The subsequent variation in Ca2+ signalling and nitric oxide synthesis could lead to the impairment of the atheroprotective function played by nitric oxide. In previous studies, it has been suggested that the reduced IP3 and Ca2+ signalling can explain the correlation of atherosclerosis with induced low WSS and disturbed flow characteristics. The massively parallel implementation described in this article provides insight into the dynamics of coupled smooth muscle cells and endothelial cells mapped onto the surface of an idealised arterial bifurcation. We show that variations in coupling parameters, which model normal and pathological conditions, provide vastly different smooth muscle cell Ca2+ dynamics and wave propagation profiles. The extensibility of the coupled cells model and scalability of the implementation provide a solid framework for in silico investigations of the interaction between complex cellular chemistry and the macro‐scale processes determined by fluid dynamics. © 2016 The Authors. International Journal for Numerical Methods in Biomedical Engineering published by John Wiley & Sons Ltd.Preferential locations of atherosclerotic plaque are strongly associated with the areas of low wall shear stress and disturbed haemodynamic characteristics such as flow detachment, flow recirculation and oscillatory flow. The areas of low wall shear stress are also associated with the reduced production of adenosine triphosphate in the endothelial layer, as well as the resulting reduced production of inositol trisphosphate (IP This research approach offers the potential of increasing the speed of knowledge discovery In silico experiments have the potential to provide insight into the observations obtained by the experimental science. In the context of biological systems dynamics, the in silico simulations enable the rapid pruning of the parameter search space for the refinement and integration of cellular‐level models into biologically realistic macro‐scale models. The large‐scale physiological simulations described here were designed to provide insight into the effects of the luminal concentration variations on adenosine triphosphate (ATP)‐dependent dynamics in the coupled endothelial cells (ECs) and smooth muscle cells (SMCs) making up an arterial wall. The simulations of this nature provide a unique opportunity to perform experiments, which would never be possible in the in vivo and in vitro settings. For example, various specific pathological conditions, as described further in the text, can be simulated by changing the homocellular and heterocellular coupling parameters. Numerical simulations of this nature have never been attempted before at the scale of millions of coupled cells.The recently coined term tunica intima, tunica media and tunica adventita. The intima consists of a single layer of ECs; the media consists of the SMCs, and the adventita is made up of connective tissue. The endothelial layer plays the role of a mediator in the transfer of chemical species to and from the surrounding tissue. The mass transport phenomenon where chemical species are actively or passively diffused within large populations of cells is made possible through cellular communication channels. These channels are known as gap junctions; they are composed of connexin protein subunits and allow direct intercellular communication. For example, the Cx37 homotypic connexins are highly selective and allow only the transfer of monovalent cationic currents; thus, they have the most influence on the membrane potential 2+ concentration current. The Cx43 connexin is the least selective, and it allows the passage of a range of large and small molecules such as ATP and inositol triphosphate (IP3) The arterial wall consists of three layers of tissue: 2+ concentration oscillations (spreading both downstream and upstream) triggered by low WSS play an important role in atherosclerotic plaque formation in vivo and in vitro experiments provide descriptive models that use cellular‐level observations, but these observations fall short of explaining the macro‐scale phenomena of plaque formation and development Recent publications on the physiology of vascular system dynamics hypothesise that cellular Ca1.13 and Ca2+ signalling induced by low WSS.On the macro‐scale level, local geometric properties of arterial trees such as curvature, branching and bifurcations determine the local haemodynamics properties such as pressure, flow velocity and WSS. Among those properties, WSS carries a special significance as it influences the localisation of atherosclerotic lesions 2+ wave propagation in the SMC layer of arterial wall. This example of cellular dynamics modelling is aligned with the work presented here because The work of 2+ signalling in arterial wall tissue. The basic EC/SMC coupled model in vivo experiments in the work of μm, which were equivalent to populations of approximately 140 SMCs. The in silico experiments reported in the work of This study investigates temporal and spatial cellular dynamics of large populations of coupled ECs and SMCs with the view of gaining insight into the Cain vitro and in vivo (cadaver) studies. Indeed, there is considerable evidence in the work of 3‐mediated channel. IP3 concentration is determined by the P2Y receptor mediated by agonists such as ATP. Finally, we note that as early as 1988 The presented work is an initial investigation into the relationship between micro‐scale cellular dynamics and the resulting ‘emergent behaviour’ that occurs on a macro‐scale exemplified by the size of atherosclerotic plaques. There is a clear correlation between the dynamics and the increase in intimal hyperplasia as found in 1.2The majority of experiments showing electrical signalling are conducted using small diameter vessels either denuded of endothelium or vessels that contain only ECs 2+ and WSS is a lot more prominent in small diameter resistance arteries. Vasomotion is not considered in this work because this phenomenon occurs more readily in resistance vessels rather than, say, the coronary tree. The focus of the paper is on much larger atherosclerosis prone vessels such as the coronary where contractility does not vary as much as that seen in smaller resistance arterioles. Fluid dynamics calculations show that WSS varies little in terms of the dilation or contraction of the artery  compared with the base WSS induced by the vascular geometry of a bifurcation.The bi‐directional relationship between Ca1.3The macro‐scale simulations reported in this work were implemented on the Blue Gene parallel architecture where a number of coupled EC/SMC units are grouped into a single task/domain in the parallel computational environment. These tasks interact with each other by exchanging state variable values for the neighbouring cells along the edge of the computational domain at every time step during the simulation process. The interaction between the groups of coupled EC/SMC units was implemented through the MPI. Full details of the computational solution are provided in Section In this article, Section The simulations reported in this work are divided into two groups. The first group is based on a synthetic ATP agonist map generated with a sigmoid function parametrised with the distance to the origin of the vessel segment, while the second group is based on the CFD‐based agonist map generated from a model of physiologically‐realistic mass transfer simulation of blood flow in a vessel. The results for both simulation groups are provided in Section 2The experimental methods described here extend the work reported previously by 2.1Agonist binds to the purinergic (P2Y) receptors on the EC surface, activating the GPCR which then activates membrane‐bound phospholipase C (PLC). PLC activation allows phosphorylation of PIP2.3 that is then released in the intracellular space. This nascent IP3 binds to IP3 receptor (IP3) on the ER/SR surface.PIP2 gives rise to IP3‐bound IP3 induces release of Ca2+ ions from the ER/SR into the cytosol.IP2+ release from intracellular store sensitises the IP3 further, which releases more Ca2+, thus making a Ca2+‐rich domain in the cytosol in both EC and SMC. The excess of intracellular Ca2+ depolarises the membrane potential.The Ca2+ encourages the replenishment of the intracellular stores via this pathway.ER/SR has low‐affinity binding sites on the cytosolic side of a channel, which constitutes a pump called the (SERCA) pump. Cytosolic Ca2+ leaks from ER/SR consistently under a concentration gradient between cytosolic and ER/SR luminal Ca2+ and keeps the Ca2+ in equilibrium during a non‐stimulated state of the cell.Ca2+ favours the influx of extracellular Ca2+ from non‐selective cation channels.In an EC, the cytosolic Ca2+ to extracellular space.The SERCA pump pushes out cytosolic Ca2+ ions intracellularly at BKCa and SKCa, let K+ ions move out of the cytosol. This hyperpolarises the membrane potential.10) In ECs, activation of KCa, upon binding to Ca+ ions efflux is the main repolarising current, residual current  also contributes to membrane potential repolarisation.Although K3 concentration increases in SMC cytosol via transmission of IP3 from coupled EC. This IP3 increase activates the downstream IP3‐induced Ca2+ release.The IP3 induced, and calcium‐induced calcium release (CICR) Ca2+ depolarises the membrane potential.The IP2+ from VOCCs, which will close upon repolarisation in the following steps.The membrane depolarisation results in the influx of Ca2+, in addition to other pathways, is pushed out via Na/Ca exchanger.Ca2+ ions to KCa opens BKCa channels in SMC causing K ions efflux and membrane repolarisation.Binding of Ca2 ions add to the repolarisation.Influx of Cl2+, IP3 or membrane potential coupling.Medium for intercellular communication via homocellular gap junctions can either be Ca2+, IP3 or membrane potential coupling. Hyperpolarised EC membrane potential can hyperpolarise SMC plasma membrane and consequently close VOCCs.Heterocellular gap junctions can couple an EC and an SMC via CaThe adhesion of agonist (ATP) to the endothelial surface starts a complex cascade of reactions in the ECs and SMCs as shown in Figure PLC conversion models reported by 3 is monotonic and increasing; thus it can be approximated by a linear function over the range of ATP. Therefore, a linear function is utilised to simulate the resulting relationship between ATP and the flux of IP3 into the EC, that is JPLC. The range of ATP concentration is represented by the equivalent values of the JPLC; the flux of IP3 into the EC is determined by the concentration of ATP in the vessel lumen. The whole range of JPLC consists of three bands, which correspond to the ATP‐dependent Ca2+ dynamics in an SMC. As shown in Figure PLC result in steady state Ca2+ concentrations, while the middle band shows the presence of Ca2+ oscillations. The oscillation envelope for this band indicates the relative high and low amplitudes for lower and higher JPLC concentrations.The ATP to J2.2s−1. The Ca2+ and IP3 homocellular coupling parameters are treated as free in a similar way to the work of s−1. The coupling of membrane potential is a combination of simple ion diffusion across the gap junction and drift given by the product of ion concentration and spatial gradient of the membrane potential. For this particular model, the electro‐diffusion drift is ignored as it has been shown to have negligible effect. Unpublished work with coupled cellular oscillatory models shows this to be the case.The variations of coupling parameters described here model the connexin gap junction permeability used in the simulations in this work. The Cx37 and Cx40 down‐regulation along with Cx43 up‐regulation in early atheroma has been reported in the work of 2+ coupling. The remaining two coupling configurations are designed to simulate pathological cases. In Case 3, IP3 coupling is added to the default homocellular EC coupling to simulate upregulation of Cx43 in lesion‐prone areas described