in turn governed by:3 or Ca2+ to IPRs, with their original values, as used by De Young and Keizer [The Caced form ,101 in tced form , namely:d Keizer . It is np upon the delivery of the stimulus, we use players P1 and P2 together with c to create the following phenomenological model:kp and Kp and Kc are the affinities of p for P2 and c. The relation between P1 and P2 in Eq (2+. This part of the model plays an important role in the "off" response. IPRs are responsible for the release of Ca2+ from intracellular stores. IP3 concentration (p) is the only free parameter in the original model of IPRs dynamics [p. However, that approach adds states to the model. Instead, P1 and P2  are used to capture the dynamics of p. Players P1 and P2 coordinate the dynamics of p in a way that the surge of p occurs when the stimulus is withdrawn, to allow release of Ca2+ from stores. Such dynamics for p can be achieved using incoherent feed-forward motifs [p by P1 and P2. Moreover, the term in c (Ca2+ concentration) accounts for the generation of p. This term saturates when the concentration of Ca2+ increases, to incorporate in the model the well-known phenomenon of Ca2+-induced Ca2+ release from intracellular stores [To model variations in Kc2−kp−p,where kpdynamics . One optd motifs  for gener stores .2+ influx through L-type voltage gated calcium channels (VGCCs) are modeled using the Goldman-Hodgkin-Katz [GVGCC is the maximum Ca2+ flux through VGCC, z is the valence of the respective ions (z = 2 for Ca2+), F is the Faraday constant, R is the gas constant, T is the temperature, V is the membrane voltage, and m is the activation variable given by:α and β are rates that vary with the membrane voltage for the L-type current as follows [The Cakin-Katz  equation follows :α(V)=1.V. Unfortunately, the available experimental data regarding changes in the membrane potential of the C. elegans ASH neuron is limited. Moreover, the ion currents, which are responsible for the voltage response of C. elegans neurons, are not completely understood. However, the available data suggests that many C. elegans neurons show a graded voltage response rather than the (better characterized and more commonly encountered) action potential response [2+ current, as follows:Vmax is the maximum membrane potential that can be reached during the graded response of the ASH neuron, and Vrest = −70mV is the resting membrane potential. α′and β′ are forward and backward rates for Eq (α′ and β′ are similar to α and β in Eqs (c (concentration of free Ca2+ in the cytoplasm) instead of V.Finally, we model the dynamics of membrane potential response ,39,40. TV−Vrest),where Vmβ in Eqs  and 20)V. Unfort2+ concentration obtained using the mathematical model can be related to signals measured in experiments [2+ interaction with an indicator genetically encoded in the C. elegans ASH neuron. The relation between the Ca2+ concentration and the measured % FRET change signal R follows an empirical form [n = 1.7, and the apparent affinity of R for Ca2+ is The Caeriments ,22. Expeeriments  are the cal form  that canvided in .2+ concentration. The use of Eq , and then calculated Rmin and Rmax. Next, we determined R0 using Eq  as a global minimizer, and trust-region-reflective nonlinear least squares (TRNLS) as a local optimizer, to determine the parameters of the mathematical model. The hybrid optimization algorithm starts with the GA, and the fittest individual found from the GA is passed to the TRNLS. The minimization problem is also subjected to constraints that ensure all parameter values are physical (namely that they are positive). The MATLAB optimization toolbox is used to perform these calculations. The maximum-likelihood of the experimental data is the objective function used in the TRNLS algorithm, namely:μexpσexp,where ReWe determined first the parameters for young unstressed worms. The solution of the GA depends on the initial population. Thus, we first applied the GA as a multi-objective optimizer to all four sets of experimental data. We then used the resulting population as an initial population for the GA applied only to the data from the young unstressed worms. Among the optimum solutions suggested by the multi-objective GA, we chose the solution for which the sum of the residuals for all experimental cases was the minimum, even though there were other solutions in which the residual for individual cases was smaller than the chosen solution. This approach allowed us to select as initial population for the GA a vicinity in the parameter space that is near an optimum solution  for all four cases.2+ dynamics for young unstressed worms. However, we sought a single set of parameters for young unstressed worms for which the sensitivity of the solution for the estimated parameters was small. To that end, we used the numerical Jacobian matrix that is obtained from TRNLS to approximate the Hessian matrix. Then, we used the Hessian matrix to construct the covariance matrix. We chose the solution that had the smallest diagonal elements of the covariance matrix. That corresponds to choosing the solution with the smallest variance of the parameters, indicating that small changes in the parameters (for that solution) do not lead to vast changes in the Ca2+ dynamics.There are more