P < .0001). The proportion of AML patients receiving chemotherapy and those of patients receiving transplants were significantly lower in the disabled population than that of nondisabled populations . This trend was more pronounced in subgroups of communication disability and major internal organ disorder. The median survival was 10.8 months for patients with disabilities, which was significantly shorter than 17.1 months for those without a disability (P = .002). Individuals with disabilities have a low diagnosis rate of AML and undergo less active treatment, which results in inferior prognosis.We analyzed the patterns of diagnosis, treatment, and prognoses of acute myeloid leukemia (AML) patients with and without disabilities. The data were collected from the National Disability Database, the Korean Central Cancer Registry, and the Korean National Health Insurance claim database. We built a cohort of 2 776 450 people with disabilities and a nondisabled cohort of 8 329 350 people who were selected at a ratio of 1:3 by matching age and sex. From this population, adult patients who were diagnosed with AML were analyzed. The number of patients with AML were 26.74 per 100 000 in people without disabilities and 20.39 per 100 000 in those with disabilities , undergo less active treatment, and have significantly lower survival rates than those without a disability. In addition to major organ dysfunction, other dysfunctions include perceptual degradation, communication problems, mobility limitation, and mental function problems. In clinical trials, inclusion criteria for study treatment generally include only the functions of major internal organs, such as cardiac, renal, and hepatic functions. However, even if physically healthy, disabilities of these patients indirectly affect cancer diagnosis and treatment. Some researchers reported that cancer screening is less commonly performed in individuals with than those without disabilities; treatment for cancer is also less active in disabled individuals.Socioeconomic problems also affect individuals with disabilities. Disabilities are commonly associated with unemployed status, low income, and poverty.Considering these facts overall, we assumed that people with disabilities are likely to have inferior outcomes compare to those without disabilities due to the inappropriate treatment AML in various ways, either due to the disability itself or due to the socioeconomic disadvantages associated with the disability. As few studies have examined how these dysfunctions affect AML diagnosis and treatment, we constructed a cohort of involving nearly two million disabled individuals using the disability registration system of the Republic of Korea and analyzed diagnosis rates, treatment patterns, and outcomes of AML. We also compared these data with those of patients without disabilities. Finally, we assessed the effects of the type and severity of disability on the AML clinical course.22.1We used three national databases; the Korean National Disability Database, Korean Central Cancer Registry (KCCR), and Korean National Health Insurance (KNHI) claims database. The National Disability Registry includes more than 90% of Korean individuals with disabilities and provides a variety of information including type and severity of disability.We compiled cases involving 2 776 450 people with disabilities using disability registration data. In addition, frequency‐matched random sampling was performed with the people without disabilities using the merged database mentioned above, grouped by age and sex at a ratio of 1:3 . We found individuals diagnosed with AML in both cohorts. Exclusion criteria included patients aged <19 years, previously diagnosed with other cancers, with no information regarding health insurance premiums, and those diagnosed with AML before 2002 (when the KNHI began collecting claims data).2.2We collected demographic data including age, sex, disease, treatment strategy, and outcomes from the KCCR database. Details of the disability in each case were sourced from the Korean National Disability Database.We classified disability types into four categories: physical, communication, intellectual, or psychological, and those affecting major internal organs Table . Disabil2.3P > .1 for all covariates). All statistical analyses were performed using SAS software ; differences were considered statistically significant when two‐sided P ≤.05 were found. The study protocol was approved by the Institutional Review Board of Chungbuk National University (CBNU‐201607‐BM‐288‐01).A chi‐squared test was used to determine discrepancies in diagnosis rate and treatment behavior according to various factors including age, sex, economic status, and type and severity of disability. The analysis of the patterns of transplantation therapy was limited to patients aged <65 years because KNHI ensures coverage for HSCT only in young patients. Survival outcomes and related risk factors were evaluated using the Kaplan‐Meier method with the log‐rank test and a multivariate Cox proportional hazards regression analysis. Before the analysis, the proportionality assumption was confirmed by plotting log hazard estimates against observation periods. Each covariate used for adjustment had Schoenfeld residual values indicating the proportional‐hazard assumption was fulfilled (33.1Baseline characteristics of the population cohort are described in Table 3.2P < .001). The percentage of AML patients in the lowest income group was higher in the people with disabilities than those without disabilities  had at least one disability. Patients with AML and disabilities had higher mean Charlson Comorbidity Index (CCI) scores compared to those in the control group . In addition, the diagnosis rate was lowest in those with disabilities of the major internal organs, followed by those with intellectual/psychological disabilities.The overall AML diagnosis rate was 25.15 per 100 000. The number of patients with diagnosed with AML was significantly lower in those with compare to those without disabilities , while the highest income group had the highest diagnosis rate (25.28 per 100 000) . The severity of the disability did not affect the chemotherapy rate. However, the rates of chemotherapy by disability type were significantly different. Patients with communication (61.9%) and major internal organ disabilities (64.7%) were less frequently treated for AML compare to those with physical (73.3%) or intellectual/psychological disabilities . The median value of anticancer drugs used for patients with communication disorders (2.61) was nonsignificantly lower than those of other disorders .Systemic chemotherapy for AML was administered to 75.9% of all patients. Patients with AML and disability had significantly lower rates of chemotherapy administration than those without disabilities . The rate of receiving allo‐HSCT was highest in those with a physical disability (18.1%), followed by those with a intellectual/psychological disability (17.2%) and a communication disability (15.6%). The rate of transplantation in patients with major internal organ disorders (12.5%) was lower compared to the proportion in the entire cohort.Among the 1243 patients who were younger than 65 years, allogeneic HSCT  was performed in 311 patients (25.0%). Among those without disabilities, 26.9% (268 patients) received allo‐HSCT, which was significantly higher than the 17.5% (43 patients) in those with disabilities (3.4P = .002). Disability severity was not associated with overall mortality, but the median OS was nonsignificantly shorter in those with a communication disability , fewer patients with disabilities underwent HSCT than did nondisabled patients. The survival rate of patients with AML and disabilities was also inferior compared to that with AML and without a disability.The diagnostic rate of AML in the present patients with disabilities was significantly lower than that in patients without a disability, and the greater the disability severity was, the greater this tendency also was. There are few previous studies of cancer incidence in individuals with disabilities. In a study of individuals with intellectual disabilities, the age‐standardized incidence of all cancers was not significantly different from that in such patients those without disabilities.For individual patients with disabilities, it could not be determined why their AML diagnosis was delayed. However, some hypotheses can be constructed based on the characteristics of individuals with disabilities. It is possible that people with disabilities did not adequately describe and/or explain the symptoms of AML due to their underlying conditions. This possibility may be especially significant for patients with intellectual/psychological and communication disabilities. People with disabilities also may have poor access to medical facilities that can provide professional care for AML. This is related to physical movement limitations, a lack of awareness of a hematologic malignancy, and a lack of a coping capacity. Finally, caregivers’ decisions may interfere with the rapid management of AML. The lives of individuals with disabilities are often influenced by the caregiver, especially for those with intellectual disabilities, where the disability is severe or rational thinking is difficult. If the caregiver is not aware of the possibility of a hematologic disease in individuals with disabilities, or is passive in management, an AML diagnosis may not be made in timely manner.It is noteworthy that the AML diagnosis rate of low‐income, nondisabled individuals is higher than the overall average, while the diagnosis rate of low‐income, disabled individuals is the lowest. In Korea, the Korea Health Insurance Corporation is responsible for all necessary medical expenses for those in the lowest income bracket; thus, the burden of the necessary costs associated with an AML diagnosis and subsequent treatment is greatly reduced. This helps reduce the risk that individuals with low incomes will not receive appropriate medical management for fatal diseases. Despite economic support, however, low incomes for people with disabilities were still a major barrier to an AML diagnosis. The current findings suggest that a policy of ensuring only the cost of medical care for people with disabilities is not sufficient to prevent missed AML diagnoses.Individuals with disabilities are also passive in their treatment of AML. Both the rate at which individuals with disabilities receive chemotherapy for AML and the number of anticancer drugs used were lower than in nondisabled individuals. In younger patients (<65 years), who could normally consider HSCT, the rate of performing HSCT was also low in patients with disabilities. Systemic chemotherapy and HSCT in AML management severely compromise the immune status of recipients and can lead to treatment‐related morbidities. Therefore, it is likely to have been difficult to undertake intensive treatments of individuals with disabilities in a poor overall condition. The treatment rate in the present study was lowest in those with major internal organ and communication disabilities, similar to a study of lung cancer treatments for individuals with disabilities.In addition to health status, nonmedical problems also affect the prognosis of AML patients with disabilities; economic problems of individuals with disabilities are also a suspected deterrent to providing adequate treatment for AML. Moreover, it is well known that a patient's economic situation is closely related to their prognosis of a malignant disease.The survival rate of AML patients with disabilities was lower than that of individuals without a disability. In addition to the underlying health problems of patients with disabilities, delayed diagnosis and passive treatment for AML may partially contribute to inferior outcomes. This is similar research results from lung cancer cases in patients with disabilities.Currently, there are various systems for the promotion of the rights and welfare of disabled people. These schemes include life support, rehabilitation, employment support, and vocational training for each type of disability. However, when people with disabilities have severe