in the work of 2+ coupling disabled to simulate the coupling in progressive atherosclerosis when Cx43 connexin is the dominant mechanism of intercellular communication because of the down regulation of Cx37 and Cx40 connexins 3 diffusion will need careful consideration due in part to its assumed smaller connexin channel permeability.Table 2+ concentration vs the IP3 flux parameter JPLC); for case 1, the range of IP3 where oscillation exists is substantial whereas case 4 has a reduced domain, and the lower bifurcation points are shifted from approximately 3.7 to 3.0. Cases 2 and 3 are identical in their bifurcation points and maximum/minimum values of Ca2+ oscillations, because of the identical heterocellular coupling parameters. The bifurcation diagrams were generated for a single EC/SMC unit without homocellular coupling.Figure 2.3Simulations of cellular dynamics even for small vessel segments require a substantial amount of computational resources because of the density and number of coupled cells comprising the endothelial and smooth muscle surfaces making up the arterial wall. The primary requirement in the development of the computation solution for the large‐scale simulations was the decomposition of the coupled EC and SMC layers into a number of ODEs systems, each corresponding to a number of ‘neighbourhoods’ of coupled cells grouped into quadrilateral surface elements later mapped to distinct computational domains. With a suitably chosen granularity of the surface‐to‐domain decomposition, the entire bifurcation surface can be mapped onto individual computational nodes in a parallel architecture.2.3.1A method for generic parameterisation of bifurcating structures published by ϕ is the space vector  and f can be either zero or some a priori defined function. For the present, we use f = 0, where u and v are orthogonal coordinates, which provide axial and circumferential directions. The Neumann and Dirichlet boundary conditions for Eq. m (u direction) and n (v direction) points on the boundary. With this method, the surface of each segment is unwrapped into a 2D m × n grid in the u and v directions, respectively; in this case, m and n represent the longitudinal and circumferential number of quadrilaterals in each semi‐tubular segment accordingly. The solution now becomes one of a 2D solution, rather than a 3D solution. To obtain a complete 3D surface, the semi‐tubular segments can be joined together at the boundaries. Figure Each Y bifurcation can be decomposed into three semi‐tubular surface segments by using the solution to a biharmonic equation, originally developed as a computer‐aided design tool by Bloor and Wilson m × n with a periodic boundary condition imposed along the longitudinal edge. In this case, the periodic boundary condition refers to the MPI‐enabled communication‐specific mapping of MPI tasks. It must be noted that the bifurcation surfaces are not constrained to symmetric or circular geometries as the solution of the biharmonic equation can be defined for any (continuous) boundary in  space.The mapping of bifurcations to 2D‐quadrilateral meshes enables the decomposition of the whole computational domain into local domains, where each domain corresponds to a single computational task in a parallel environment. Furthermore, this type of decomposition enables the use of MPI‐specific virtual Cartesian topologies for efficient inter‐processor communication. Each tubular segment in the computational solution presented here is mapped to a 2D Cartesian grid of size Q = 4080 quadrilateral elements, where each element represents a cell neighbourhood on the bifurcation surface. With this type of surface decomposition, each quadrilateral domain corresponds to a parallel computational task running on a single core. The SMCs and ECs within each domain can then be placed into the quadrilaterals with little complexity because the  directions provide physiologically correct axial and circumferential alignments for ECs and SMCs, respectively. The SMC and EC cells are aligned orthogonally with respect to each other where ECs align axially with the flow, whilst SMCs align circumferentially. Figure μm and 65 × 10μm, respectively, the SMCs and ECs are arranged into ‘fundamental’ surface units, where the area covered by five ECs corresponds to the area of 13 SMCs; this arrangement corresponds to the layer arrangement used by Figure ‡http://http://www.netlib.no/netlib/bihar/dbihar.f.Bjorstad, P. E. (1980). Numerical Solution of the Biharmonic Equation. Retrieved from . The Fortran code was integrated into a C++ ‘wrapper’ as a part of a small library for generating surfaces from vessel centre lines; the library is using the relevant functionality of the (VTK) library An implementation of the biharmonic equation solver in Fortran is freely available through online resourcesTwo synthetic arterial bifurcation surface meshes were generated for the simulations reported in this work. The geometric configuration properties, such as proportional length and angle of the branches, were similar in both cases; however, the second mesh was generated for the vessel surface approximately twice the size of the first mesh. The physical dimensions of the cells are only relevant when generating the surface from a centreline with a specified vessel diameter; the knowledge of the physical dimensions of cells enables the generation of meshes with the correct cell density for a given centreline and vessel diameter. For example, a surface mesh for a bifurcation vessel segment with a parent and two branch arteries of 8.8 mm length and 2.6 mm diameter each is covered by about 1.2 million cells, where there are 326400 ECs, 848640 SMCs, with a total of 1175040 cells. If each EC and SMC are modelled by four and five ODEs, respectively, then the total number of ODEs for this mesh is 10575360, while the larger mesh requires 20901888 ODEs. These numbers of ODEs need to be solved thousands of times even for a short temporal domain. Table 2.3.2The simulations were performed on the IBM BlueGene P architecture at the University of Canterbury High Performance Computing Centre. The implementation also has been tested on the BlueGene Q architecture at the Argonne National Laboratory.For each quadrilateral domain, the system of ODEs was solved using an explicit Runge Kutta  scheme initially implemented in Fortran as rksuite_90 component of the (NAG) Fortran Library A parallel simulation requires as input the basic geometric configuration the surface for a given bifurcation in terms of the number of quads along axial and circumferential dimension; in addition, each simulation requires the configuration of the coupled EC and SMC in a quad. The quads are grouped into 2D Cartesian meshes, each representing a tubular segment/branch of a bifurcation. Within each 2D Cartesian mesh, the quads correspond to computational domains, which are mapped to distinct MPI tasks. In addition, each simulation requires an explicit agonist (ATP) map, where each EC cell is assigned a surface layer ATP value. The agonist maps were generated from the corresponding EC surfaces in order to preserve the implicit topology of the cells. The types of agonist maps used in this work are described in Sections The state variables' values for ECs and SMCs were written to HDF5 format after every elapsed physiological second. In the post‐processing step, the HDF5 output data for every second was mapped onto the surface/cell mesh and written out in VTK's VTU format, which is an XML‐based format with binary data for unstructured grid data. The visualisation and analysis were performed with the ParaView 2.4Work by PLC values in the endothelial layer defined as a function of axial distance only. As noted previously, ATP concentration is linearly related to the rate of IP3 production given as JPLC. Figure Q = 4080.Synthetic agonist maps for the simulations presented here were generated with a sigmoid‐like distribution of J2.5The CFD simulations for generating physiologically realistic ATP maps follow the algorithm described in the work of u represents the blood velocity vector field u = , p the kinematic pressure field and ν the kinematic viscosity. The species mass transport was solved simultaneously via the conservation of species equation given by ϕ is the species ATP concentration and D is the isotropic diffusion coefficient. The walls of the artery are assumed to be rigid and stationary. Due to the very high Péclet number, the time‐averaged nucleotide concentration does not differ significantly from the steady state concentration Here, A parabolic Dirichlet velocity profile was implemented on the inlet  and zero velocity gradient Neumann boundary conditions on the outlets. The pressure field values were specified as zero‐pressure gradient Neumann boundary conditions for the inlet and zero‐pressure Dirichlet boundary condition on the outlets.μM was specified on the inlet and zero gradient Neumann boundary conditions on the outlets. As noted in Section 3 production rate is monotonic and increasing, which can be approximated by a linear function. Therefore, a linear relationship between ATP and the IP3 flux into the EC (JPLC) is deemed sufficient. Following the work of For the species boundary conditions, a constant Dirichlet value of 0.1 S(x) represents the production of ATP as a function of WSS in the form of: K and τm values as reported in the work of Here, 3 concentration in the EC. Figure Q = 4080.All other parameters for mass transport were taken from the work of 3Q = 4080 mesh (corresponding to 4080 cores of Blue Gene/P). For the Q = 8064 mesh (corresponding to 8064 cores of Blue Gene/P), only Case 1 coupling with a synthetic agonist profile was performed. This simulation was designed to verify that a larger population of cells did not change the dynamics of the simulations in a significant way. All simulations were run for 1000 physiological seconds, where output was recorded after every physiological second. The small mesh bifurcation simulations with Q = 4080 cores on Blue Gene/L on average took approximately 20.3 h to complete, while the large mesh bifurcation simulations with Q = 8064 cores on Blue Gene/P took approximately 15.1 h. The visualisations for all simulations are available on the UC HPC YouTube Channel.The simulations were performed with both synthetic and CFD‐based ATP agonist surface concentration profiles. In both cases, the simulations were carried out for all four cell‐coupling cases. All simulation runs were executed for the 3.1Q = 4080 mesh along with the propagating Ca2+ oscillations in the SMC layer at two simulation steps of t=60 and 120 s, respectively. The oscillations originate in the downstream branches of the bifurcation at the upper end of the JPLC‐dependent Ca2+ oscillatory domain §In the simulation snapshots the ‘upstream’ direction is defined from right to left (in opposition to blood flow) and ‘downstream’ to be from left to right .. As the oscillations propagate upstream, the wave amplitude increases, while the wave period decreases. This behaviour continues with the time progress of the simulation.Figure 2+ waves travel past the oscillatory domain because the system is excitable for a range of low JPLC in the non‐oscillatory domain close to the Hopf bifurcation. This excitability is found in similar Ca2+ SMC models such as the model by Figure Figure In order to generate 2D space/time plots for visualising and comparing temporal wave propagation patterns, a subset of SMCs was extracted from all time steps for a given simulation. This subset was defined by plotting time‐dependent SMC concentrations along a line extending along the outer surface of the bifurcation from inlet to outlet.2+ oscillation dynamics for the two mesh sizes. The 2D wave plots show that the overall simulation results show significant similarities for both bifurcation meshes. These differences are explained by the following: Q = 8064 mesh was not an exact scaling of the Q = 4080 mesh.The Q = 8064 