than one parameter sets that can result in the measured Ca2+ signaling mechanism–TRPV channels being activated/deactivated, IP3, IPRs, PMCAs, SERCAs–and they have been selected based on discussions in the existing literature [For the other three experimental cases, we start with the parameters found for young unstressed worms to create the initial population for the GA in the hybrid optimization. Next, we limit the algorithm to change only 13 selected parameters of the total of 23. These 13 parameters  and unstressed C. elegans of various ages was stimulated by hyperosmotic solution of 1M glycerol [C. elegans: i) young (L4+1/Day 1 of adult life) unstressed animals , ii) young (L4+1/Day 1 of adult life) oxidative-stressed animals, iii) aged (L4+5/Day 5 of adult life) unstressed animals, and iii) aged (L4+5/Day 5 of adult life) oxidative-stressed animals. It is noted that Day 5 worms can be considered of middle age [All experimental results presented in Figs glycerol ,22 were glycerol , where eglycerol . Experimddle age , but for2+ entering the cell and consequently accounts for the amplitude of the cell's response to the applied stimulus, whereas the slope corresponds to the time rate of the Ca2+ influx.The peak and the slope of the rising phase of the "on" response in the ratio traces were calculated at the onset of stimulus  of the cytoplasmic Ca2+ after 30 sec of the response onset and until the stimulus is withdrawn. The maximum peak of the "off" response /(Tminplateau − Tmax), where Tmax is the time when onmax is observed, and Tminplateau is the time when the plateau starts.The decaying slope of the "on" response can be defined as the difference between the maximum value of the FRET ratio change onmax of the "off" response is calculated as the % peak FRET ratio change upon withdrawal of the stimulus. The rising slope of the "off" response is the difference between the average stabilized FRET signal Aplateau at the plateau between 30 and 40 sec and the maximum peak offmax observed upon withdrawal of the stimulus, over the time needed to reach this maximum, counting from the stimulus offset. The rising slope can be expressed as (offmax − Aplateau)/(Tmax − 40).The maximum peak offmax observed upon withdrawal of the stimulus over the time needed to reach the minimum value observed after the stimulus offset, namely offmax/(Toffmax − Tmin).The decaying slope of the "off" response is the maximum value of the % FRET ratio change offt-test. Statistically significant differences were considered the ones with p-value < 0.05. Exact p-values are provided for each comparison on the respective plot. Statistical analyses were performed in Excel  and Minitab .All comparisons in S1 Fig2+ transient induced by the pulse (30sec) delivered in the experimental data and the model results, presented here for comparison; (C) A long pulse of 50 sec results in a Ca2+ transient of similar shape with the one shown in (B). The response to the shorter stimulus in (A) includes an “off” response stronger than the one observed in (B) and (C), although still smaller than the “on” peak. All three Ca2+ transients are generated using the parameters estimated for young unstressed worms (reference case).(A) A short pulse of 10sec still results in distinct peaks for “on” and “off” responses of different magnitudes, without the plateau region; (B) The Ca(PPTX)Click here for additional data file.S2 Figc in 2+ is released from the ER during the “on” response, then there is more available to be released from ER during the “off” response. Kc* corresponds to the value of this parameter used in the model for young unstressed worms (reference case).K(PPTX)Click here for additional data file.S1 TableWith bold are the selected parameters which are investigated for the aging and oxidative stress effect in the next three worm populations.(PPTX)Click here for additional data file.S2 Table2+ transients in young (Day 1) stressed worms compared to the reference case, either due to their change compared to the reference case, or due to their abundance in the plausible solutions.For each parameter, are indicated the mean of the values it takes in all sets of plausible solutions in which it appears, standard deviation, the difference of the mean with the parameter value for young unstressed worms , the % m(PPTX)Click here for additional data file.S3 Table2+ transients in aged (Day 5) unstressed worms compared to the reference case, either due to their change compared to the reference case, or due to their abundance in the plausible solutions.For each parameter, are indicated the mean of the values it takes in all sets of plausible solutions in which it appears, standard deviation, the difference of the mean with the parameter value for young unstressed worms , the % m(PPTX)Click here for additional data file.S4 Table2+ transients in aged (Day 5) stressed worms compared to the reference case, either due to their change compared to the reference case, or due to their abundance in the plausible solutions.For each parameter, are indicated the mean of the values it takes in all sets of plausible solutions in which it appears, standard deviation, the difference of the mean with the parameter value for young unstressed worms , the % m(PPTX)Click here for additional data file.