illnesses, they are more vulnerable than nondisabled people, but additional support policies are inadequate. For fatal diseases such as AML, it is absolutely crucial to recognize the onset immediately and to undertake an active treatment. Therefore, when people with disabilities are diagnosed with a serious disease such as AML, a system should be in place to provide basic economic support and to ensure the mobility of the patient, hospital visits, and long‐term nursing care. For example, a system should be in place to nurture health‐care professionals specialized in treating each type of disability and to allow them to be dedicated to persons with disabilities who are in need of medical treatment. In addition to disabled people, caregivers should also have the means to provide adequate education, economic, and emotional support.Our research has several limitations. First, this was a retrospective study using big data; it is not possible to obtain detailed personal information such as causes of disabilities, the characteristics of AML, or causes of death. Originally, the prognosis of AML depends on the characteristics of AML itself, such as the presence of a hematologic disease before the AML diagnosis, the subtype of AML, the presence/absence of chromosome abnormalities, and the mutation profile. Depending on these characteristics, the intensity of treatment may differ. Our study could not determine whether there was a difference in the disease characteristics of AML with or without a disability present due to methodological limitations. Thus, the poor prognosis of AML in people with disabilities is not entirely excluded from the possibility that it is due to the nature of the disease itself, not to external factors such as the timing of diagnosis or the intensity of treatment. Another limitation of our study was that we were unable to determine the type or intensity of the combination therapy because the treatment information was extracted from claims information for insurance benefits. As a result, the use of low‐intensity anticancer drugs for conventional purposes was considered here as a case of chemotherapy, and the proportion of people receiving chemotherapy may be higher than previously known. However, patients who are aged or in a poor general condition often do not use anticancer drugs at all; therefore, even if it is a temporary low‐intensity regimen, determining whether or not they have been prescribed may be appropriate for evaluating the aggressiveness of a treatment. In addition, this analysis did not include information about anticancer drugs or treatments not covered by medical insurance. Accordingly, care is needed when interpreting the results, as drugs provided through clinical trials and the effects of treatments paid by the patient are not included. Finally, due to data access limitations, we could not obtain information about the number of disabilities that each patient had. It is possible that more patients with multiple disabilities have more disadvantages with regard to an AML prognosis. In this study, only one major disability for each patient was analyzed. In order to establish AML treatment strategies that take into account the characteristics of disabled patients more accurately, further research based on more detailed patient classification techniques is necessary.Nonetheless, to the best of our knowledge, this is the first study to use data from a large national registry to analyze the AML diagnosis and treatment patterns of individuals with disabilities. A disability is an obstacle preventing proper treatment of AML for a variety of reasons; thus, efforts to overcome the adverse effects of a disability are necessary. In addition, it is essential to consider measures for economic problems and education, as well as social support for patients and caregivers. Further research is needed to develop public health policies for patients with AML and disabilities.None declared.Study design: Jihyun Kwon, So Young Kim and Jong Hyock Park; Investigation: Kyoung Eun Yeob, So Young Kim and Jong Hyock Park; Resources: Yeon‐Yong Kim, Jong Heon Park and Jong Hyock Park; Analysis and interpretation: Kyoung Eun Yeob; Writing of manuscript: Jihyun Kwon and So Young Kim; Review & Editing: Hye Sook Han, Ki Hyeong Lee, Dong Wook Shin, Jong Hyock Park and Ichiro Kawachi; Supervision: Jihyun Kwon, So Young Kim, Jong Hyock Park and Ichiro Kawachi.This article does not contain any studies with human participants or animals performed by any of the authors.Supplementary MaterialClick here for additional data file.


Because of the less well-developed T-tubular system in atrial compared to ventricular cardiomyocytes, spatial gradients in intracellular calcium concentrations may play a more significant role in atrial cardiomyocyte pathophysiology, rendering common-pool models less suitable for investigating underlying electrophysiological mechanisms. In this study, we developed a novel computational model of the rabbit atrial cardiomyocyte incorporating detailed compartmentalization of intracellular calcium dynamics, in addition to a description of membrane currents and intracellular processes. The spatial representation of calcium was based on dividing the intracellular space into eighteen different compartments in the transversal direction, each with separate systems for internal calcium storage and release, and tracking ionic fluxes between compartments in addition to the dynamics driven by membrane currents and calcium release. The model was parameterized employing a population-of-models approach using experimental data from different sources. The parameterization of this novel model resulted in a reduced population of models with inherent variability in calcium dynamics and electrophysiological properties, all of which fall within the range of observed experimental values. As such, the population of models may represent natural variability in cardiomyocyte electrophysiology or inherent uncertainty in the underlying experimental data. The ionic model population was also able to reproduce the U-shaped waveform observed in line-scans of triggered calcium waves in atrial cardiomyocytes, characteristic of the absence of T-tubules, resulting in a centripetal calcium wave due to subcellular calcium diffusion. This novel spatial model of the rabbit atrial cardiomyocyte can be used to integrate experimental findings, offering the potential to enhance our understanding of the pathophysiological role of calcium-handling abnormalities under diseased conditions, such as atrial fibrillation.Models of cardiac electrophysiology are widely used to supplement experimental results and to provide insight into mechanisms of cardiac function and pathology. The rabbit has been a particularly important animal model for studying mechanisms of atrial pathophysiology and atrial fibrillation, which has motivated the development of models for the rabbit atrial cardiomyocyte electrophysiology. Previously developed models include detailed representations of membrane currents and intracellular ionic concentrations, but these so-called “common-pool” models lack a spatially distributed description of the calcium handling system, which reflects the detailed ultrastructure likely found in cells  Mathematical models of cardiac electrophysiology (EP) have advanced significantly over the past decades, and are valuable tools for gaining physiological insight from the expanding pool of experimental data  at the cell periphery and subsequently propagates toward the center of the cell in a saltatory manner through diffusion and Ca2+-induced Ca2+ release  system  may contribute to alterations in radial Ca2+ flux. We then used the control population to (1) assess if changes in current conductances have an effect on Ca2+ propagation dynamics; (2) to quantify possible correlations between Ca2+ properties at the membrane and at the center of cell; and (3) to query the underlying mechanisms of the differences observed in the Ca2+ propagation patterns across the control population.In this paper, we describe the development of a novel model of the rabbit atrial CM with spatial description of ionic species and the CaThe rabbit atrial CM model was developed based on the previously published human atrial CM model by Voigt & Heijman  through an implementation of Ca2+ diffusion terms. Thus, a total of 18 domains represent a one-dimensional cross-section of the cell, where the two outermost domains (1 and 18) correspond to the region close to the cell membrane. These domains therefore include the sarcolemmal currents, Ca2+ release units (CRU), and Ca2+ buffers. In contrast, the inner domains (2–17) are not in direct contact with the cell membrane and contain only the CRUs and Ca2+ buffers. The inner domains contain a cytosolic space, the sarcoplasmic reticulum (SR), and a sub-SR space (SRS), while the membrane domains also include a subsarcolemmal (SL) space. In domains 1 and 18 the SRS represents a junctional space in which LTCC and the ryanodine receptors (RyR) interact, which is indicated with “junc.” This corresponds to the dyadic space in other CM models. We used a cell volume of 16 pL based on an estimated cell length of 130 μm  direction. This architecture allows simulating the radial flux of Ca2+ enters the cell only through the membrane domains, CaTs in the inner domains are dependent on diffusion from the periphery. Thus, the model allows to reproduce radial gradients and possible heterogeneities in CaTs arising from local control mechanisms. The model also includes implementations of both deterministic and a stochastic RyR models. However, for the sake of simplicity, we have employed the deterministic formulation of the RyR, and thus in our simulations only one segment was simulated, since segments are identical when using the deterministic model.Because in this model Ca2+ currents ICaL and ICaT; the fast Na+ current INa; repolarizing K+ currents Ito1, IKr, IKs, and IK1, as well as three background currents ICab, INab, and IClb. Additionally, the model includes the ionic currents of the Na+-Ca2+ exchanger (INCX); the Na+-K+ pump (INaK); and the plasmalemmal Ca2+ pump (ICaP). The total membrane current in the model is the sum of all these currents:The membrane model includes CaClb) from the Lindblad et al. model, since a chloride current has been reported in rabbit atrial CMs  for 12 s (6 beats), and then caffeine application was simulated by increasing RyR2 activation to 1.0 over the course of 10 ms, and keeping the channels fully open for another 10 s. The effect of the caffeine was then removed by unclamping the RyR activation for 3 s. This protocol is illustrated in NCX closely matched experimental data in Greiser et al. , and bulk cytosolic spaces, but the cytosolic Na+ trace is common to all domains, so that only this is updated in the inner domains. The concentrations and fluxes of Ca2+ and Na+ across the different model compartments in a membrane domain are schematically shown in 2+ can be found in the supplementary material of Voigt et al. . The source code of the model is available at 90) was 143 ms, which is longer than the reported 120 ms, and the resting membrane potential (RMP) was around −74 mV as compared to the −80 mV reported in literature. Additionally, the CaT amplitude (CaT-A) in the baseline model was only about 0.09 μM, compared to the ~1.0 μM amplitude in simulated CaTs by Lindblad et al.  that was morphologically similar to experimentally measured APs, but did not match in terms of quantifiable metrics. The action potential duration at 90% repolarization .90 values of 93 and 103 ms, respectively, while Yamashita et al.  has been reported at around 100 mV  in the membrane domains (domains #1 and #18), and in the central domains (domains #9 and #10). The membrane and central CaTs are denoted as CaTm and CaTc, respectively. When using deterministic RyR formulations, the model is symmetric about the cell center, resulting in identical CaTs in domains 1 and 18, as well as in domains 9 and 10. Whole-cell CaTs were also obtained as the mean bulk cytosolic CaTs of the 18 domains.The primary focus of this paper is on Cam and CaTc traces at steady state (20 min pacing). Specifically, we analyzed the