bifurcation is at a different location in the parent artery segment compared with the Q = 4080 mesh.The Hopf bifurcation point (lower bound of oscillations for a single SMC) in the ATP profile for the Figure 2+ oscillations in the SMC under the Case 1 coupling conditions are initially ‘extinguished’ at the lower border of the oscillatory JPLC concentration range. However, in both cases the oscillations propagate upstream past the lower boundary  at approximately 200 and 250 s for the Q = 4080 and Q = 8064 meshes, respectively.The Ca¶https://www.youtube.com/watch?v=ax8I6rZG-6Q,Coupling Case 2—https://www.youtube.com/watch?v=HfMHsZrM8bw,Coupling Case 3—https://www.youtube.com/watch?v=lx-Pza_{Z}HVk,Coupling Case 4—https://www.youtube.com/watch?v=5JpfDcs5GTA.Coupling Case 1—.Figure 3.2Q = 4080 bifurcation surface mesh described in Section 2+ oscillations in the SMC layer at time t = 300 s. The oscillations originate largely in the branches of the bifurcation where the JPLC concentration is high compared with the rest of the vessel segment.CFD‐based agonist maps were generated only for the t = 300 s. One commonality between all cases is that similar to the synthetic agonist map the oscillations propagate along the direction of the decreasing ATP gradient. Although not shown in the static figures, waves propagating on the surface of the inlet cylinder seem to do so in a circumferential direction. Variations in Ca2+ are determined from the small neighbourhood of the lower bound of the JPLC‐dependent Ca2+ oscillatory range, as shown in Figure 2+ concentration. The visualisations for all four coupling cases with the CFD‐based ATP map are available online∥https://www.youtube.com/watch?v=UPZZdbF8De0,Coupling Case 2—https://www.youtube.com/watch?v=QH9gyH5s1fo,Coupling Case 3—https://www.youtube.com/watch?v=yj_{q}1QIbrdg,Coupling Case 4—https://www.youtube.com/watch?v=8q6hrFsUy4s.Coupling Case 1—.Figure 3.3Figure 43 diffusion will require a close review due in part to its relatively small homotypic connexin channel permeability even though Ca2+ buffering is taken into account.Although the analysis of the model is qualitative in nature, the simulations results allow us to investigate the differences among coupling cases for a particular region in the ATP map. The visualisations (figures in the text as well as supplementary videos) are designed to demonstrate the emergent cell behaviour between healthy and pathological conditions. As it has been noted earlier, the four coupling cases represent simple approximations to a complex physiological phenomena and should not be seen as a complete description of the mechanisms involved. However, they are put forward as an initial framework from which further more complex pathways can be simulated. In particular, the role of IP4.12+ waves for the synthetic agonist concentration maps. Figure 2+ whilst remaining in the oscillatory regime. Overall, waves do not propagate into areas of high ATP/JPLC/IP3 concentrations defined by the upper bound of the JPLC‐dependent Ca2+ oscillatory range shown in Figure We first turn to discussion and analysis of Ca2+ moving into neighbouring cells and being either taken up by the SR or diffused out to other nearby cells whose Ca2+ concentration is of a size that will allow ions to move across the cell boundary through gap junctions. The second feature is that of how waves terminate: as the concentration of ions in the cells decreases due to diffusion, the waves become weak and narrow in width and eventually are extinguished by seemingly curving back upon themselves. Spiral waves are known to occur in a number of biological conditions as reported in the work of 2+ is dispersed in wave packets. The blocks of Ca2+ concentration are not single cells . The initial examinations indicate that these packets of cells have similar cytosolic Ca2+ concentrations and are in phase in terms of their oscillatory dynamics. However, it is as yet unclear as to the exact reason for this.Figure 2+ coupling. Thus, any oscillations produced in the ECs do not acquire fully propagated to the SMCs, although because of the heterocellular IP3 diffusion, cytosolic Ca2+ is propagated from cell to cell in only small amounts. It should be noted that if the sensitivity of the IP3 receptors on the SR is increased then this may very well produce profiles similar to Case 2 because of increased cytosolic Ca2+ effluxed from the SR.Figure 2+ coupling. The agonist‐induced increase in IP3 and subsequent Ca2+ efflux from the ER is now diffused to the SMC via gap junctions. The increase in SMC Ca2+ causes oscillations (via the CICR mechanism) to occur, and further diffusion allows cells not normally participating in an oscillatory regime to become activated. The curvature of the waves seen in  in the daughter artery whereas for Cases 2, 3 and 4, the daughter arteries are relatively free of oscillation. For Case 2, compared with Case 1, the dynamics are substantially different due to the inclusion of heterocellular Cat = 100 s, the upstream Ca2+ dynamics initiate very quick ‘flashes’ of intense concentration just downstream of the upstream wave domain border; these ‘flashes’ are evident for approximately 5 to 6 s and leave a band of inactive Ca2+ as shown in Figure t = 600 s. These can be seen in the supplementary video material provided online.Case 4 coupling parameters results are shown in Figure 2+ waves along the outer edge from inlet to upper daughter outlet indicate a strong similarity between the Q = 4080  indicates that this is caused by the diffusion of Ca2+ to the extent where the cytosolic Ca2+ is too low to cause the CICR mechanism to initiate oscillations. A finite time is needed for diffusion to build up the cell Ca2+ again to renew oscillatory behaviour.In the space/time concentration plots shown in Figure 2+ concentration plots for all four coupling cases. Case 1 coupling simulation results have been discussed earlier. Case 2 coupling plot indicates the effect of ‘wave packets’ propagating in both upstream and downstream directions. This points to distinct ‘communities’ of cells whose Ca2+ oscillations are in phase and hence do not provide sufficient diffusion between them. Only when the boundaries of these packets allow significant Ca2+ into the community does the cell packet breaks up. This phenomena need further analysis to confirm our initial explanation. Case 3, as mentioned previously is similar to Case 2. Case 4, however, presents a combination of phenomena where packets of cells exist throughout specific positions on the surface. At the later simulation stages, the waves propagate further upstream in a well‐defined manner. For Cases 2, 3 and 4, the propagation beyond the bifurcation point starts immediately in contrast to Case 1, where it takes a substantial time to produce upstream wave propagation past into the non‐oscillatory region. It is clear that the diffusion of Ca2+ through heterocellular gap junctions is the dominant mechanism for all wave profiles, although gap junction transmission of IP3 does have an effect through the action of IP3 receptors on the SR of an SMC. However, this may be a function of the IP3 permeability value being relatively small.Figure 4.22+ concentration at time t = 300 s. Although this ATP map is substantially different from the synthetic condition, the waves still propagate in a direction towards low ATP (or JPLC rate) concentration and, for this particular, ATP concentration profile waves move downstream into the daughter artery. Low ATP concentration occurs upstream in the parent artery segment. Here, complex waves and patterns occur, but this is due to the low levels of ATP.We now turn to the case for the CFD‐based ATP agonist map. Figure 4.3in vivo domain localised cell‐coupling parameters could change in response to the haemodynamic‐dependent agonist concentrations and stress on the cyto‐skeleton over long periods of time. This agonist‐induced change in coupling conditions could be one of the triggers linked with the transition from healthy to diseased state. This hypothesis requires further investigation with more complex coupling models. It may be suggested that the transition from healthy to diseased state is a multi‐stage process where a response to the changing haemodynamic conditions results in coupling condition changes followed by the corresponding change in the oscillatory behaviour. Figure A comparison with the simulation results for the CFD‐based ATP map in Figure 4.4Limitations of the model include only one‐cell‐deep layer of SMCs. Future work will extend the simulations to include a model of SMC layer of variable thickness in terms of the number of cells. Additionally, Figure 52+ wave propagation profiles determined by the coupling configurations. The simulations demonstrate that heterocellular diffusion of both IP3 and Ca2+ through gap junctions plays an important role in information transfer over large spatial scales. Zero membrane potential coupling does not eliminate Ca2+ oscillations but provides a significant difference in the oscillatory dynamics over scales much larger than a single cell. Indeed the simulations show that IP3 is an important transport mechanism for oscillations to occur and one that should not be neglected over and above that of Ca2+.We have developed a massively parallel framework for performing 3D simulations of coupled EC/SMC dynamics in a bifurcating artery. The simulations reported in this work include up to 2.3 million cells modelled by more than 20 million ODEs. The results of the time‐dependent simulations show a radically varying range of Ca2+ oscillation propagation is strongly dependent on the ATP gradient along the EC layer and the Ca2+ cellular dynamics. Although the bifurcation meshes used in this work are symmetric, and the use of symmetric boundary conditions could have been used to reduce the computation time, the simulations were performed with full bifurcation meshes in preparation for large‐scale simulations with meshes modelling realistic arterial segments where daughter arteries are of different diameters, and the bifurcation angles change depending on the location of the arterial segment in the human vasculature. We believe that the coupled EC/SMC dynamics modelling framework is an invaluable tool capable of providing deep insights into the cellular dynamics and the development of vascular disorders in connection with the local properties of arterial geometries. We would like to emphasise that the relationship between Ca2+ dynamics (in terms of cellular oscillations and wave propagation) and vascular disease is tentative at present, while considerable further research is required to advance the understanding of the interplay between vascular geometry, fluid dynamics and cellular function. It is our intention to extend the parallel implementation of the model to simulate patient‐derived geometry at a realistic scale. This work will involve the use of substantially larger computational resource. The presented framework is a foundation step towards simulating a full coronary tree, which involves the solution of billions of ODEs, and is considered to be a classic exascale problem.The results of the reported simulations show that for both the synthetic and CFD‐based agonist maps the phenomena of macro‐scale CaThe authors would like to thank the members of the University of Canterbury High Performance Computing Centre, Tony Dale and François Bissey for their enthusiastic support and expert advice on the technical front. We also thank Allanah Kenny for the dynamical system behaviour analysis and the final manuscript review.The authors wish to acknowledge the contribution of NeSI to the results of this research. New Zealand's national compute and analytics services and team are supported by the New Zealand eScience Infrastructure (NeSI) and funded jointly by NeSI's collaborator institutions and through the Ministry of Business, Innovation and Employment.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.