Mitochondria participate in Ca2+ cycling. We studied the role of mitochondrial Ca2+ flux in ventricular spontaneous electrical activity.Ca2+ transients were determined by fluorescent imaging.Spontaneously contracting mouse embryonic stem cells (ESC)-derived ventricular cardiomyocytes (CMs) were differentiated from wild type and ryanodine receptor type 2 (RYR2) knockout mouse ESCs and differentiated for 19–21 days. Automaticity was also observed in human induced pluripotent stem cell (hiPSC)-derived ventricular CMs differentiated for 30 days, and acute isolated adult mouse ventricular cells in ischemic simulated buffer. Action potentials (APs) were recorded by perforated whole cell current-clamp. Cytoplasmic and mitochondrial Ca2+ channel, cytoplasmic NCX and mitochondrial NCX. Spontaneous beating was modulated by SR Ca2+ release from RYR2 or inositol trisphosphate receptors (IP3R), the pacemaker current (If) and mitochondrial Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU). In RYR2 knockout mouse ESC-derived ventricular CMs, mitochondrial Ca2+ flux influenced spontaneous beating independently of the SR Ca2+ release from RYR2, and the mitochondrial effect was dependent on IP3R SR Ca2+ release. Depolarization of mitochondria and preservation of ATP could terminate spontaneous beating. A contribution of mitochondrial Ca2+ flux to automaticity was confirmed in hiPSC-derived ventricular CMs and ischemic adult mouse ventricular CMs, confirming the findings across species and cell maturity levels.In mouse ESC-derived ventricular CMs, spontaneous beating was dependent on the L-type Ca2+ uptake plays a modulatory role. Mitochondrial Ca2+ uptake through MCU is influenced by IP3R-dependent SR Ca2+ release.Mitochondrial and sarcolemma NCX fluxes are required for ventricular automaticity. Mitochondrial Ca This Ca2+ release activates the Na+/Ca2+ exchanger (NCX) to produce an inward current that can accelerate the rate of spontaneous membrane depolarization.2+ transients, mouse ESC-derived CMs and acutely isolated mouse ventricular CMs were loaded with Indo-1 AM  for 40 min followed by 20 min of de-esterification at room temperature[2+ transients were acquired at a rate of 1 kHz. Analysis of the signals was performed using IonWizard. Indo-1was excited at 405 nm and emitted fluorescence was collected at wavelengths of 470–490 nm. Selective experiments using Fluo-4 as the fluorescent reporter with mESC-derived cardiomyocytes showed comparable results to Indo-1 (data not shown). Fluo-4 AM (ThermoFisher Scientific), 3 μmol/L, was loaded in hiPSC-derived CMs for 20 min followed with 20 min de-esterification. The excitation wavelength was 488 nm and the absorption wavelength was 500 nm. Cells were transferred onto the stage of a real-time florescence microscope . Analysis of the signals was performed with the software MetaMorph . Cytoplasmic Ca2+ transients were presented as the ratio of intensities normalized by background fluorescence (F/F0).To visualize changes in the intracellular Camperature. The CMs2+ transients were monitored by loading cells with Rhod-2 AM , washing the cells twice, and allowing for de-esterification for 1 h in Rhod-2-free DMEM. In these measurements, the ruptured current-clamp technique was used with 20 μM EGTA added in the pipette solution. Minor cytosolic traces of Rhod-2 were eliminated by whole-cell dialysis by the pipette solution[2+ contaminate. Rhod-2 intensity was sampled at a rate of 1 kHz. Mitochondrial Ca2+ transients were presented as background-subtracted normalized fluorescence (F/F0).Mitochondrial Ca solution, 24. We 5’-GCCAGAGACAGACAAUACUtt-3’ and 3’-ttCGGUCUCUGUCUGUUAUGA-5’. siRNA-MCU2 targets nucleotides 360–378 of the corresponding mRNA (5’-GGGAAUUGACAGAGUUGCUtt-3’ and 3’-ttCCCUUAACUGUCUCAACGA-5’). The non-targeting siRNA (scrambled) was used as a control (5’-GCCUAAGAACGACAAAUCAtt-3’ and 3’-ttCGGAUUCUUGCUGUUUAGU-5’). Cells were transfected by Lipofectamine RNAiMAX reagent (ThermoFisher Scientific) for 72 h with a siRNA