rise time (measured from onset of the CaT to peak), duration at 50% decay (CD50), and amplitude, in addition to the time difference between peaks of CaTc and CaTm, as measured from AP onset (CaTdelay), represented schematically in 2+ metrics presented were again determined as the average of the values calculated for the last 5 beats.Additionally, we extracted and analyzed a number of relevant metrics that characterize CaTSR and [Na+]i traces to analyse in relation to the other CaT metrics. In our analyses, we determined CaSR and [Na+]i as the mean value during each beat, with CaSR corresponding to the average over all cell domains.We additionally extracted Ca2+ metrics defined above, as well as correlations between the Ca2+ metrics in the membrane domains and in the central domains, and between CaSR and [Na+]i and the central Ca2+ metrics. The correlations were determined using Kendall's tau (τ), since the variables followed discrete distributions. Significance was determined with 95% confidence, and all data analyses were performed in Matlab R2017a employing custom routines.Finally, we performed correlation analysis to identify significant correlations between selected model parameters and output variables. We analyzed the correlations between the maximum ion channel conductances and the Ca90, APD40, and CaT-A was responsible for excluding the majority of models, with 175 models satisfying the requirements for these three parameters. Mean and standard deviation of APD90, APD40, APA, RMP, CaT-A, [Na+]i, and CaT rise time for the original and control populations are shown in Varying the maximum conductances between 25 and 400% resulted in an initial population of 3,000 models with a large degree of variation in AP and CaT properties. Calibration of the population by constraining output values to the ranges in CaL, Na, GKr, GKs, and GClb). The reduced variability in these six parameters was found to be significant , and the distributions are illustrated in The APs and CaTs of the control population are shown in m and CaTc traces of the control population. As expected, the Ca2+ signals at these two different locations showed differences in morphology, resulting from the differences in the underlying mechanisms driving the signal. The membrane signal CaTm results from a combination of Ca2+ entering the cell via ICaL and Ca2+ released from junctional CRUs, while the central signal CaTc is the Ca2+ released from non-junctional CRUs via CICR as a result of Ca2+ diffusing from neighboring domains. The CaTc is therefore initiated with a time delay in comparison to CaTm, which corresponds to the diffusion time of Ca2+ from the periphery to the central regions of the cell.2+ metrics for 16 models are shown in m and CaTc traces , with no significant differences in the amplitudes of CaTm and CaTc, which is consistent with findings in control atrial CMs  and a longer CD50 than CaTm. Furthermore, CaTdelay in the population was 42 ± 12 ms, which matches the 52 ms time delay measured from line scans of rabbit atrial CMs , [Na+]i showed a large degree of variation among models.The values of the Ca2+ wave propagation patterns with physiological characteristics under normal conditions, including full regenerative propagation, delayed CaTc, and steady CaTs over time. However, despite the similarities there were also measurable differences in the propagation patterns, as characterized by the Ca2+ metrics defined above and by plots of the spatio-temporal Ca2+ dynamics. Three representative Ca2+ wave propagation patterns  are highlighted in bold. Our analyses reveal that the Ca2+ wave properties of these 16 models were primarily sensitive to GCaL, 2+ channel conductance GCaL showed a negative correlation with CD50m, but not with CaTAm and CaTAc. m and CD50m, which is the expected given role of INCX in Ca2+ extrusion. m, possibly because larger CaTs increase NCX activity. However, delay. Finally, we found c and CD50c, indicating that a larger INaK current resulted in a smaller CaTc signal.We next analyzed the correlations between the maximum conductances of individual currents and the Ca2+ metrics, [Na+]i, and CaSR are compiled in p-value < 0.05) highlighted in bold. We observe that CaTdelay which was not sensitive to any of the ion current conductances, was strongly correlated with CaSR. We also found [Na+]i to be correlated with tpeakm, CaTAm, and CD50c. Furthermore, we observe that the individual Ca2+ metrics related to CaTm and CaTc were in general not correlated, except for CaTAm which was correlated with tpeakc, and CD50c, while CaTdelay was strongly correlated with all CaTc properties, tpeakc, CaTAc, and CD50c. The three Ca2+ metrics trise, CD50, and amplitude, were correlated for both CaTm and CaTc, as expected (data not shown).The correlation among the seven Ca2+ dynamics. The central motivation for developing the model was to be able to describe radial diffusion of calcium, which is important for the investigation of the effects of asynchronous Ca2+ release on arrhythmic activity in atrial CMs lacking TTs. We used a population-of-models approach to parameterize the maximum conductances of sarcolemmal ion currents to produce a pool of models that matched reported experimental data from the rabbit atria. The result was a population of 16 models that were all consistent with observed experimental values, but still recapitulated observed variability in Ca2+ wave characteristics.We have developed a model of a healthy atrial CM with rabbit-specific EP and spatially distributed Ca2+ metrics used in the model selection step were, in general, sensitive to these maximum conductances.The small number of models selected by the experimental data shows that imposing a sufficiently large number of constraints in model outputs  can reduce the parameter space to discrete sets of parameterizations, each following a unique trajectory. This shows that rather different parameter combinations can result in models with very similar behaviors see . NonetheIt is worth noting that the data used here to constrain the model were obtained from a large assortment of published experimental data. Therefore, the 16 models of the control population reflect not only the natural variability observed within different atrial regions, but also experimental uncertainties inherent to methodologies used by different research groups. The population-of-models-based approach we used here contrasts with the more standard approach, wherein a computational model is fitted to a small set of experimental observations, often obtained from a single atrial region, to yield a single model parameterization that captures the average behavior in the experimental data. Although useful for assessing the mechanisms underlying general characteristic behaviors of the model, the single-parameter approach lacks the ability to reproduce experimental observations from a range of data. In contrast, incorporating variability into the model via the population-of-models-based approach employed here allows a generalization of model results to a wider set of conditions and phenotypes.2+ metrics from CaTm and CaTc quantify differences in the Ca2+ wave properties across the 16 models. Correlation analysis showed that GCaL and 2+ wave propagation in the model. This result is consistent with the known role of these ionic currents in intracellular Ca2+ regulation. For instance, experimental observations have shown the role of increased sarcolemmal Ca2+ in modulating the regenerative propagation of the Ca2+ signal toward the inner locations of the cell , and thus reduce CaTdelay, while simultaneously promoting a longer CaT duration (larger CD50c). The observed correlation between Ca2+ metrics and [Na+]i is also expected since [Na+]i plays a significant role in the modulation of NCX function, and therefore in the regulation of the sub-sarcolemmal Ca2+ signal , in turn, uses a bi-directional Hill equation with affinity for cytosolic and SR Ca2+, although the formulation used here (from the Voigt & Heijman model) had been adjusted to human atrial CM data. Additionally, Ca2+-release in Hilgemann and Noble , and down-regulation of Serca (together with increased [Na+]i), with alternans amplitude increasing with increasing percentage of TT disruption or [Na+]i. This study emphasized not only the role of the spatial distribution of LTCCs in the generation of alternans, and thus the importance of considering spatial heterogeneity of Ca2+ cycling, but also the role of regulating [Na+]i on abnormal intracellular Ca2+ propagation.Another important consideration is the effect of TTs on atrial CM Caa et al.  studied 2+ cycling with variable TT density, showing that decreased TT density correlated with increased propensity for Ca2+ alternans. Their simulations with patchy TTs also showed observable spatial Ca2+ gradients leading to the formation of intracellular Ca2+ waves at the sites with no TTs and of delayed after depolarizations. In a similar study, Song et al.  and to (3.4 to 16.6 pA/pF) are approximately within ±50% of the experimental values  as compared to the experimentally reported value of −70 pA/pF. We note, however, that peak dynamic INa we obtained from the Aslanidi model was −120 pA/pF. Peak IKr values (0.45–2.1 pA/pF) in our population were also in general considerably larger than the reported experimental value of 0.7 pA/pF (Muraki et al., Kr in the Aslanidi model was 0.43 pA/pF, which is close to the experimental value.Given the wide range of variation of the maximum conductances in the control population see , and thei et al. , and Muri et al. , as wellWe also note that variability in experimental recordings of ion currents is typically very large, and ideally this should have been taken into account in the validation of the population. Nevertheless, there are observable discrepancies between peak currents in our control population and in experiments, which are limitations of the model, and we recognize that these should be further investigated. Future iterations in the development of the control population should include fine-tuning of the maximum conductances of the 16 models in order to produce a control population that better matches experimental data.2+ wave propagation dynamics. Future work could also address the effects of AF-induced remodeling on Ca2+ wave propagation, and in particular look at the role of RyRs and Serca2a parameters in abnormal behaviors, such as failed Ca2+ propagation, Ca2+ alternans, and afterdepolarizations.Finally, a relevant model improvement would be to include the calcium-dependent component of the chloride current, which has been shown to be involved in APD alternans in rabbit atria (Kanaporis and Blatter, 2+ wave propagation patterns. These differences were directly linked to two Ca2+ currents, ICaL, and INCX. However, the study also showed the Ca2+ wave patterns to be a complex interplay among different components, including CaSR and [Na+]i.This paper presented a novel model of the rabbit atrial CMs, and provides a framework for analysing cardiac cell models based on correlation analysis. We have shown that the model is able to reproduce experimentally observed physiological Ca2+ description in the model, along with the methodology presented here can be used as a tool to study sub-cellular mechanisms, and their implication in the arrhythmogenesis in diseased condition, such as atrial fibrillation. This work can therefore be extended to assess such mechanisms under altered conditions, such as electrical remodeling. In particular, the framework can be useful for querying the drivers of arrhythmogenic Ca2+ cycling, such as Ca2+ alternans, and to formulate hypothesis on new targets to restore normal cell function. In conclusion, the study provides a population of spatial models of the rabbit atrial cardiomyocyte that can serve as a starting point for future studies employing the commonly used experimental model.The spatial CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MV, JH, and US conceived the study. MV performed the computational simulations and data analysis, and drafted the manuscript. HA, JS, and JH provided the critical revision to the manuscript. All authors 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.