Flight controllers of the International Space Station (ISS) are engaged in shift work to provide 24-h coverage to support ISS systems. The purpose of this study was to investigate the prevalence and associated factors of shift work sleep disorder (SWSD) among Japanese ISS flight controllers.A questionnaire study was conducted using the Standard Shiftwork Index to evaluate sleep-related problems and possible associated variables. Among 52 respondents out of 73 flight controllers, 30 subjects were identified as night shift workers who worked 3 or more night shifts per month. Those night shift workers who answered “almost always” to questions about experiencing insomnia or excessive sleepiness in any case of work shifts and days off were classified as having SWSD. Additionally, 7 night shift workers participated in supplemental wrist actigraphy data collection for 7 to 8 days including 3 to 4 days of consecutive night shifts.Fourteen of 30 night shift workers were classified as having SWSD. Significant group differences were observed where the SWSD group felt that night shift work was harder and reported more frequent insomniac symptoms after a night shift. However, no other variables demonstrated remarkable differences between groups. Actigraphy results characterized 5 subjects reporting better perceived adaptation as having regular daytime sleep, for 6 to 9 h in total, between consecutive night shifts. On the other hand, 2 subjects reporting perceived maladaptation revealed different sleep patterns, with longer daytime sleep and large day-to-day variation in daytime sleep between consecutive night shifts, respectively.As the tasks for flight control require high levels of alertness and cognitive function, several characteristics, namely shift-working schedule (2 to 4 consecutive night shifts), very short break time (5 to 10 min/h) during work shifts, and cooperative work with onboard astronauts during the evening/night shift, accounted for increasing workloads especially in the case of night shifts, resulting in higher or equal prevalence of SWSD to that among other shift-working populations. Further studies are required to collect more actigraphy data and examine the possibility of interventions to improve SWSD. The International Space Station (ISS) is a manned space facility where various scientific activities are conducted for scientific and technological advancement. By utilizing unique features of the space environment, a broad range of scientific experiments in fields including astronomy, materials science, life science, and space medicine have been performed in the ISS . In addiAmong the issues in space mission operation, sleep loss has been considered a primary concern affecting onboard astronauts as well as flight controllers on the ground , 3. BecaSleep disturbances in onboard astronauts are thought to involve a combination of several factors including circadian misalignment due to relatively low light levels in the space vehicle, psychological excitation, space motion sickness, and temporal shifting in scheduled sleep time due to operational demands . By contCurrently, in developed countries, shift work is a common system of work adopted for various types of jobs such as in medical, security, and transportation services; essential infrastructure maintenance; and industrial activities . The behIn this study, we aimed to examine sleep patterns and conditions among the shift workers belonging to the Japan Aerospace Exploration Agency’s (JAXA) Flight Control Team (JFCT), along with their life habits and personality traits. In addition to the behavioral characteristics of work among ISS flight controllers, the shift work conducted by the JFCT includes three major considerations. First, because ISS operation is based on Greenwich Mean Time (GMT), Japan Standard Time (JST) for cooperative work between the JAXA mission control center and the ISS astronauts corresponds to the time from evening through midnight to early morning. Second, breaks during a work shift are limited to the time when telecommunication between the ISS and the JAXA mission control center is operationally interrupted, for 5 to 10 min/h. Third, in their rotating shift work schedule, the number of consecutive night shifts ranged from 2 to 4 days, which might increase the circadian stress compared to a rapid-rotation schedule. Thus, compared to other types of shift work, this combination of situations is both unique and challenging, especially during the night shift. We initially attempted to investigate the prevalence rate and associated factors of SWSD using a questionnaire. Subsequently, supplemental data collection using wrist actigraphy was conducted to obtain objective and detailed sleep information in order to explore the possible relation of SWSD with perceived adaptation to shift work.Members of the JAXA Flight Control Team (JFCT)  who had During the period of the present study, there were 73 total registered members of the JFCT, with 7 to 13 members assigned to respective sections. The period for shifts under a 3-shift-per-day schedule was from 8 a.m. to 5 p.m. (day shift), from 4 p.m. to 1 a.m. (evening shift), and from midnight to 9 a.m. (night shift). The JFCT has been engaged not only in mission control but also in office work, training, and preparing for specific mission operations. The rotating shift schedule of the JFCT members was arranged differently in each of the 7 sections. One section adopted a schedule of 4 consecutive day shifts, evening shifts, and night shifts that were rotated at 8-day intervals. The schedule for the other 6 sections set 2 or 3 consecutive days for each work shift at relatively irregular intervals. Occasionally, the consecutive days of each work shift were decreased to 1 day or increased to 4 days due to vocational or personal reasons. Among those 6 sections, 1 section adopted a schedule where 2 or 3 consecutive days of night shifts were preceded by 1 to 3 consecutive days of evening shifts. The total number of night shifts among the JFCT members ranged from 0 to approximately 7 days/month.A questionnaire study was conducted using the Standard Shiftwork Index . AlthougThe presence/absence of SWSD was judged based on criteria defined by the International Classification of Sleep Disorders (ICSD-2) . AccordiWrist actigraphy is an established procedure to investigate objective sleep-wake patterns using a watch-like device measuring wrist activity, which can record continuously for 24 h/day over long durations . After tThe measurement period for actigraphy was planned from 2 days before the first night shift through 3 or 4 consecutive night shifts until the second day after the last night shift. As the shift schedule of 2 subjects included 1 or 2 days of consecutive evening shifts preceding 3 consecutive night shifts, the actigraphic recordings began 2 days before the first evening shift. Therefore, the planned measurement period ranged from 7 to 9 days for each respective subject. Within 2 weeks after the measurement, the subjects were individually debriefed on the actigraphic results with discussion of their actual circumstances.Actigraphic recordings were analyzed with commercial software  using the Cole-Kripke algorithm  for scorU tests were used for pair-wise post hoc comparisons for continuous and ordinal variables, respectively. The Pearson chi-square test and Fisher’s exact test  were used for categorical variables.Statistical analysis was conducted using PASW Statistics software 17.0.2 (IBM Corporation) and “R” . As multp < 0.05.The actigraphic sleep parameters were analyzed by one-way repeated measures ANOVA to detect the effect of day-to-day variations. The level of significance was considered after the Greenhouse-Geisser correction for repeated measures. Bonferroni tests were used for pair-wise post hoc comparisons. Data on the initial night for 1 subject were missed due to unknown hardware trouble. In addition, the number of consecutive days of night shifts differed among the subjects . Therefore, statistical analysis was conducted for all 7 subjects for the day before the first night or evening shift, the days from the first to third night shifts, and the days after the last night shift. Statistical significance was defined as p = 0.043) and the number of days of night and evening shifts per month . The DW group had a higher ratio of women (38 %) than the other groups. As the number of night shifts was used to determine subjects’ groups, significant differences were observed between night shift workers (SWSD+ and SWSD−) and the other two groups. Regarding the number of evening shifts, post hoc comparisons revealed no significant difference among the ONEW, SWSD−, and SWSD+ groups. The SWSD− and SWSD+ groups were similar in all measured variables, with no significant differences.Fifty-two  of 73 total members of the JFCT completed the questionnaire, yielding a response rate of 71.2 %. Based on the records from work attendance logs for the past 2 months, 30 of 52 respondents were regarded as night shift workers who had worked at least 3 night shifts per month. The remaining 22 members consisted of 8 DW and 14 ONEW subjects. Among 30 night shift workers, 14 members (46.7 %) were identified as SWSD+, who answered “almost always” to any of the questions about experiencing symptoms related to insomnia or excessive wake time sleepiness. While DW subjects did not answer “almost always,” 7 ONEW members (50 %) answered “almost always” to any of those questions. Table χ2 = 7.53, p = 0.02). Post hoc comparisons revealed significantly higher workloads in the SWSD+ group than those in the SWSD− group. In addition, the SWSD+ group demonstrated tendencies to be subjectively unadapted and higher perceived risk of human error in the case of night shifts compared to the SWSD− group. There was no significant group difference in subjective workloads and perceived risk of human error for day and evening shifts.Table χ2 = 12.18, p = 0.002) and “feeling unrestored after sleep”  in the case of night shifts and “difficulty falling asleep”  in the case of evening shifts. Post hoc comparisons revealed that those symptoms were significantly more frequent in the SWSD+ group than in the SWSD− group. Furthermore, the ONEW group had a significantly higher reported frequency of “difficulty falling asleep”  in the case of evening shifts compared to the SWSD− group and a significantly lower reported frequency of “waking up earlier than one’s intention” in the case of night shifts compared to the SWSD+ group.Figure Table According to responses to the questionnaire, 6 of the 7 subjects were identified as SWSD− because they did not answer “almost always” to items regarding insomnia symptoms and excessive wake time sleepiness. Among those 6 SWSD− subjects, 1 subject answered “somewhat unadapted” to the item asking about subjective adaptation to shift work, whereas the other 5 subjects answered “adapted” or “somewhat adapted.” One of the 7 subjects was identified as SWSD+ due to answering “almost