concentration of 30 pmol/L. MCU mRNA was measured after 72 h by qPCR. Single Cell-to-CTTM Kit (PN 4458237) and primers  were from ThermoFisher Scientific.To silence MCU, specific MCU siRNAs were synthesized by Life Technologies. siRNA-MAll data are presented as mean ± SE. The t-tests and Chi-tests were employed for statistical analysis. All experiments were carried out at 37°C.2+ oscillations. For example, RYR2 loss contributes to bradycardia, arrhythmia, and sudden death in adult mice[+ channel, L-type Ca2+ channel, T-type Ca2+ channel, HCN channel, K+ channel and NCX1[Ventricular spontaneous electrical activity or abnormal automaticity is thought to be a cause of arrhythmias, especially after ischemic insult. Automatdult mice. Major i and NCX1.2+ oscillations[2+ cycling modulates cardiac pacemaker and atrial cell automaticity. In pacemaker cells, inhibition of mitochondrial Ca2+ influx into (Ru360) or Ca2+ efflux from (CGP) mitochondria modulated the spontaneous AP frequency in opposing directions (111 ± 1% control or 89 ± 2% control), respectively. In atrial cells, NCLX knockdown using siRNA results in slowing of spontaneous electrical activity, an effect similar to that seen in pacemaker cells[2+ affect spontaneous activity and whether these mechanisms are similar in pacemaker, atrial, and ventricular cardiomyocytes are unknown.Mitochondria are known to participate in Caillations, 29. Mitker cells, 13. The2+ flux since blocking the L-type Ca2+ channel with 10 μmol/L nifedipine for 2 min or blocking the sarcolemmal NCX with the NCX1 specific blocker[2+ channel activity, it is unclear whether the L-type Ca2+ channel provides directly Ca2+ to the NCX or indirectly through the SR. Spontaneous beating was modulated by the pacemaker current through hyperpolarization-activated cyclic nucleotide (HCN)-gated channels, designated as If. The specific blocker, 10 μmol/L ivabradine[p<0.05, 2+ cycling in the modulation of spontaneous electrical activity[2+ release pathway through the RYR2, and by treating with 2 μmol/L 2-aminoethyl diphenylborinate (2-APB) for more than 15 min, which inhibited SR Ca2+ release pathway through IP3Rs. The spontaneous beating rates were greatly reduced to 54% and 71% by ryanodine and 2-APB, respectively (Wild type (Wt) ESC-derived ventricular-like CMs showed spontaneous beating . Spontanc blocker, KB-R794vabradine, had a m activity, the beaectively .2+ flux also influenced spontaneous beating in Wt CMs. Inhibiting mitochondrial Ca2+ uptake by intracellular application of a MCU-specific blocker, 1 μmol/L Ru360, had a similar effect on beating rate as inhibiting SR Ca2+ release from IP3Rs  CMs showed the expected reduction in spontaneous beating similar to the effect of ryanodine on Wt CMs , and Ru360 showed a similar effect to that in Wt CMs, reducing the spontaneous beating rate by 45.0%  was significantly reduced from 2.7 ± 0.6 to 1.3 ± 0.4 while the Ca2+ transient rise time constant substantially increased from 78.8 ± 19.5 to 155.0 ± 24.4 ms and the decay time constant increased from 129.8 ± 25.4 to 224.3 ± 31.7 ms. Basal mitochondrial Ca2+ (F/F0) was slightly decreased from 2.3 ± 0.3 to 1.7 ± 0.9  phenylhydrazone (FCCP), a protonophoric uncoupler, and oligomycin, an inhibitor of F1F0-ATP synthase[2+ transients and spontaneous activity  CMs showed a smaller AP amplitude, a shorter AP duration, a more depolarized minimum diastolic membrane potential, a smaller cytoplasmic Ca2+ transient amplitude, a slower rate of rise of the Ca2+ transient, and a slower Ca2+ decay rate. The basal Ca2+ concentrations were similar, however.The features of APs and cytoplasmic Cap<0.05, -/- ESC-derived CMs, the average beating rate was 1.39 ± 0.15 Hz and decreased to 0.52 ± 0.20 Hz after 2 μmol/L 2-APB was applied, suggesting that both SR Ca2+ release pathways are involved in generating spontaneous electrical activity. Cytoplasmic Ca2+ transients were similarly affected .The average effect of FCCP (w/wo oligomycin) on [Mg(PDF)Click here for additional data file.