In particular, the Ca2+-mediated communication between the ER and mitochondria plays a crucial role in paraptosis. Mitochondrial Ca2+ overload from the intracellular Ca2+-flux system located at the ER–mitochondrial axis can induce mitochondrial dilation during paraptosis, while the accumulation of misfolded proteins within the ER lumen is believed to exert an osmotic force and draw water from the cytoplasm to distend the ER lumen. In this process, Ca2+ release from the ER also critically contributes to aggravating ER stress and ER dilation. This review focuses on the role of Ca2+ transport in paraptosis by summarizing the recent findings related to the actions of Ca2+-modulating paraptosis-inducing agents and discussing the potential cancer therapeutic strategies that may effectively induce paraptosis via Ca2+ signaling.Paraptosis is a type of programmed cell death that is characterized by dilation of the endoplasmic reticulum (ER) and/or mitochondria. Since paraptosis is morphologically and biochemically different from apoptosis, understanding its regulatory mechanisms may provide a novel therapeutic strategy in malignant cancer cells that have proven resistant to conventional pro-apoptotic treatments. Relatively little is known about the molecular basis of paraptosis, but perturbations of cellular proteostasis and ion homeostasis appear to critically contribute to the process. Ca Paraptoor cells . In addior cells . Paraptoor cells , 2004. Achondria . Paraptochondria , suggestchondria . The actchondria , whereaschondria . The terchondria . Recentlhemicals . Here we2+ homeostasis due to their ability to store Ca2+ and respond to cytosolic Ca2+ signals. Under normal physiological conditions, the concentrations of both cytosolic Ca2+ and Ca2+ stored within the ER lumen are strictly regulated. The cytosolic Ca2+ concentration [(Ca2+)c] is approximately 0.1 μM, compared to an extracellular (Ca2+) of ∼1 mM and an ER Ca2+ concentration [(Ca2+)er] of ∼0.5 mM (2+ into the ER is governed by sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps  and  inositol 1,4,5,-triphosphate receptors (IP3Rs) (2+ by sequestering and releasing Ca2+. The Ca2+ concentration (Ca2+) inside the mitochondria has similar values measured in the bulk cytoplasm (0.1–0.2 μM) under resting conditions; however, mitochondrial Ca2+ concentration increased 10—20-fold more than the cytosolic compartment during stimulation with (Ca2+)-increasing agents  mainly through the voltage-dependent anion channel (VDAC)  mainly through the mitochondrial Ca2+ uniporter (MCU) complex  and the mitochondrial H+/Ca2+ exchanger (mHCX) play major roles in mitochondrial Ca2+ efflux mechanisms. The stoichiometry of mNCX-driven transport is electrogenic, with three (or four) Na+ for one Ca2+ (+ for one Ca2+) (2+ efflux pathway under certain conditions when the mPTP is transiently opened (The ER and mitochondria both participate in regulating intracellular Ca ∼0.5 mM . The impA) pumps . In resp (IP3Rs) ; the lat (IP3Rs) . The mitg agents . In partl (VDAC) . After r complex . This MC complex  and esse complex ; the dom complex , and the complex . The act complex  and its  complex ; they totrations . Therefoone Ca2+ , whereasne Ca2+) . The mity opened .2+ homeostasis at the ER by altering Ca2+ store content and Ca2+ dynamics  and/or at the mitochondria by affecting the activities of MCU complex components to regulate the mitochondrial Ca2+ levels. In addition, recent studies have shown that treatment of cancer cells with drugs that perturb Ca2+ homeostasis [CGP-37157 , which further impair proteasome activity. This proteasome-inhibiting activity of celastrol may stabilize IP3R and MCU to reinforce the Ca2+-mediated effects of celastrol, leading to ER stress, ER vacuolation, and subsequent cell death  in these cells. These morusin-induced mitochondrial Ca2+ influx, ROS generation, MMP depletion, cytoplasmic vacuolization, and cell death were suppressed by a pretreatment with 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of anion channels, including VDAC  of this agent in these cells. Pretreatment with the VDAC inhibitor, DIDS, significantly attenuated the effects of δ-TT on cell viability (paraptosis and apoptosis), suggesting that mitochondrial Ca2+ overload is critical for the anticancer activity of δ-TT in CRPC cells (δ-Tocotrienol (d-TT) induces paraptosis together with autophagy and apoptosis in castration resistance prostate cancer (CRPC) cells. d-TT-induced paraptosis was demonstrated by the reported dilation of ER cisternae and swollen and damaged mitochondria with disintegrated cristae . δ-TT-inPC cells .2+ and the activation of the Ca2+-activated protease, calpain, preceded CMR-induced cytoplasmic vacuolation, and a pretreatment with the intracellular Ca2+ chelator, BAPTA-AM, blocked CMR-induced cytoplasmic vacuolation. These findings together suggest that ROS-mediated impairment of MMP and Ca2+ homeostasis may contribute to CMR-induced paraptotic cell death , one of the major secondary metabolites found in fungus-infected mulberry leaves, induces mitophagy and a paraptosis that is accompanied by extensive cytoplasmic vacuolation originated from the ER in PC-3 and MDA-MB-231 cells . In thisll death .2+ mobilization, was involved in the F2-mediated cell death of U-87 cell (Oligomeric procyanidins (F2), which are abundant in grape seeds, can induce paraptosis in U-87 cells . Apoptot-87 cell .Bacillus subtillis were reported to simultaneously induce paraptosis, apoptosis, and autophagy in heterogeneous human epithelial colorectal adenocarcinoma (Caco-2) cells  cells . Inductiin cells .2+ from the ER to the mitochondria and induced the subsequent loss of MMP (ΔΨm), resulting in vacuolization of the cytoplasm and intracellular organelles . The resultant disruption of thiol redox homeostasis in the ER, in turn, activates protein kinase R (PKR)-like endoplasmic reticulum kinase signaling and the release of Ca2+ ions from the ER. This prolonged Ca2+ imbalance triggers the swelling of the ER and the mitochondria as well as mitochondrial membrane depolarization, leading to cell death , a third-generation 1,4-dihydropyridine (DHP) used to treat high blood pressure, potentiates the antitumor effects of various PIs  in many solid cancer cell lines by inducing paraptosis, a cell death mode accompanied by extensive vacuolation derived from the ER and the mitochondria . Ler enh cytosol .2+ levels. An increase in Ca2+ (possibly by an influx of extracellular Ca2+) appears to play a critical role in the anticancer effects of Lop/Btz by affecting ER-derived vacuolation. Mitochondrial Ca2+ overload also contributes to Lop/Btz-mediated cytotoxicity, although it does not affect the dilations of the ER and the mitochondria  induced by curcumin /glucose-regulated protein 78 (GRP78), PDI, GRP94, and ERp57/PDIA3, are regulated by the binding of Ca2+  was shown to be involved in the paraptosis induced by a purified resin glycoside fraction (RFP) of Pharbitidis Semen (In addition to the disruption of Cauolation . Conversuolation . These ais Semen . BlockadThe inherent or acquired resistance of tumor cells to apoptosis is a major hurdle limiting successful anticancer therapy and the cause of many cancer-related deaths . Therefo2+ overload was reported to be able to induce apoptosis or necrosis, depending on the availability of ATP (2+ imbalance and the integrity of mitochondrial membrane during the progression of paraptosis will be helpful to clearly understand paraptosis and clarify the differences among various cell death modes accompanied by mitochondrial Ca2+ overload and swelling.Although various models have been proposed to classify programmed cell death, exclusive definitions are difficult to conceptualize and probably factitious, given the crosstalk between cell death signaling pathway . In addiy of ATP . In apopy of ATP . Althougy of ATP  and/or Ay of ATP  into they of ATP . Mitochoy of ATP . Howevery of ATP . MMP wasy of ATP . Reducedy of ATP . Therefo2NNMe2 (While curcumin , celastr2NNMe2  induce p2NNMe2 , 2020a, 2NNMe2 , and itu2NNMe2 , induce 2NNMe2 . The dom2NNMe2 . Further2NNMe2 . We now 2NNMe2 . Thus, t2+ imbalance-mediated paraptosis. Interestingly, these Ca2+ homeostasis-disrupting drugs also induced proteostatic impairment, leading to the accumulation of poly-ubiquitinated proteins and ER stress marker proteins. Genetic or pharmacological inhibition of Ca2+ imbalance effectively blocked this proteostatic impairment and subsequent paraptosis (2+ homeostasis may negatively affect cellular proteostasis and enhance proteotoxicity, contributing to paraptosis. Pretreatment with CHX almost completely inhibited Ca2+ release from the ER, mitochondrial Ca2+ overload, disruption of proteostasis, and subsequent paraptotic events (2+ imbalance and proteostatic disruption are interdependently regulated during the progression of paraptosis. In addition, simultaneous disruption of Ca2+ homeostasis and proteostasis, rather than induction of Ca2+ imbalance or proteostatic disruption alone, may be an effective means to kill cancer cells by inducing paraptosis (Although the molecular basis of paraptosis needs further investigation, many reports have indicated that impaired proteostasis is critical for the induction of paraptosis, as evidenced by the accumulation of poly-ubiquitinated proteins and ER stress marker proteins as well as the ability of the protein synthesis blocker to effectively inhibit vacuolation and cell death . As showraptosis , 2014. Tc events . Taken traptosis .2+ homeostasis-perturbing drugs, such as lercanidipine, loperamide, and CGP-37157, act together with PIs to induce paraptosis (2+ homeostasis-perturbing drugs alone slightly and transiently induced mitochondrial dilation without dilation of the ER, whereas treatment with the PIs alone was not enough to induce paraptosis in these studies. It is possible that either Ca2+ imbalance or proteasome inhibition may trigger cellular adaptive responses, including the autophagy, UPR, and reactivation of the ubiquitin–proteasome system, contributing to the restoration of cellular homeostasis and viability (3R (2+ channels may induce the enhanced and sustained release of Ca2+ from the ER in cells treated with these Ca2+ homeostasis-perturbing drugs, contributing to the effective induction of paraptosis. A combination of Ca2+ homeostasis-perturbing drugs and PIs may therefore effectively induce paraptosis by triggering a vicious cycle of ER–mitochondria Ca2+ signaling. Furthermore, the combined regimen of a PI plus Ca2+-modulating clinical drugs may provide us many advantages, including enhanced therapeutic effectiveness, better safety due to the reduced side effects of a PI by the use of its lower dose, and the potential to expand the applicability of PIs to solid tumors (2+ signaling may provide a novel therapeutic strategy to overcome the resistance of solid tumors against PIs in the future. It will be intriguing to explore the processes/regulatory pathways underlying the differences between ER and mitochondrial Ca2+ transfer in cancer cells vs. normal cells under paraptosis-inducing conditions. Further investigation of the regulatory mechanisms of Ca2+ in paraptosis may provide new insights into paraptosis and potential therapeutic strategies for the treatment of resistant cancer.Btz, the first PI approved by FDA for clinical application, has shown efficacy in both front-line and relapsed/refractory settings, but its use can be limited by the development of resistance and/or side effects. The principal dose-limiting toxic effects of Btz are peripheral neuropathy, thrombocytopenia, neutropenia, fatigue, and diarrhea . In addiraptosis . Treatmeiability . Additiolity (3R  and RyR lity (3R ; thus, td tumors . TherefoEK and KSC wrote the manuscript. KSC supervised the preparation of the manuscript. DML created the figures. MJS and HJL created the table. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intracellular calcium dynamics play essential roles in the proper functioning of cellular activities. It is a well known important chemosensing and mechanosensing process regulated by the spatio-temporal microenvironment. Nevertheless, how spatio-temporal biochemical and biomechanical stimuli affect calcium dynamics is not fully understood and the underlying regulation mechanism remains missing. Herein, based on a developed microfluidic generator of biochemical and biomechanical signals, we theoretically analyzed the generation of spatio-temporal ATP and shear stress signals within the microfluidic platform and investigated the effect of spatial combination of ATP and shear stress stimuli on the intracellular calcium dynamics. The simulation results demonstrate the capacity and flexibility of the microfluidic system in generating spatio-temporal ATP and shear stress. Along the transverse direction of the microchannel, dynamic ATP signals of distinct amplitudes coupled with identical shear stress are created, which induce the spatio-temporal diversity in calcium responses. Interestingly, to the multiple combinations of stimuli, the intracellular calcium dynamics reveal two main modes: unimodal and oscillatory modes, showing significant dependence on the features of the spatio-temporal ATP and shear stress stimuli. The present study provides essential information for controlling calcium dynamics by regulating spatio-temporal biochemical and biomechanical stimuli, which shows the potential in directing cellular activities and understanding the occurrence and development of disease. Upon stimulation of these physiochemical signals, the modulating signals are encoded in the complex spatio-temporal dynamics of cytosolic Ca2+ concentration, ranging from calcium spiking and oscillations to complex Ca2+ waves, which ultimately lead to the diversity and specificity of cellular behaviors as well as dysfunctions [2+ dynamics in vascular endothelial cells (VECs) [2+ microdomains and Ca2+ waves. Unimodal and oscillatory Ca2+ responses can respectively contribute to cell migration and cell proliferation [2+ responses by regulating spatio-temporal physiochemical stimuli is crucial for ensuring the proper functioning of cellular activities and biological processes.Calcium ion Ca+ is a hipoptosis ,4,5,6. Cocessing ,8,9. In unctions ,13,14,15s (VECs) ,19,20,212+ signal transduction pathways and corresponding regulation mechanisms have been carried out. Due to the complexity of human body and the existence of various interference factors, the quantitative characterization of Ca2+ responses in vivo is challenging. Therefore, a lot of efforts were made in vitro to mimic the spatio-temporal physiochemical extracellular microenvironment, and to probe Ca2+ responses induced by the spatial-temporal physiochemical stimuli. Recently, with the developments in microfabrication and microfluidic techniques, the microfluidic system has become an ideal platform for quantitatively probing Ca2+ dynamics in response to biochemical and biomechanical stimuli [2+ dynamics through the stimulation of extracellular biochemical  or biom factors , electro factors  and opti factors ) signalso et al.  and Jafao et al.  demonstrt levels ,31. Howet levels ,33.2+ signaling pathways and making substantial predictions. Considering diverse Ca2+ signaling pathways and extracellular physiochemical stimuli, the multiple Ca2+ signal patterns observed at the single cell level have been finely modeled, including calcium spikes, regular oscillatory, and complex waves [2+ responses to continuous and pulsed biochemical stimulations in both the oscillatory (low ligand concentration) and acute (high ligand concentration) regimes have been analyzed. The Ca2+ responses synchronized to periodic stimuli (phase-locking feature) have also been theoretically proved, as well as the dependence of Ca2+ oscillatory frequency on stimulation concentrations [2+ signaling model elicited by the activations of both ATP and shear stresses. The results reveal the fidelity of Ca2+ responses to stepwise increasing shear stress combined with ATP stimuli. The frequency of Ca2+ oscillatory increases with the increase in ATP concentration. These theoretical studies provide new insights into Ca2+ signaling pathways and the corresponding regulation factors. Nevertheless, based on our knowledge, the existing models at the single cell level focus on Ca2+ dynamics regulated by temporal physiochemical signals, the spatial characteristics of extracellular stimuli are often ignored. In addition, little information exists on how spatio-temporal and amplitude varying extracellular stimuli modulate Ca2+ response, and how Ca2+ signals encode the frequency and amplitude information of modulating signals are not clear.Along with experimental approaches, theoretical modelings have been a valuable method for understanding Caex waves ,41,42,43trations ,34,35. Qtrations ,37,38 ha2+ responses in VECs. ATP and shear stress, the important and ubiquitous extracellular stimuli of VECs [2+ dynamics induced by both ATP and shear stress stimuli. By considering the transmission characteristics of microchannels, the capacity of the microfluidic generator and the transmission of ATP signal within the microchannel are analyzed. Exposed to the transmitted spatio-temporal ATP and shear stress stimuli, the intracellular Ca2+ dynamics of adherently cultured cells are theoretically investigated. The influence of spatio-temporal characteristics of ATP and shear stress stimuli on the intracellular Ca2+ responses are discussed. Consequently, this study provides crucial information needed to regulate intracellular Ca2+ dynamics for further controlling cellular functions and biological activities.In the present study, we construct a theoretical model to investigate the effects of spatio-temporal coupling biochemical and biomechanical signals on the intracellular Ca of VECs ,27,37,442+ dynamics) in response to spatio-temporal ATP and shear stress signals. It should be noted that the theoretical modeling is nevertheless developed based on this proposed microfluidic system, the modeling situation can be generally applicable to universal microfluidic systems [In this study, the theoretical modeling is established based on a developed microfluidic generator of biochemical and biomechanical signals for probing cellular activities ,46 Figu. The mic systems ,25,48,49Based on the proposed microfluidic system, an initial linear ATP concentration profile is introduced into Inlet-Y1 at a constant flow rate te Q2(t) . Within D is the diffusivity of ATP molecule and Simultaneously, the soluble ATP molecules are convected and diffused with the flow, wherein the concentration of diffusible ATP Based on the quasi-steady assumption , the sheBy considering the time-dependent Taylor–Aris dispersion and taking the average along heightwise, the governing equation of ATP concentration (Equation ) can be According to the experimental conditions, the boundary conditions of Equation  are deri2+ dynamics of adherently cultured VECs is theoretically modeled. As shown in 2+ signaling pathways through P2Y receptors and P2X4 Ca2+ channels (indirect mechanism) [2+ signaling induced by shear stress through TRPV4 and/or TRPV4-C1 compound channels in VECs (direct mechanism) [2+ change is negligible due to the minimal effect. According to these conditions, intracellular calcium homeostasis is maintained by balancing multiple Ca2+ signaling cues, including the Ca2+ release from internal calcium stores to the cytosol, the Ca2+ restore into calcium stores from the cytosol, the Ca2+ inflow from extracellular fluid into the cytosol, the extrusion and exchange of Ca2+ to the extracellular environment, and the Ca2+ combined with soluble cytosolic proteins. The dynamic of cytosolic-free calcium ion can be thus described asC is the cytosolic-free Ca2+ concentration, 2+ from the calcium stores, 2+ back into calcium stores from the cytoplasm, 2+ inflow through Ca2+ channels, 2+ outflow to the extracellular environment refer to the Ca2+ clearance mechanism, and 2+ by soluble cytosolic proteins.Induced by the spatio-temporal extracellular ATP and shear stress see , the intchanism) ,38 but achanism) . Extracei is the concentration of IP3, an intracellular signaling mediator. 2+ concentration in the calcium stores, and 2+. 2+-induced Ca2+ release (CICR) mechanism. 2+ buffering sites on proteins in the cytosol. 