always” to items asking about waking earlier than one’s intention and feeling unrestored after sleep in the case of night shifts. This SWSD+ subject answered “unadapted” to the question asking about subjective adaptation to shift work.Figures F  = 12.50, p < 0.001] and STST . As the subjects took daytime naps before reporting to the first night shift, STIB and STST were significantly increased on the day before going to the first night shift (p < 0.05). Significant increases in STIB and STST were also observed on the day after the last night shift because the subjects took daytime naps and/or had a long nocturnal sleep period (p < 0.05). Although those increases in STIB and STST on the days before and after the night shift period were consistently observed among all subjects, the values on the days between consecutive night shifts revealed individual characteristics suggesting possible association with subjective adaptation to shift work. Among the SWSD− subjects who answered “adapted” or “somewhat adapted” to shift work, consisting of 3 evening nappers and 2 non-nappers, STIB on the days between consecutive night shifts was relatively stable, ranging between 6 and 9 h  and the ISS (GMT), the work for the JFCT during evening and night shifts includes direct communication with the onboard astronauts for performing real-time operations including the experimental implementation and troubleshooting of problems or anomalies. By contrast, JAXA day shifts (from 8 a.m. to 5 p.m. JST) correspond to the time for astronauts’ nocturnal sleep and post-sleep activities . These differences in work characteristics between the day and evening/night shift could account for increased subjective workloads during evening/night shifts. It is conceivable that the SWSD+ subjects with sleep deficiency during the night shift periods felt the impact of night shifts more profoundly than the SWSD− subjects.The DW group, consisting of 8 subjects who worked day shifts only, did not answer “almost always” to any of the items concerning insomnia symptoms or excessive wake time sleepiness. Previous studies investigating general populations have demonstrated that 10 %  and 18 %The present study identified SWSD+ subjects based on the criteria of answering “almost always” to any of the questions concerning insomnia or excessive wake time sleepiness. While no validated instrument is currently available to screen for SWSD, several major studies investigating the prevalence of SWSD in nurses , 10 and n = 1968) found associations between SWSD and older age, male sex, and number of nights worked using logistic regression analysis. Comparison between the SWSD+ and SWSD− groups in the present study revealed no difference in those variables, suggesting that those factors might not be associated with SWSD in this specific population. Among demographic and lifestyle variables, the only variable associated with SWSD was the later bedtime of SWSD− subjects in the cases of day shifts and days off, suggesting that SWSD− subjects might tend to favor late hours. However, this speculation is inconsistent with the diurnal score results, which showed no difference in chronotype among the groups. As diurnal score was not a factor associated with SWSD in a study that examined nurses [A previous study  investigd nurses , chronotAs we could not identify definitive associated factors of SWSD in the questionnaire study, an actigraphic study was additionally conducted to obtain objective, real-world information on sleep patterns to examine possible associations with SWSD or subjective adaptation to shift work. Because of the busy and important vocational duties in the JFCT under a nonstandardized shift schedule, this study was limited by issues including the small number of subjects, different night shift schedules among the subjects, lack of objective measurements evaluating cognitive functions, and possible factors under real-life conditions that might affect the results. However, the results may suggest characteristics in sleep patterns for coping with consecutive night shifts. As shown in Fig. It is conceivable that the measured sleep patterns of the remaining 2 subjects between consecutive night shifts were related to their subjective evaluations of SWSD+ and/or lower adaptation. The long daytime TIB with relatively long period of wakefulness  in the SWSD+ subject might induce feelings of waking earlier than one’s intention and/or less refreshment after sleep. Short TIB after the first night shift in the other subject might bring about severe sleepiness and fatigue during the second night shift. As the debriefing confirmed that short TIB after the first night shift occurred because this subject worked on other tasks, such experiences could be likely to induce a feeling of maladaptation to shift work. Because 14 of 30 JFCT night shift workers were identified as SWSD+, there might be other sleep patterns specific to SWSD or low subjective adaptation to shift work. Therefore, further actigraphy data collection and examination of the possibility of interventions to improve SWSD is required in future studies.Considering possible interventions for the JFCT, current working conditions involve several operational constraints. It has been suggested that rapid shift rotation is preferable to reduce the circadian stress associated with night shift work , but sucIn conclusion, the present study demonstrated the possibility of a higher or equal prevalence of SWSD among JFCT shift workers compared to other populations engaged in shift work. As insomnia symptoms and declining working competency were observed especially in the case of night shifts, a combination of several factors including the number of consecutive night shifts and working conditions are considered to make night work more challenging. While the questionnaire results indicated almost no difference in demographic and lifestyle variables between SWSD+ and SWSD− subjects, the results of actigraphic recordings indicated that subjects who were subjectively adapted to shift work maintained more regular and appropriate daytime sleep durations between consecutive night shifts. Those sleep patterns seemed to vary according to individual circumstances, which may reflect genetic characteristics and social situations. Given the small number of subjects who participated in the actigraphic study, a larger sample size is required to identify broader trends in sleep patterns.The present results represent the first reference data on sleep patterns and conditions of the ISS flight controllers. As ISS mission control is operated internationally by mission control centers located in the USA, Russia, Germany, and Japan, investigating and comparing sleep conditions in shift-working situations across these mission control centers could contribute to establishing an appropriate shift work system according to the circumstances of each country.


We integrated, for the first time, postmyocardial infarction electrical and autonomic remodeling in a detailed, validated computer model of β-adrenergic stimulation in ventricular cardiomyocytes. Here, we show that β-adrenergic stimulation inhibits alternans and provide novel insights into underlying mechanisms, adding to a recent controversy about pro-/antiarrhythmic effects of postmyocardial infarction hyperinnervation. 2+ and/or repolarization alternans and may differentially affect ventricular arrhythmogenesis. Here, we used a detailed computational model of the canine ventricular cardiomyocyte to study the determinants of alternans in the BZ and their regulation by β-adrenergic receptor (β-AR) stimulation. The BZ model developed Ca2+ transient alternans at slower pacing cycle lengths than the control model, suggesting that the BZ may promote spatially heterogeneous alternans formation in an infarcted heart. β-AR stimulation abolished alternans. By evaluating all combinations of downstream β-AR stimulation targets, we identified both direct (via ryanodine receptor channels) and indirect [via sarcoplasmic reticulum (SR) Ca2+ load] modulation of SR Ca2+ release as critical determinants of Ca2+ transient alternans. These findings were confirmed in a human ventricular cardiomyocyte model. Cell-to-cell coupling indirectly modulated the likelihood of alternans by affecting the action potential upstroke, reducing the trigger for SR Ca2+ release in one-dimensional strand simulations. However, β-AR stimulation inhibited alternans in both single and multicellular simulations. Taken together, these data highlight a potential antiarrhythmic role of sympathetic hyperinnervation in the BZ by reducing the likelihood of alternans and provide new insights into the underlying mechanisms controlling Ca2+ transient and repolarization alternans.The border zone (BZ) of the viable myocardium adjacent to an infarct undergoes extensive autonomic and electrical remodeling and is prone to repolarization alternans-induced cardiac arrhythmias. BZ remodeling processes may promote or inhibit CaNEW & NOTEWORTHY We integrated, for the first time, postmyocardial infarction electrical and autonomic remodeling in a detailed, validated computer model of β-adrenergic stimulation in ventricular cardiomyocytes. Here, we show that β-adrenergic stimulation inhibits alternans and provide novel insights into underlying mechanisms, adding to a recent controversy about pro-/antiarrhythmic effects of postmyocardial infarction hyperinnervation.http://ajpheart.podbean.com/e/%CE%B2-ar-stimulation-and-alternans-in-border-zone-cardiomyocytes/.Listen to this article’s corresponding podcast at  Anith (SCD) , 35. Morth (SCD) , which ith (SCD) .2+ handling processes (2+ release-reuptake mismatch (2+ release (2+ transient (CaT) alternans formation cellular mechanisms controlling Calternans , 31, 62.lternans , 62. How2+ release, both through activation of RyR2 channels and indirectly through regulation of SR Ca2+ load, in the suppression of alternans. The tools and insights resulting from this work facilitate a more comprehensive understanding of the complex pathophysiology involved in ventricular arrhythmogenesis post-MI.Here, we hypothesized that β-AR stimulation would inhibit CaT and repolarization alternans in BZ cardiomyocytes and multicellular tissue. We characterized the interplay of BZ electrophysiological remodeling and β-AR stimulation on the likelihood of alternans by fusing an existing state-of-the-art computational model of β-AR stimulation in the canine ventricular cardiomyocyte  with a m2+ current (ICaL), phospholamban (PLB), slowly activating delayed rectifier K+ current (IKs), RyR2, troponin I (TnI), fast Na+ current (INa), Na+-K+ pump current (INaK), and ultrarapid plateau K+ current (IKur). A diagram of the model is provided in appendix a. The model can reproduce, among other things, ISO-dependent changes in cAMP levels, target phosphorylation, and electrophysiological properties, including action potential (AP) duration (APD) and CaT amplitude. We simplified the activation of RyR2 by β-AR stimulation in the model to facilitate its analysis while maintaining all relevant properties of the original model behavior. In particular, the number of parameter differences between phosphorylated and nonphosphorylated formulations has been reduced from four to three ; full details and motivation are provided in appendix b.We used the canine cardiomyocyte model by Heijman et al. , includi2+/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation rate (+425%), ICaL (−36%), INa (−61%), transient outward K+ current , background Ca2+ current , and time-independent inward rectifier K+ current . BZ cells maintain the original model of β-AR stimulation unless indicated otherwise.A BZ cell was modeled using the Heijman et al. model by applying BZ electrophysiological remodeling described in Hund et al. , changin2+ release due to β-AR stimulation-dependent RyR2 regulation to study the influence on alternans formation, as provided in appendix c.To demonstrate the generality of the results, key experiments were repeated in the O’Hara-Rudy human ventricular cardiomyocyte model  by impleappendix d). To determine the impact of downstream effects of β-AR stimulation separately , the signaling cascade of the model was turned off and the phosphorylation levels of various sites were manually clamped to either 0 (nonphosphorylated) or to 1 (fully phosphorylated). The model was modified to similarly allow isolating the distinct effects of RyR2 activation via β-AR stimulation.Simulated fixed-rate pacing was used to study the formation of repolarization alternans and underlying CaT alternans at a given basic pacing cycle length (BCL). The pacing stimulus was a 1-ms-long pulse at −80 pA/pF, because in less excitable BZ cells the duration of 0.5 ms common in in silico studies failed to trigger an AP at fast pacing rates. Amplitude of repolarization alternans was defined as maximum APD − minimum APD of the last 10 APDs of each simulation. The alternans ratio of APD or CaT amplitude was defined as 1 – minimum/maximum, reflecting the minimum and maximum values of two successive beats, respectively, in line with previous experimental studies , 18. A sAmerican Journal of Physiology-Heart and Circulatory Physiology website.).Single cell simulations were performed using the previously published MATLAB  code of the Heijman et al. model  using thE and F). In contrast, the magnitude of maximal APD and CaT alternans was slightly smaller in BZ  compared with NZ  models. The β-AR stimulation abolished both APD and CaT alternans in NZ and BZ models at all BCLs.Our model replicated characteristics of the experimentally observed alternans formation and its relationship to β-AR stimulation , as showTo discern which of the eight downstream targets of β-AR signaling incorporated in the model are responsible for the abolishment of CaT alternans, we analyzed CaT alternans formation at bcl of 260 ms for all 256 combinations of either dephosphorylated or fully phosphorylated targets. The strongest alternans-abolishing effect was achieved with the simulated phosphorylation of RyR2, preventing CaT alternans in all 128 combinations involving this downstream effect (not shown).7) possible combinations of β-AR signaling phosphorylation targets in the absence of RyR2 phosphorylation, 88 of which showed alternans at a BCL of 260 ms  and 40 of which did not . D and E, shows the amount of APD and CaT alternans as a function of the total amount of Ca2+ released from the SR during two BCLs  for each of these 128 combinations. Combinations without RyR2 phosphorylation that did not manifest APD, and CaT alternans could be divided into two major groups. The first group involved those combinations of downstream effects that diminished SR Ca2+ release . Alternans formation in the family of models used in this study is predominantly due to a mismatch between Ca2+ release and Ca2+ reuptake at rapid pacing rates, causing the next Ca2+ release to be smaller, which, in turn, makes the next release larger, etc. , optionally with other β-AR-mediated effects that do not decrease SR Ca2+ release. In this case, the overall SR Ca2+ load increased and the junctional SR (JSR) emptied almost completely during releases. In this setting, increased loading of the network SR (NSR) increased Ca2+ diffusion from the NSR to JSR, preventing CaT alternans, since even after a large Ca2+ release, the reuptake and NSR-to-JSR-transport mechanisms were fast enough to maintain a normal subsequent release. To illustrate this effect, we considered two combinations of downstream effects: C1, free of alternans due to large Ca2+ release (simulated phosphorylation of ICaL + PLB + TnI + INa), and C2, manifesting alternans (phosphorylation of ICaL + IKur), with the large Ca2+ release in C2 closely matching the Ca2+ releases of C1. The Ca2+ handling properties of these variants during two APs at a BCL of 260 ms are shown in 2+ release was nearly identical despite the different NSR contents, which affected the speed of JSR transport (Jtr) and thus JSR refilling. Because in the C2 model the JSR was not refilled sufficiently after the first release, and given that the SR Ca2+ load-release relationship is steep, the next Ca2+ release is comparatively small and a relatively large amount of Ca2+ remains in the JSR. More Ca2+ is then added to the JSR during and after the second release, but the process is limited by the high level of Ca2+ in the JSR, reducing the concentration gradient between NSR and JSR (2+ release flux (Jrel) at the beginning of the shown interval. In this case, the second release was almost identical to the first release, supporting the importance of NSR loading (promoted by a high Ca2+ reuptake rate) in alternans prevention.The second major group of β-AR stimulation-dependent effects preventing the formation of alternans was the group with the largest SR Ca and JSR . As a thJrel over two APs (ranging from 5.2 to 16.4 mmol/l). Thus, the total amount of Ca2+ released is not the key mechanism behind the abolishment of alternans under these conditions. To discern the mechanism behind the antialternans effect of RyR2 activation, we isolated and separately simulated the three downstream effects of β-AR stimulation on RyR2 biophysical properties. Under maximal β-AR stimulation, the time constant of RyR2 activation and deactivation was reduced to ~21% of the original value, the SR Ca2+ release magnitude was reduced to 65%, and the JSR Ca2+ leak was increased as in the original model. All effects of β-AR stimulation other than RyR2 activation were disabled, unless stated otherwise, to isolate the effects of altered RyR2 dynamics from other downstream effects.With simulated downstream effects of β-AR stimulation on RyR2, alternans was never present, independent of the integrated 2+ load.We simulated all eight possible combinations of the three β-AR-stimulation-induced changes to release dynamics . Most co2+ release  produced an immediate decrease in the duration of SR Ca release C, which  release F. With reds Jrel D, resulteds Jrel E. There 2+ leak was an increased Ca2+ concentration in the subspace between RyR2 and L-type Ca2+ channels  was chiefly due to limiting the amount of Ca2+ released, which acted first during the large SR Ca2+ release, thereby providing more Ca2+ for the next release, similar to an increase in SR Ca2+ leak.Finally, the antialternans effect of decreasing the RyR2-mediated amplitude of SR Ca2+ released over two consecutive APs , suggesting that the accelerated SR Ca2+ release dynamics are the most important factor limiting alternans.To investigate the robustness of these results, we evaluated a range of RyR2_Amp and RyR2_τ values, measuring the APD alternans amplitude and the integral of SR Cative APs . A wide 2+ release time constant on alternans formation in another computational model, the state-of-the-art human ventricular cardiomyocyte model by O’Hara et al. , which was not sufficient to produce APD alternans (right). Increasing the Na+ current amplitude by 25% resulted in robust CaT and APD alternans throughout the strand, which could be inhibited by simulated β-AR stimulation (A and B). We investigated the mechanism underlying the modulation of alternans by cell-to-cell coupling and found that the reduced AP upstroke due to the electrotonic load resulted in a smaller peak ICaL, which triggered a smaller SR Ca2+ release  and more than four times that of NZ cells when taking into account the remodeling of β-AR expression (−25%) that has been reported to occur in BZ tissue .Numerous studies have investigated the subcellular determinants of CaT and repolarization alternans , 46, 62.aT decay . The antaT decay , 18. How2+-independent mechanism that may also be affected by altered cardiac metabolism during long-term sympathetic stimulation. Spatially heterogeneous alternans is significantly more proarrhythmic than spatially homogeneous (concordant) alternans because of the presence of steep spatial repolarization gradients .Arrhythmias are an intrinsically multicellular phenomenon, and the effects of sympathetic stimulation on alternans in tissue appear more complex. Huang et al.  found thradients . Restoriradients . Hammer radients  showed t2+ release via RyR2 or other downstream targets of β-AR stimulation can inhibit alternans formation.We identified a central role for the modulation of SR calcium release properties in the inhibitory effects of β-AR stimulation on CaT alternans. In particular, either direct or indirect modulation of SR Ca2+ release   . There, 2030 and Ser2809 by PKA and on Ser2815 by CaMKII in response to sympathetic stimulation. The exact pathways and phosphorylation sites mediating β-AR stimulation-dependent modulation of RyR2 and their relevance for cardiac (patho)physiology remain controversial . Furthermore, β-blockers can, surprisingly, increase cardiac innervation density  and, in 2+ handling is spatially heterogeneous and subject to strong local positive feedback systems. Previous work has shown that stochastic channel gating and local Ca2+ handling produce complex temporal repolarization variability , the British Heart Foundation Centre of Research Excellence in Oxford (RE/13/1/30181), an NC3R Infrastructure for Impact award (NC/P001076/1), an Engineering and Physical Sciences Research Council Impact Acceleration Award (EP/K503769/1), and the ComBioMed project funded by the European Union’s Horizon 2020 research and innovation program (Grant Agreement 675451). J. Heijman was supported by the Netherlands Organization for Scientific Research (NWO/ZonMW Veni 91616057).No conflicts of interest, financial or otherwise, are declared by the author(s).J.T., B.R., G.B., and J.H. conceived and designed research; J.T. and J.H. performed experiments; J.T., B.R., G.B., and J.H. analyzed data; J.T., B.R., G.B., and J.H. interpreted results of experiments; J.T. and J.H. prepared figures; J.T. and J.H. drafted manuscript; J.T., B.R., G.B., and J.H. edited and revised manuscript; J.T., B.R., G.B., and J.H. approved final version of manuscript.