2+, 2+ efflux of the Ca2+ clearance mechanism.Based on the existing models ,39,40,51Equation  are as f2+ in calcium stores and buffering proteins are governed bySimilarly, the balance of Ca2+ signaling pathways, such as the synthesis of IP3 and the Ca2+ influx through Ca2+ channels. Among these processes, it is well known that the concentration of IP3 as a second messenger is dependent on the extracellular ATP concentration, the relationship can be expressed by [3, respectively. As shear flow with soluble ATP molecules are transported to cells cultured at the bottom of the microchannel see , cells cessed by ,51,52. Therefore, 2+ in the no-load case. 2+ inflow rate through the TRP channels. The TRPVC4 and TRPC1 channels are both mechanosensitive, which can be directly activated by shear stress. For ease of reference, the expressions of Ca2+ inflow through TRPV4 and TRPC1 channels are given by the following form [2+ inflow when all channels are open. The parameter 4 channels induced by shear stress, which is expressed as [2+ channels, k is the Boltzmann constant, T is the temperature, and N is the Ca2+ channels density per unit area. l is the length of the cell in the flow direction, and Except for ATP-gated P2XEquation , is maining form :(20)qin_essed as (21)p1=14-C1 channels activated by the binding of three IP3 molecules to IP3R type 3 in the membrane of the Ca2+ stores, which is presented asThe parameter 4-C1 channels decayed by intracellular Ca2+ concentration, which satisfies the following exponential function,The parameter 2+ dynamics model are sampled similarly from the previous models [2+ dynamics, where the system of ordinary differential equations (ODEs) is solved with the stiff solver ode15s.Dimensions of the microfluidic chip and default parameters used in the sub-model of extracellular ATP transport are listed in s models ,51. All The generation of spatio-temporal ATP in the microchannel is demonstrated in ter ∼4 s c, showinter ∼4 s a. Howeveter ∼4 s a, pulsatlitude δ b. For a litude δ b. It shoExcept for the spatio-temporal ATP signals, within the microchannel, a pulsatile shear stress originated from the pulsatile flow is uniformly distributed simultaneously. The shear stress is proportional to the total flow rate Equation , thus it2+ dynamics induced by the spatio-temporal ATP and shear stress signals, the proposed model of Ca2+ dynamics is firstly validated by comparing with the existing experimental results [2+ initially increases sharply (around 25 s) and then oscillates for a few minutes with a decreasing plateau level and oscillatory amplitude. The Ca2+ oscillation is subject to the extracellular sinusoidal ATP stimulus and varies at the same frequency (∼0.1 Hz) as the ATP waveform. Comparing the modeling and experimental results, the differences in plateau levels and oscillatory amplitudes (after 100 s) may be caused by the nonlinear relation between Ca2+ and fluorescence intensity [2+ concentration predicted by the model . Therefore, at a high ATP level, a weak pulsation of ATP has no influence on cytosolic Ca2+, the oscillations is dominated by pulsatile shear stress. With moderate ATP stimulus of high pulsatile amplitude, both pulsatile ATP and shear stress contribute to Ca2+ oscillations, leading to synchronous changes in Ca2+ responses at the pulsatile frequency of ATP and shear stress. When the ATP stimulus reaches a low level or no ATP stimulus, Ca2+ oscillations emerge at the minimal level . Theref2+ dynamics have been unexpectedly observed  can modulate the distinct modes of Ca2+. As reported in previous studies, unimodal Ca2+ responses and Ca2+ oscillatory can accordingly regulate and promote cell migration, cell proliferation, and tube formation, critical phases involved in angiogenesis and vasculogenesis [2+ responses by spatio-temporal physiochemical modulating stimuli would be essential for regulate angiogenesis, which also provides potential alternative strategies to inhibit tumor vascularization [Exposed to the spatial combination of ATP and shear stress stimuli, two main response modes of intracellular Caobserved . At the mulation : an acutogenesis ,19,20. Trization ,20,21.2+ dynamics. The proposed mathematical model integrates a module of extracellular signals transmission within the microchannel and a module of intracellular Ca2+ dynamics, which mimics the universal conditions for probing Ca2+ dynamics based on microfluidic platforms. The results show that the features of pulsatile flows and the transmission characteristics of microchannels greatly affect the generation and transmission of spatio-temporal ATP and shear stress stimuli, resulting in different spatial combinations of stimuli within the microchannel. In response to the various stimuli combinations, the intracellular Ca2+ dynamics of cultured cells indicates two Ca2+ response regimes depending on the characteristics of stimuli. Overall, the findings of this research provide insights for the intracellular Ca2+ dynamics regulated by the combination of spatio-temporal ATP and shear stress stimuli, which have significant implications for understanding the underlying signaling transduction mechanisms and directing cellular behaviors. In conclusion, based on a developed microfluidic generator of biochemical and biomechanical signals, theoretical modeling has been carried out to investigate the combination effects of spatio-temporal ATP and shear stress stimuli on the intracellular Ca


Recently, increasing experimental evidence, as well as insight through mechanistic in silico modeling demonstrates the crucial role of the exchanger in sinus node pacemaking. NCX had a key role in the exciting story of discovery of sinus node pacemaking mechanisms, which recently settled with a consensus on the coupled-clock mechanism after decades of debate. This review focuses on the role of the Na+/Ca2+ exchanger from the early results and concepts to recent advances and attempts to give a balanced summary of the characteristics of the local, spontaneous, and rhythmic Ca2+ releases, the molecular control of the NCX and its role in the fight-or-flight response. Transgenic animal models and pharmacological manipulation of intracellular Ca2+ concentration and/or NCX demonstrate the pivotal function of the exchanger in sinus node automaticity. We also highlight where specific hypotheses regarding NCX function have been derived from computational modeling and require experimental validation. Nonselectivity of NCX inhibitors and the complex interplay of processes involved in Ca2+ handling render the design and interpretation of these experiments challenging.The electrophysiological mechanism of the sinus node automaticity was previously considered exclusively regulated by the so-called “funny current”. However, parallel investigations increasingly emphasized the importance of the Ca Year by year, several studies were published supporting both theories. Thus, the exciting exploration of SN pacemaking mechanisms has been full of debates for over a half century. Numerous mechanisms have been suggested, tested, refuted, accepted, tested, and retested again, updated and after intense point–counterpoint public debates finally settled at a delicate balance known as the coupled-clock mechanism that includes both membrane and sarcoplasmic reticulum (SR) linked contributions. The general field of SN pacemaking (Few areas of cardiac cellular electrophysiology had more intense debate over the years than ‘what makes our hearts beat'—the electrophysiology of the sinus node (SN). In the last two decades, we could witness the struggle of two competing oscillator concepts explaining SN automaticity: in essence, the funny-current  are mainly active before , whereas ICaL (L-type Ca2+-current) and IKr (rapid delayed rectifier potassium current) are mainly active thereafter (at more positive Vm), while NCX is active throughout the entire AP cycle. Early studies suggested a major role of a decaying delayed rectifier potassium current (IKr)  generated by the SR via ryanodine receptors (RYRs) during the DD  starting from the maximal diastolic potential (MDP) and ending at the action potential (AP) threshold of about −40 mV. This threshold is called takeoff potential . At the nt (IKr)  and the IKr)  superfamily, as the exchanger carries three Na+ in exchange for one Ca2+. Thus, the exchanger is electrogenic and affects the transmembrane voltage. The first data regarding the exchange ratio of NCX was published by Reuter and Seitz describing the Na+/Ca2+ exchange as neutral, i.e., changing two Na+ ions for one Ca2+ , where ENCX is determined by the transmembrane Na+ and Ca2+ gradients (ENCX = 3ENa − 2ECa). When the difference between Vm and the equilibrium potential is positive, the exchanger acts in the reverse mode; however Ca2+ extrusion is favored when Vm is more negative than the equilibrium potential (vs. reverse), the Na+/Ca2+ exchanger can generate either inward or outward current, it can depolarize or repolarize the cell membrane, respectively.Naone Ca2+ . Later, one Ca2+ . Chapmanone Ca2+ . Nowadayer ratio . The eleotential . Dependiintracellular factors, the Ca2+i, ATP, phosphatidylinositol-di-phosphate-2 (PIP2), NO and extracellular factors such as monovalent cations, PKC activators, and agents that induce proteolysis or protein phosphorylation can activate the exchanger , selective inhibition of the exchanger became one of the most intensive research areas demanding the development of properly selective agents. In this chapter, we summarize the NCX inhibitors from the early nonspecific substances to novel, highly selective agents.Since NCX plays a crucial role under physiological circumstances in the maintenance of Ca2+, Sr2+, Mg2+, Cd2+, Ba2+, Mn2+) and trivalent  cations have a nonspecific inhibitory effect on the NCX current phenyl]ethyl]isothio-urea-methanesulfonate, carbamidothioic acid) were developed as novel NCX inhibitors with promising blocking potency; however experimental studies reported that KB-R7943 exerts complex nonspecific effects on other transmembrane ion currents including Irse mode . SEA-040reported . The revectively .2+-handling could not be investigated directly. Efforts were made continuously to develop a potent, selective inhibitor to clarify the role of this highly complex transport system in cardiac function. Recently, two novel NCX blocking compounds were identified: ORM-10103 and ORM-10962. The selectivity of both agents was intensively tested by , IK1 (inward rectifier potassium current)) ) . The effurrent)) .2+ itself. The activation is strongly promoted by elevated Na+i and increased by higher pacing frequency  and an oxidant  factor is required to enhance NCX. Diethylpyrocarbonate increased Na+-dependent Ca2+ uptake  and a slow component: NCX triggered by the release of stored intracellular Ca2+ (INaCa). Satoh et al.  in detail but demonstrated that cellular Ca2+ overload was induced during exposure of SNCs to caffeine , the Dokos et al. and Kurata et al. models did not exhibit sustained pacemaking behavior upon complete NCX block. The effect of clamping the intracellular  Ca2+ concentration to the initial value  is shown in The first computational pacemaking model of mammalian SNCs including NCX was proposed by  current . Upon coby e.g., ] where I2+-dynamics in the spontaneous firing rate of APs in sinus venosus of cane toad and provided quantitative data regarding the magnitude of the NCX current during the SN AP. They measured 20–27 pA NCX current in early diastole (Ca2+i: 250–300 nM) and 12 pA (Ca2+i: 200 nM) in late diastole in toad isolated sinus venosus cells. The authors emphasized that the net inward current  required during DD to generate a pacemaker potential is less than 1 pA  can cause substantial changes in the firing rate. m of −40 mV in the presence of 500 nM Ca2+i  of NiCl2 suppressed the local release, thus they suggested the T-type Ca2+ channels triggered subsarcolemmal Ca2+ sparks, which in turn