Recently, we identified a Ca2+ channel (TcIP3R) associated with intracellular Ca2+ stores in Trypanosoma cruzi, the parasitic protist that causes Chagas disease. In this study, we measured [Ca2+]i during the parasite life cycle and determined whether TcIP3R is involved in the observed variations. Parasites expressing R‐GECO1, a red fluorescent, genetically encoded Ca2+ indicator for optical imaging that fluoresces when bound to Ca2+, were produced. Using these R‐GECO1‐expressing parasites to measure [Ca2+]i, we found that the [Ca2+]i in epimastigotes was significantly higher than that in trypomastigotes and lower than that in amastigotes, and we observed a positive correlation between IPTc3R mRNA expression and [Ca2+]i during the parasite life cycle both in vitro and in vivo. We also generated R‐GECO1‐expressing parasites with TcIP3R expression levels that were approximately 65% of wild‐type (wt) levels (SKO parasites), and [Ca2+]i in the wt and SKO parasites was compared. The [Ca2+]i in SKO parasites was reduced to approximately 50–65% of that in wt parasites. These results show that TcIP3R is the determinant of [Ca2+]i in T. cruzi. Since Ca2+ signaling is vital for these parasites, TcIP3R is a promising drug target for Chagas disease.Regulation of intracellular Ca In mammals, [Ca2+]i is regulated by several factors, including Ca2+ influx into the cytosol through voltage‐gated Ca2+ channels (VGCCs), receptor‐operated Ca2+ channels (ROCs), and store‐opened Ca2+ channels (SOCs); buffering of Ca2+ with plasma membrane and cytosolic proteins; accumulation of Ca2+ in intracellular Ca2+ stores through the sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA); and efflux of Ca2+ from stores through Ca2+ channels, such as inositol 1,4,5‐trisphosphate receptors (IP3Rs), and ryanodine receptors (RyRs) Calcium ion Ca+ is the eptors IPRs, and rTrypanosoma cruzi is the parasitic protist that causes Chagas disease in Latin America. At present, only two drugs are available for Chagas disease (benznidazole and nifurtimox), and these often induce severe side effects and are effective for only the acute phase of the disease. Since no practical drug or vaccine for Chagas disease is available, new treatments are greatly needed 2+]i regulation is vital for T. cruzi, and the molecular mechanisms of [Ca2+]i regulation in the parasite are thought to be quite different from those in mammalian cells 2+ transporters—ROCs, SOCs, or Na+/Ca2+ exchangers, have been detected in Trypanosomes. The results of a proteome analysis of T. brucei suggested that a putative VGCC is localized to the flagellum 2+ ATPase (PMCA) have also been reported; one is localized on the plasma membrane, and the other is localized to the acidocalcisome of T. bruceiT brucei like mammalian cells 3R homolog in T. cruzi (TcIP3R), and showed that it is mainly localized to the ER. When the expression level of TcIP3R is reduced to less than one‐half of that in wild‐type (wt) cells, the parasite cannot grow 3R may be a promising drug target 3R regulates parasite growth, transformation, infectivity, and virulence in mammalian hosts, indicating that TcIP3R is an important regulator of the parasite life cycle 2+ indicators, such as Fura‐2, showed that Ca2+ signaling is important for host cell invasion i in the parasite changes significantly during its life cycle, and that TcIP3R is the determinant of [Ca2+]i in T. cruzi.In this paper, we reported the successful preparation of parasites expressing R‐GECO1  using specific primers  and KOD‐Plus Neo . The PCR‐amplified gene was inserted into pENTR/D‐TOPO . The resultant plasmid, pENTR/R‐GECO1, was converted to a pTREX vector R)‐GW; Life Technologies) using the Gateway recombination system, to generate pTREX (neoR)‐GW/R‐GECO1.The R)‐GW/R‐GECO1, we used PCR with pTREX (neoR)‐GW/R‐GECO1 as the template and the primers (forward: 5′‐GGGGATCGATCCGGAACAA‐3′ and reverse: 5′‐ATTGGCTGCAGGGTCGCT‐3′). These two PCR fragments were ligated with DNA Ligation Kit Ver. 2.1 , to generate pTREX (purR)‐GW/R‐GECO1.The puromycin resistance gene was amplified by PCR using pBApo‐CMV Pur DNA plasmid vector  as the template with the specific primers (forward: 5′‐ATGACCGAGTACAAGCCCAC‐3′ and reverse: 5′‐TCAGGCACCGGGCTTGC‐3′). To remove the neomycin resistance gene from pTREX , which is a cell‐impermeant Ca2+ chelator and reduces the levels of extracellular Ca2+, and 1,2‐Bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic Acid, tetraacetoxymethyl ester (BAPTA‐AM), which is a cell‐permeant Ca2+ chelator thereby reduces [Ca2+]i, were purchased from Dojindo Molecular Technologies, Inc. , and an IP3R inhibitor, 2‐aminoethoxydiphenyl borate (2‐APB), was purchased from Sigma‐Aldrich .Epimastigotes of the 7 epimastigotes were resuspended in Amaxa Basic® Parasite Nucleofector Kit 2 solution . The resuspended wt or TcIP3R SKO parasites R)/R‐GECO1 or pTREX (purR)/R‐GECO1, respectively, and then electroporated with an Amaxa Nucleofector Device (Lonza) using program U‐033. Stable wt transformants expressing R‐GECO1 were selected by incubating the cells for 30 days in LIT medium containing 0.5 mg·mL−1 G418, and then clonal derivatives were isolated by limiting dilution. The stable SKO transformants expressing R‐GECO1 were selected by incubating the cells for 30 days in LIT medium containing 0.25 mg·mL−1 G418 and 3 μg·mL−1 puromycin. Amastigotes or trypomastigotes stably expressing R‐GECO1 were transformed from the epimastigotes expressing R‐GECO1 using the methods described above.A total of 1 × 10Fmax) of R‐GECO1 in individual parasites was determined by treating them with high Ca2+ solution (10 mm) and ionomycin , which increase the cytosolic Ca2+ content of parasites so as to saturate R‐GECO1 with Ca2+. To convert fluorescence intensity to Ca2+ concentration, the following formula was used:Kd is the dissociation constant for R‐GECO1 (482 nm); n is the Hill coefficient of R‐GECO1 (2.0); F is the fluorescence intensity in each parasite; FMAX is the maximal fluorescence intensity of R‐GECO1 in the parasite (see above); and FMIN is the minimal intensity of R‐GECO1 calculated using the ratio change value obtained by Zhao et al. (= 1/16 of FMAX).Fluorescence images of the parasites were acquired using a fluorescent microscope  or a laser confocal microscope . After acquiring fluorescence images in normal culture medium, the maximal fluorescence signal  using one‐way ANOVA or Student's t‐test.Statistical analysis was performed with 2+]i in T. cruzi during its life cycle, parasites expressing R‐GECO1 were prepared. The R‐GECO1 gene was cloned in the T. cruzi pTREX expression vector R‐GECO1 is expressed under the ribosomal promoter; therefore, R‐GECO1 was constitutively expressed throughout the parasite life cycle. We at first investigated whether the fluorescence signal in the parasites expressing R‐GECO1 was reduced after treatment with a cell‐permeant Ca2+ chelator BAPTA‐AM. After replacement of the parasite medium with PBS, BAPTA‐AM  was added to the PBS to reduce Ca2+, and [Ca2+]i was measured after 3 min  and epimastigotes (327 ± 44 nm) than in trypomastigotes (85 ± 39 nm). These results indicated that the [Ca2+]i changes significantly during the progression of the parasite life cycle. Ca2+ oscillation was not detected at any stage.To exclude variations in signal intensity among the parasite clones, a clonal derivative was isolated and used for the experiments. Figure 2+]i of parasites during intracellular growth. 3T3‐Swiss cells were infected with R‐GECO1‐expressing trypomastigotes, and then the growth of one parasite was monitored under a fluorescent microscope . These results suggest that TcIP3R is important for the regulation of [Ca2+]i in T. cruzi.Previously, we reported that 3R is involved in the regulation of [Ca2+]i in T. cruzi, the level of TcIP3R was reduced in parasites expressing R‐GECO1, and the [Ca2+]i in wt and mutant parasites was measured  parasites, in which one of the TcIP3R genes was disrupted by homologous recombination TcIP3R gene by Southern blot analysis and an approximately 35% reduction in TcIP3R expression levels in the SKO parasites, and these parasites show various phenotypes, such as inhibition of epimastigote growth R‐GECO1 gene was cloned into an expression plasmid vector for T. cruzi containing a puromycin resistance gene (pTREX(purR)), and then this plasmid was transfected into the SKO parasites. SKO parasites expressing R‐GECO1 were selected in culture medium containing G418 and puromycin. For the control, wt Tulahuen strain parasites were transfected with pTREX(purR)/R‐GECO1 and selected in culture medium containing puromycin. Since the expression level of R‐GECO1 among the parasite clones might vary, the fluorescence intensity in the parasites was randomly measured without cloning. The fluorescence intensity in the SKO parasites was significantly lower than that in the wt parasites. Importantly, the TcIP3R expression level in the SKO parasites was reduced to approximately 65% of wt levels To investigate whether TcIP2+ influx from the extracellular fluid or efflux to the extracellular fluid is important for maintenance of [Ca2+]i  into the cytosol by TcIP3R should be effectively returned to the store(s) by SERCA Next, we investigated whether Ca+]i Fig. C. Excess2+]i are kept at low concentrations (~ 100 nm) in the absence of extracellular stimuli 2) to generate IP3, which activates IP3R and transiently increases [Ca2+]i2+]i in epimastigotes and amastigotes is constitutively high. Recently, it has been reported that Trypanosoma brucei PI‐PLC may be constitutively activated T. cruzi PI‐PLC have been reported to be similar to that of T. brucei PI‐PLC T. cruzi PI‐PLC might maintain high [Ca2+]i through constitutive TcIP3R activation.In animal cells, [Ca2+]i is determined by the balance between Ca2+ influx and efflux, and Ca2+ release via IP3R does not result in higher [Ca2+]i2+ influx increases through the SOC mechanism activated by Ca2+ release from the ER, thereby resulting in an increase of [Ca2+]i2+]i in T. cruzi is not always increased through TcIP3R directly but the parasites have some unknown mechanism(s) to increase Ca2+ influx. Interestingly, since amastigotes parasitize the host cell cytoplasm, where the concentration of Ca2+ is much lower than the [Ca2+]i in amastigotes, the parasites may not receive Ca2+ from outside through a SOC‐like mechanism. However, how amastigotes maintain high [Ca2+]i within the host cells remains unknown at present.According to the cell boundary theorem, [Ca2+]i levels in T. cruzi are determined by the level of TcIP3R expression. Since Ca2+ signaling is essential for the parasite and the primary structure of TcIP3R shares low similarity with that of mammalian IP3Rs, TcIP3R, the key Ca2+ signaling molecule, is a promising drug target for Chagas disease.In conclusion, our present study revealed that basal i within host cells. 3T3‐Swiss albino cells were infected with R‐GECO1‐expressing trypomastigotes. A bright‐field movie of cells that are heavily infected with R‐GECO1‐expressing amastigotes and trypomastigotes is shown (A). A representative microscopic movie was obtained with an inverted microscope . Note that the trypomastigotes in the host cell move intensely. A fluorescent image of the same field is also shown (B).Click here for additional data file. Click here for additional data file.