stimulated the inward NCX to depolarize the pacemaker potential to threshold.A seminal study was published by Huser et al. in 2000 . By usin2+ releases . In turn, the negative chronotropic effect of ryanodine is the result of disappearance of localized pre-AP Ca2+ releases. Substituting Na+ for Li+ to inhibit NCX caused sinus arrest leading to the conclusion that Ca2+ release-NCX cross-talk has crucial importance in pacemaking. The authors provided a possible scenario for the current interactions underlying spontaneous automaticity: after reaching the maximal diastolic potential, If activates to depolarize the membrane and activates ICaT and ICaL causing LCR from the SR, which in turn activates NCX and the inward current augments to activate the remaining L-type Ca2+ channels. These results were interpreted such that SR Ca2+ release has a pivotal impact in defining the actual firing rate and inactivation of either the RyR or NCX activity lead to frequency slowing or abolishment.One year later, the Lakatta group  further 2+ concentration in the subspace and Vmvia NCX for spontaneous depolarizations in SNCs was numerically reproduced and mechanistically underpinned by 2+ clock, which yielded rhythmic spontaneous depolarization over a wider range of parameters of the currents involved in the M-clock  , i.e., arguing against an independent Ca2+ clock, thus underlining the coupled nature of the pacemaking mechanism. In line with this finding, up = 20 mM/s)  . By moduductance . However20 mM/s) . HoweverVinogradova et al. explored in 2004 whether the LCRs require previous membrane depolarization as was suggested so far . Experim+ solution abolished spontaneous firing in the presence of CPA, an inhibitor of SR uptake. On the other hand, if NCX would be entirely a consequence of SR Ca2+ release, the CPA should have a similar effect as a low Na+ solution. However, the SR inhibition only slowed the heart rate, while low Na+ abolished spontaneous pacemaking within one beat, with concomitant decrease of Ca2+i. However, considering the rather complex effects of reduced Na+ solution, the results could not unequivocally support the authors' hypothesis. The application of KB-R7943 to inhibit NCX has to be considered problematic because of its nonselectivity.In contrast to the increasing body of evidence, Sanders et al. claimed in 2006 that the contribution of NCX to spontaneous depolarization does not exclusively depend on the intact SR function in guinea pig SNCs . They de2+ imaging, Yaniv et al. (2013) could show that the coupled clocks also control beat-to-beat regulation of the spontaneous beating rate  and refined current formulations based on more recent experimental data  rabbit SN vs. ventricular cells. They found that in the presence of similar physiological Ca2+ concentrations, the SNCs generate large rhythmic spontaneous Ca2+ releases in contrast to ventricular cells in which they found Ca2+ sparks with small amplitude and stochastic nature, as was originally described  reduced the AP firing rate, partially by inhibiting If and by decreasing the SR Ca2+ content in isolated SNCs. This means that despite the fact that ivabradine has no direct effect on intracellular Ca2+ cycling and does not exert effects on surface membrane channels other than If, it still suppresses the SR Ca2+ load and the LCR events. These novel findings further supported the hypothesis that cross-talk between the membrane and Ca2+ clock regulates SN automaticity.In 2013, Yaniv et al. found new evidence regarding the membrane and Cacoupling . Applica+/Ca2+ gradients in the pacemaker function of the NCX  enhanced the LCRs, shortened the CL, which was interpreted as an extension of normal chronotropic response. Further increase of Na+i by up to 15 mM caused reduction of LCRs, large CL variability as a consequence of Vm-Ca2+ clock uncoupling. In parallel with the biphasic CL changes, the NCX current also showed biphasic alterations: initially the INCX increased due to the high amplitude LCRs, then declined as ENCX reduced. The authors claimed that ENa, ECa, and ENCX tightly regulate the SN automaticity via influencing several ion channels and components of the SN clocks. the NCX . They us2+ release propagation patterns that was employed in 2+ pumping prepared the cell for stronger (higher amplitude) and more synchronous LCRs (more subcellular Ca2+ wave collisions) in the next cycle, which elicited a higher NCX current.2+ channels (Cav1.3) in the generation of the LCRs and Ca2+i dynamics in SNCs from wild-type and Cav1.3 knockout mice  enzyme, which restricts local RyR Ca control . Also invates AC . This fe2+ signaling cascade in mouse SNCs, and they demonstrated that the Ca2+ handling proteins are abundantly expressed, and LCRs were also detected in skinned cells. They showed that inhibition of intrinsic PKA activity reduces PLB phosphorylation and prolongs the LCR period causing reduction in the SNCs firing rate. The PDE inhibition resulted in the opposite effects: increased PLB phosphorylation, shortened LCR period, and accelerated firing. In the same study the authors also demonstrated that β-adrenergic activation requires intact Ca2+ handling, indicating a pivotal role of AC-cAMP-PKA-Ca2+ signaling cascade in maintaining the normal automaticity in mouse SNCs   . Howeverctively) . An ampling rate . In thisal cAMP' .2+ release during DD as the specific link between β-adrenergic stimulation and increased firing rate in rabbit SNCs  caused immediate changes of spontaneous beating rate, which could be traced back to changes of local NCX using mechanistic computational modeling  reduced the number and size of LCRs and lengthened the LCR period with concomitant decrease of the beating rate. Numerical modeling indicated that cholinergic-modulation of the beating rate is integrated into the Ca2+ cycling via LCR-mediated function of the NCX. The authors concluded that in the presence of low doses of carbachol, the muscarinic activation induced beating rate reduction is mediated by suppression of cAMP-PKA-Ca2+ signaling, while IK(Ach) activation contributes only under higher carbachol concentrations.2+ pumping rate, which in turn affect diastolic NCX  hyperpolarized MDP via a reduction of NCX due to lower Ca2+i  The more negative Vm increases the magnitude of If and ICaT during early diastole. iii) The enhanced Ca2+ entry during DD increases intracellular Ca2+ and CICR causing a higher NCX current.In a theoretical analysis of the relative importance of the membrane and Caer Ca2+i . This meincomplete NCX1 knockout mice where the baseline sinus frequency was completely identical between control and NCX1-deleted mutants  having amplitudes larger than the Ca2+ sparks and smaller than global Ca2+ transients. Since the NCX extrudes Ca2+ from the vicinity of RyRs it attenuates the spread of Ca2+ leading to weaker recruitment. However, when NCX is genetically decreased (i.e. incomplete NCX1 knockout), the Ca2+ spread and recruitment are enhanced providing larger Ca2+ and driving force for the remaining NCX molecules. These results indicate that NCX has a crucial role under the normal heart rate, and a small fraction (i.e. 20%) of NCX is able to produce sufficient current under DD. However, when NCX expression is low, its capacity is fully used for Ca2+ extrusion under rest and there is no more support for frequency increase under β-adrenergic response.In response to that paper, Maltsev et al. in 2013 provided rabbit SN numerical simulations  to addre2+ extrusion is carried by NCX. At the same time, it is an interesting finding that the Ca2+ transient amplitude was decreased and the decay was slowed while the SR Ca2+ content was unaffected. The authors claimed that the NCX1 deleted cells are not able to compensate the NCX1 deletion. They argue for a fundamental role of NCX in pacemaking even under resting conditions, and the normal pacemaking function is not possible without proper NCX function.Herrmann et al. in 2013 used inducible and SN specific Cre transgenic mice lacking NCX1 selectively in SN pacemaking cells . The NCXf and intact Ca2+ stores. They claim that NCX has a crucial role in normal pacemaking. They observed LCRs but no Ca2+ transients in NCX deleted cells. They also conclude that If is not sufficient to depolarize the cell membrane if NCX is completely lacking, interestingly not even if isoproterenol was added. Torrente et al. in 2015 created an atrial-specific NCX knockout mouse where NCX was totally eliminated from the atria including the SN , there is no indication of spontaneous atrial depolarizations on the ECG and the animals exerted junctional escape rhythm . Furtherg the SN . These ccurrents .2+ releases could be eliminated by blocking NCX (with 1 µM SN-6 and 10 µM KB-R7943) but not by blocking If (with 3 mM ivabradine). Their observations suggest that in these hESC-CMs, the M-clock and the voltage clock act as two redundant pacemaker mechanisms which can work independently.In 2016, Choi et al. observed regular oscillations of inward currents in voltage-clamped human embryonic stem cell-derived cardiomyocytes, which exhibit spontaneous APs when not being voltage clamped . The oscβ-adrenergic stimulation elicited higher heart rates in NCX-overexpressing mice, suggesting more inward NCX current which enhances the speed of DD.f function were published where autonomic rate modulation was preserved  activate inward NCX, which defines the ignition onset. If, and ICaT also contribute to early depolarization to reach the ICaL threshold. The physiological role of the low-voltage activation threshold L-type Ca2+ channel isoform (Cav1.3) may have more importance to regulate the early ignition phase. However, the details of the specific contribution of Cav1.3 require further investigation. The ICaL-mediated Ca2+-influx recruits more LCRs via diastolic CICR, and this positive feedback further increases NCX. It was also shown that ICaT also has a role in the diastolic CICR, similarly as early results suggested . The underlying mechanism of ryanodine induced decrease in the firing rate could be driven by the marked decrease in DDR, which was observed in isolated rabbit sinoatrial node cells by 2+-clock (in case of ryanodine) is perturbed, it will indirectly affect the other clock via the coupling mechanism. As a coupling, similar responses happened, the ignition potential become depolarized, and time-to-ignition phase becomes longer. This provides delay in clock-coupling by CICR delay.In 2018, a new model was proposed by the Lakatta group extending SN electrophysiology by an integrated regulatory mechanism including close interactions of LCRs, NCX, and Ileration . In contuggested . Howevere.g. extracellular Ca2+ concentration , data from human SNCs or tissue are scarce. Considering the vast difference in the main quantity of interest, the beating rate, between commonly used laboratory animals and humans , it however cannot be assumed that the details of pacemaking mechanisms and most importantly the delicate balance of competing effects can be transferred from animal models to human in general. Indeed, it has been shown that computational models of SNCs of different species  react markedly different to changes of ntration . Given tntration , speciespig SNCs . Moreovepig SNCs . Regardifference .2+ clock are coupled to electrogenic surface membrane molecules to initiate rhythmic APs, and that Ca2+-cAMP-protein kinase A (PKA) signaling has a regulatory role in the clock coupling in human SNCs  models, which becomes more pronounced only slightly before AP ignition. In the Yaniv et al. and Maltsev–Lakatta models in contrast, NCX is apparent markedly earlier. A current integral reflects the total charge transported across the membrane during a specific period of time. For a current normalized to the membrane capacitance (pA/pF), the integral is identical to the potential difference caused by the charge accumulated on the capacitive membrane due to the current (units: ms * pA/pF ≡ mV) is a measure of how much of the DD (in terms of mV) can be attributed to NCX. To quantify the role of NCX to DD in the models of different species, we computed the NCX integral during DD:takeoff was defined as the first time step for which d2Vm/dt2 exceeded 15% of the maximum d2Vm/dt2 in the time interval between MDP and max(Vm). The curvature based measure was inspired by the shape of the SN APs with the threshold empirically chosen to strike a balance between a detection early in the upstroke and robustness to minor increases of d2Vm/dt2 during DD. m during DD and gives an idea about how much of the total DD can be attributed to NCX in each of the models across different species. Under the simplifying assumption of independent currents, almost the entire DD can be attributed to NCX in the Severi et al. rabbit and Kharche et al. mouse model. In the Yaniv et al., LLFS, and Maltsev–Lakatta models, NCX alone would cause more DD than the net ΔVm observed, i.e., outward currents partly compensate the NCX-induced depolarization whereas the role of NCX is almost negligible in the Garny et al. and Kurata et al. rabbit models.where tm oscillations for several seconds were observed  compared to the parent model. Interestingly, NCX block experiments caused an increase in beating rate in the Fabbri et al. model.  mediated by a synergistic acceleration of DD caused by elevated Ca2+i, shortening of APD and less negative maximum diastolic potential. Upon 90% NCX block, automaticity ceased. The β-adrenergic rate modulation experiments by Fabbri et al. did not include any direct effects on NCX but the SR Ca2+ pumping was modeled as a target of isoproterenol. The application of 1 µM isoproterenol yielded a 27.9% increase in beating rate. If only SR Ca2+ pumping was modeled as a target of isoproterenol, this increase was markedly attenuated and even slightly reversed with a remaining effect of −0.4%. Loewe et al. proposed the Loewe–Lutz–Fabbri–Severi model (LLFS) (f block (CL +25.9%) was in accordance with the available experimental human data [+26% reported by The first human SNC model originating from a SNC model rather that an atrial model was presented by l (LLFS)  as an exl (LLFS) . In the 2+ within the cell following release from the SR has been found and the results were interpreted in terms of distinct release sites under different control mechanisms . RyRs are activated by an increase of local Ca2+i resulting in Ca2+-induced Ca2+ release (CICR). Each CRU generates small, temporal Ca2+ release events, called Ca2+ sparks which have a radius of 1.5 µm (via fire-diffuse-fire propagation (β-adrenergic response via variation of single CRU Ca2+ currents (Ispark). The LCR controls SN chronotropy (acceleration and slowing) via changing the LCR size, amplitude, and number per cell surface area.In SNCs, the diastolic NCX is not only regulated by Vf 1.5 µm . Therefopagation . Spark c2+ measurements on rabbit SNCs and via numerical modeling, that increasing the Ispark amplitude results in local synchronization of CRU firing yielding increasing LCR size, rhythmicity, and occurrence rate  in turn increases the number of sparks (nsparks) since there is more Ca2+ available for diffusion and activation of additional RyRs. As the above formula predicts, the larger number of sparks tends to increase the NCX current, and finally, these opposite changes are able to largely compensate each other providing marginal change in the NCX. Stern et al. in 2014 provided three-dimensional SNC computational modeling including diffusion and buffering of Ca2+ in the cytosol and SR but omitted the submembrane space and inactivation of RyRs because of the lack of experimental validation data  correlated with cell capacitance in rabbit sinoatrial node, and larger cells showed greater density  and flexible (i.e. adaptation to a given vegetative stimulus) pacemaker function . The minf, without correlation between If density and cell size. In the absence of If, the diastolic NCX in response to Ca2+ release  could drive the pacemaking. Wide ranges of ICaL and IK densities were found in these cells. Functional heterogeneity of SNCs was recently described by β-adrenergic stimulation synchronized LCRs in all dysrhythmic cells and in the 1/3 of the dormant cells. Furthermore, the dormant cells developed automaticity in response to β-adrenergic stimulation. These results indicate partial or complete uncoupling between Ca2+ and M-clocks in the dysrhythmic and dormant cells.Monfredi et al. examined the major ionic currents in intercaval pacemaker cells (IPCs) obtained from rabbit heart and found marked electrophysiological heterogeneity in IPCs . Experim2+ extrusion to maintain the Ca2+ flux balance  or incomplete transgenic knockout mutants are not able to unequivocally pin down NCX's function in pacemaking. Theoretically, partial NCX suppression  may lead to 3 different outcomes, characterized by the intracellular Ca2+ concentration since NCX is the major pathway for Ca2+ extrusion:When active in forward mode, NCX generates inward current and at the same time represents the most important pathway for Ca balance . Since t2+i because of activation of other compensatory mechanisms (such as plasma membrane ATPase). In this fortunate case, all NCX inhibition is pure current inhibition with minimal secondary, indirect mechanisms through changes of Ca2+i. However, this scenario may have the lowest probability.i) NCX inhibition does not increase Ca2+i because the  uninhibited fraction is able to remove the Ca2+. This suggests a non-linear relationship between the degree of NCX inhibition and reduction of NCX function in terms of Ca2+ extrusion during the DD, making the interpretation difficult.ii) NCX inhibition does not increase Ca2+i. In this case, the Ca2+ gain will enhance the Ca2+-dependent inactivation of the ICaL, which may shorten the AP. However, also the AP threshold is expected to be higher, requiring more time to be reached. According to the ignition theory . These indirect mechanisms may counteract the effect of NCX inhibition.iii) NCX inhibition elevates Can theory , the decf and intact Ca2+ stores. Current mechanistic models of both human , transgenic animals and numerical modeling clearly indicate the fundamental role of NCX in maintaining the resting heart rate.Groenke et al. demonstrated that NCX1 KO mice show no spontaneous atrial depolarization ; only juth human  and rabbth human  SNCs alsβ-adrenergic response involves the increase of intracellular cAMP and PKA, increases CaMKII activity and increases the intracellular Ca2+ load via phosphorylation of various channels . These results indicate a pivotal role of NCX not only in maintaining the basal SN automaticity but also in the sympathetic stimulus-mediated heart rate acceleration.The CaL (m is depolarized (more than 0 mV) and Na+i is high. Experimental results as well as modeling data demonstrate that SN APs peak around 20 mV, which could be considered high enough to “switch on” reverse mode; however the time at depolarized levels is very short. During normal Na+i, mechanistic modeling predicts only marginal reverse mode at the beginning of the AP, thus the NCX current is mainly inward throughout the entire AP. The Yaniv et al.  are a matter of debate. Vinogradova et al. showed that ryanodine has a marked effect on the DD by decreasing DDR, thus causing significant CL prolongation  but it becomes persistent. In this case, the diffusional resistance between the network and junctional SR limits intra-SR flux and preserves the Ca2+-load of the network SR. This maintains SR Ca2+ load and drives persistent release flux , accelerated the DD, augmented the LCRs, and reduced the CL variability  and even 20 mM caesium was unable to terminate spontaneous SN beating , slow activation kinetics, and moderate effects of inhibitors (2 and ivabradine cause incomplete and voltage-dependent block. A further issue with ivabradine is the significant IKr block even at the widely used concentration of 3 μM (2+. As discussed above (What is the Role of NCX in Pacemaking Under Resting Heart Rate)?, increased Ca2+ limits the effect of NCX inhibition via secondary effects and covers the exact role of NCX during DD. Taking together these results, it seems likely that complete pharmacological inhibition of If or NCX is currently not feasible using the available compounds, impeding the identification of the role of NCX and If in SN pacemaking.Important pharmacological problems arose regarding I beating . Therefohibitors . However of 3 μM . SimilarAn indirect experiment was also published in 2015 by in vivo, however within the experimental variance . While this pronounced bradycardic effect of hypocalcemia was mediated primarily by ICaL, NCX was subsequently attenuated due to the reduction of Ca2+i. CaL and NCX as well as INaK due to ankyrin-B dysfunction increase variability in SN automaticity , the shortcomings of transgenic animal models, and the nonselectivity and/or indirect effects of parallel Ca2+ increase of selective inhibitors indicate a clear demand of further experimental research to fully reveal the role of NCX in SN pacemaking.A large body of experimental evidence indicates and mechanistic AL—preparing the manuscript: in silico mechanistic modeling, preparing the figures. AV—supervising the study. NN—preparing the manuscript: experimental results of the NCX function in SN. ZK, NT—searching of relevant publications, preparing subchapters of the manuscript.We declare that all sources of funding received is submitted.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.