The importance of epigenetic dysregulation to acute myeloid leukemia (AML) pathophysiology has become increasingly apparent in recent years. Epigenetic regulators, including readers, writers, and erasers, are recurrently dysregulated by way of chromosomal translocations, somatic mutations, or genomic amplification in AML and many of these alterations are directly implicated in AML pathogenesis. Mutations in epigenetic regulators are often discovered in founder clones and persist after therapy, indicating that they may contribute to a premalignant state poised for the acquisition of cooperating mutations and frank malignancy. Apart from the proto-oncogenic impact of these mutations, the AML epigenome is also shaped by other epigenetic factors that are not mutated but co-opted by AML oncogenes, presenting with actionable vulnerabilities in this disease. Targeting the AML epigenome might also be important for eradicating AML leukemia stem cells, which can be critical for disease maintenance and resistance to therapy. In this review, we describe the importance of epigenetic regulators in AML. We also summarize evidence implicating specific epigenetic regulators in AML pathobiology and discuss emerging epigenome-based therapies for the treatment of AML in the clinic. Acute myeloid leukemia (AML) is a clonal malignancy resulting from the transformation of hematopoietic stem and progenitor cells. AML is marked by enhanced proliferation and impaired differentiation of immature myeloid progenitors. Over the past few decades, strategies for treating AML have remained largely unchanged, although survival outcomes have improved, especially in younger patients . DespiteAcute myeloid leukemia is highly heterogeneous in terms of its underlying genetics, pathobiology, and clinical manifestation. Even though the morphological and cytogenetic heterogeneity of AML has been recognized for several years, the marked molecular heterogeneity has only come to the fore recently. Emerging evidence from genome-scale studies propelled by advances in next-generation sequencing (NGS) has substantially broadened our knowledge of the spectrum and frequency of mutations in AML. Characterization of the genomic AML landscape has led to the identification of recurrent mutations in a number of previously uncharacterized genes in AML. The classes of genes mutated in AML include transcription factors, kinases, cell cycle regulators, spliceosomal genes, and epigenetic regulators. The observation that genes encoding epigenetic regulators are among the most commonly occurring mutated factors in AML, strongly points to a role of epigenome dysregulation in AML pathogenesis. These mutations in epigenetic regulators encompass a broad spectrum of epigenetic writer, eraser, and reader proteins which will be the focus of this review. The epigenome is dynamically regulated through chemical modification of DNA and RNA as well as the histone proteins around which DNA is packaged. Our genomes harbor a number of enzymes that deposit these chemical marks (writers), or remove them (erasers), dedicated to specific modifications of DNA or chromatin. Proteins with specialized domains that can selectively bind to specific DNA, RNA, or histone modifications (readers) also abound, indicating a well-orchestrated mechanism for relaying epigenetic marks to downstream effectors. The coordinated action of epigenetic reader, writer, and eraser proteins is important for regulation of various cellular processes, including transcription, DNA replication, cell cycle control, and the DNA damage response. Recurrent genomic alterations in epigenetic writer, reader, and eraser proteins, such as DNA methyltransferase 3A (DNMT3A), TET1/2, IDH1/IDH2, EZH2, mixed-lineage leukemia (MLL), NSD1/3, AF10, ENL, and other epigenetic regulators have been cataloged in AML, inspiring a wave of preclinical studies aimed at uncovering causal links between epigenome dysregulation and leukemogenesis , FLT3-ITD , and KRAS/NRAS, are acquired later during the development of AML , isocitrate dehydrogenases (IDH1/IDH2), methylcytosine dioxygenases of the ten-eleven-translocated (TET) family, and human homologs of the Drosophila polycomb complex such as Enhancer of Zeste 2 (EZH2) and additional sex-combs like genes (ASXL1/2) have been discovered in AML and myelodysplastic syndromes and myeloproliferative neoplasms (MDS/MPN), and many of these mutations have been causally linked to myeloid transformation in murine models. The role of these epigenetic modifiers in AML pathobiology and studies exploring these proteins as druggable targets will be described in detail below. Apart from genes mentioned above, there are a number of examples of epigenome modulators that are not directly mutated but nevertheless implicated in AML pathogenesis. Several chromatin modifiers have been discovered as selective dependencies in specific AML subtypes as discussed in the Section “Ever since chromosomal translocations and fusion oncogenes were discovered in AML several years ago, it was apparent that chromatin modulators such as the “writers” MLL1/KMT2A, CBP/p300, and NSD1/KMT3B might have causative roles in AML pathogenesis. MLL1, CBP, and NSD1/3 are involved in recurrent chromosomal translocations in a fraction of AML patients. These translocations were discovered early because they could be observed using methods, such as karyotyping and fluorescence DNMT1, DNMT3A, and DNMT3B encode methyltransferases that catalyze this reaction. DNMT3A and DNMT3B are largely de novo DNMTs, whereas DNMT1 predominantly plays a role in the maintenance of DNA methylation  interacts with the Polycomb repressive complex 1 (PRC1) to silence genes, suggesting that PRC1 activity could be an attractive target in DNMT3A-mutant tumors . The DNMT family, including hylation . CpG clusurvival . A majorent cell , 35. TheHSC pool , myeloidt tumors .IDH1 and IDH2 mutations are found at a frequency of 10–20%, and these mutations are more common in the cytogenetically normal sub-group of AML. IDH1 and IDH2 mutations are mutually exclusive and result in a gain of neomorphic activity . 2-HG acts as an “oncometabolite” since its accumulation in leukemic cells interferes with the enzymatic functions of several chromatin modifiers that use α-KG as a cofactor. Mechanistic investigations into the model of action of IDH mutations have shown that hematopoietic specific IDH1 (R132H) mutation using a conditional knock-in strategy expands HSC and myeloid progenitor compartments but fail to show signs of overt AML . The IDH1 and IDH2 proteins are nicotinamide adenine dinucleotide phosphate (NADP+)-dependent enzymes that mediate a number of important cellular processes including lipid metabolism, glucose sensing, and oxidative phosphorylation . IDH1 anactivity . Specifivert AML . SimilarIDH mutations is the TET family of methylcytosine dioxygenases. Normally, TET2, with the cofactor molecule α-ketoglutarate (α-KG), converts 5-mC to 5-hydroxymethylcytosine (5-hmC), which can then be demethylated back to cytosine via a series of intermediate steps  genes and their cofactor MEIS1. These HOX/MEIS genes are crucial for perpetuating the highly self-renewing state that is triggered by MLL-fusion protein expression in transformed hematopoietic progenitors. Indeed, several recent studies have shown that oncogenesis by MLL-fusion proteins requires the coordinate action of a number of chromatin factors that are essential and rate limiting for the transcriptional activation of HOX/MEIS genes. A prime example of this is the histone methyltransferase (HMT) DOT1L. The DOT1L protein biochemically interacts with several of the most common MLL-fusion partners, including AF4, AF9, ENL, AF10, and AF17 –NSD1 fusions, NPM1 mutations or mutations in the DNMT3A gene , embryonic ectoderm development, suppressor of zeste homolog, and RbAp46/48, also known as RBBP4. PRC1 composition is more variable with only two core components RING1A and RING1B which complex together with the proteins BMI1, MEL18, or NSPC1  proteins are transcriptional repressors that regulate key fundamental processes, including cellular identity, differentiation, and stem cell plasticity . PcG proor NSPC1 . The PRCor NSPC1 –16. Thesutations , suggest effects , whereasisorders . These rBMI1 oncogene is implicated in the self-renewal of normal as well as leukemic stem cells in AML  H3K4 demethylase is fused to NUP98 in approximately 10% of pediatric acute megakaryoblastic leukemia resulting in the cytogenetically cryptic NUP98–JARID1A translocation. These fusions are believed to compromise normal functions both of NUP98 as well as JARID1A, leading to leukemogenesis  , 84. In d in AML , furthermonocytic leukemia zinc-finger MOZ (MYST3) protein and its paralog MORF (MYST4) are HATs involved in recurring chromosomal rearrangements in AML. The balanced chromosomal translocation t, which is found in <1% of AML patients, leads to in-frame fusions of MOZ with the HAT CBP  inhibitors—has been shown to reverse the commonly observed silencing of tumor suppressor genes (TSG) and restore normal differentiation. Since these epigenetic processes are involved in both silencing as well as activation of transcription dependent on the epigenetic mark and the chromatin context, it may be very difficult to identify which subset of AML may benefit most from broad-based epigenomic reprogramming therapies. More targeted therapies require the identification of specific silenced TSG or activated oncogenes for targeted therapeutics Figure . NeverthDNA methylation is dysregulated in most cancers including leukemia and has been the preferred target for cancer therapy since the development of hypomethylating agents (HMA). The HMAs AZA and DAC are nucleoside analogs and inhibitors of the DNMT enzymes DMNT1 and DMNT3. Investigational treatment with AZA and DAC in AML started more than 40 years ago [reviewed in Ref .]. AZA ain vitro and in vivo  2, BRD3, and BRD4, which are universally expressed, while BRDT expression is limited to the testes  protein family serve as transcriptional adapter molecules that facilitate transcription –160. Thee testes . Diversee testes  and are e testes –166. Thee testes , 168. Ree testes , 169–172e testes , 172. OTe testes . BETi hain vitro cytotoxic effects and significantly improved the survival of mice engrafted with AML cells without overt toxicity  was developed that was shown to confer significant survival benefits in a mouse model of IDH mutant leukemia and also in a xenografts model of primary human AML  was developed by Epizyme Inc. as a potent and selective inhibitor of the methyltransferase activity of DOT1L . Using tic drugs , 191. EPngements , 187. EPin vivo  and Salgomed Inc. (San Diego). Y-CS and B-RC declare no conflicts of interest.