Liver cancer (hepatocellular carcinoma) is a significant health burden worldwide. It is often not detected until at an advanced stage when there are few treatment options available. Changes in calcium concentrations within liver cancer cells are essential for regulating their growth, death, and migration (metastasis). Our aim was to review published papers which have identified proteins involved in calcium signaling as potential drug targets for the treatment of liver cancer. About twenty calcium signaling proteins were identified, including those involved in regulating calcium concentrations in the cytoplasm, endoplasmic reticulum and mitochondria. A few of these have turned out to be sites of action of natural products previously known to inhibit liver cancer. More systematic studies are now needed to determine which calcium signaling proteins might be used clinically for treatment of liver cancer, especially advanced stage cancers and those resistant to inhibition by current drugs.2+-signaling proteins identified as potential targets for therapeutic treatment at different stages of HCC. These potential targets include inhibition of the self-renewal properties of liver cancer stem cells; HCC initiation and promotion by hepatitis B and C and non-alcoholic fatty liver disease ; and cell proliferation, tumor growth, migration and metastasis. A few of these Ca2+-signaling pathways have been identified as targets for natural products previously known to reduce HCC. Promising Ca2+-signaling targets include voltage-operated Ca2+ channel proteins (liver cancer stem cells), inositol trisphosphate receptors, store-operated Ca2+ entry, TRP channels, sarco/endoplasmic reticulum (Ca2++Mg2+) ATP-ase and Ca2+/calmodulin-dependent protein kinases. However, none of these Ca2+-signaling targets has been seriously studied any further than laboratory research experiments. The future application of more systematic studies, including genomics, gene expression (RNA-seq), and improved knowledge of the fundamental biology and pathology of HCC will likely reveal new Ca2+-signaling protein targets and consolidate priorities for those already identified.Hepatocellular carcinoma (HCC) is a considerable health burden worldwide and a major contributor to cancer-related deaths. HCC is often not noticed until at an advanced stage where treatment options are limited and current systemic drugs can usually only prolong survival for a short time. Understanding the biology and pathology of HCC is a challenge, due to the cellular and anatomic complexities of the liver. While not yet fully understood, liver cancer stem cells play a central role in the initiation and progression of HCC and in resistance to drugs. There are approximately twenty Ca Intracll death ,32,49.2+-signaling tool-kit” in the adult hepatocyte is similar to that in many other non-excitable mammalian cells . Howeve2+-signaling in hepatocytes have been conducted using isolated rat or mouse hepatocytes, liver cell lines established from rat or mouse liver tumors and in liver cell lines established from human liver tumors. Ca2+-signaling proteins and pathways in normal human hepatocytes are less well characterized. However, most of the studies reported in this review involve human liver tissue and liver cell lines derived from human liver. While the Ca2+-signaling pathways in normal human adult hepatocytes are likely to be broadly similar to those in normal rat and mouse adult hepatocytes, there are some differences.Most studies of Ca3 receptors (InsP3R) are expressed in adult rat and mouse hepatocytes, although the level of expression of type 3 InsP3R (InsP3R3) is low. The roles of type 2 InsP3 receptors (InsP3R2) in generating oscillations in [Ca2+]cyt and in the regulation of bile secretion and Ca2+-signaling in the hepatocyte nucleus have been well characterized cyt and maintain adequate Ca2+ in the ER lumen during hormonal Ca2+-signaling. Thus, Ca2+ entry through store-operated Ca2+ channels replenishes intracellular Ca2+ which is extruded from the cytoplasmic space via the plasma membrane (Ca2++Mg2+)ATP-ase following an increase in [Ca2+]cyt. In hepatocytes isolated from human liver, TRPC6 polypeptides may also make a contribution to store-operated Ca2+ entry cyt following the addition of extracellular Ca2+ to the culture medium of cells previously incubated in the absence of extracellular Ca2+ plus an agent such as thapsigargin or a generator of InsP3 to release Ca2+ from the ER. While this is a useful and valuable technique, measurement of Ca2+ entry by patch clamp recording is needed to confirm the presence of functional store-operated Ca2+ channels. These are normally defined as possessing the properties of Ca2+-release-activated Ca2+ channels (CRAC) channels, originally identified in lymphocytes and mast cells cyt play a role in maintaining the ability of liver cancer stem cells to self-renew and form tumors and identified InsP3R2 as a contributor to these [Ca2+]cyt oscillations [2+]cyt in Hep12 cells, but not in Hep-11 cells, suggesting that oscillations in [Ca2+]cyt are an important characteristic of liver cancer stem cells. The authors also employed the genetically encoded GCaMP-ER2 Ca2+ sensor targeted to the ER to investigate oscillations in [Ca2+]ER and the role of the ER and InsP3Rs in the generation of [Ca2+]cyt oscillations. Using RNA-seq, 10 genes encoding Ca2+-signaling proteins which were expressed at higher levels in Hep-12 cells compared with Hep-11 cells were identified cyt oscillations in Hep-12 cells. Knockdown of InsP3R2 in Hep-12 cells reduced their capacity to form spheroids and reduced their capacity to form tumors in NOD/SCID mice.Further investigations using Hep-12 (subset of HCC cells enriched in liver cancer stem cells) and Hep-11 (HCC cells) provided additional evidence that oscillations in MT decreases the mitochondrial membrane potential, inhibits the electron transport chain and oxidative phosphorylation and increases the production of ROS and reactive nitrogen species by components of the electron transport chain cyt enhance tumor cell growth.In studies employing human HepG2 HCC cells and a xenograft nude mouse model of HCC, Li and colleagues showed that hypoxia induced the expression of STIM1, increased cyt in HCC cells. The use of inhibitors of TRPC6 channels and of the (Na+-Ca2+) exchanger or shRNA to knockdown the expression of these proteins, showed that they are required for TGFβ -induced migration, invasion and metastasis in HCC cells. Co-immunoprecipitation experiments provided evidence that TGFβ increased the association of TRPC6 protein with the (Na+-Ca2+) exchange protein. In human HCC tissues, the expression of both TRPC6 and the (Na+-Ca2+) exchanger was higher in human HCC tissue compared to that in non-tumor tissue. Moreover, higher levels of expression of these proteins correlated with a higher tumor grade cyt and ROS and activation of the STAT3 pathway MT while deletion of InsP3R3 impaired the increase in [Ca2+]MT and caused cell death. The results of these studies with both HCC and cholangiocarcinoma suggest that targeting InsP3R3 with a pharmacological inhibitor would inhibit progression of HCC MT plays important roles in the regulation of the citric acid cycle and ATP production and in the apoptotic pathway. Under normal conditions, Ca2+ is transported from the cytoplasmic space into the mitochondrial matrix via the mitochondrial Ca2+ uniporter. Ca2+ can also be transferred directly from the ER to the mitochondrial matrix via the mitochondrial-associated membranes (MAMs) and the voltage-dependent anion channel (VDAC). Ca2+ is normally transported out of mitochondria by the (Ca2+ - H+) exchanger [2+]cyt and/or [Ca2+]ER is chronically elevated the mitochondrial matrix may become overloaded with Ca2+. This then triggers the release of Ca2+ via the mitochondrial permeability transition pore  and mitochondrial Ca2+ uptake 1 and 2 (MICU1 and 2) MT, which in turn increases ROS and promotes HCC metastasis. Buffering of [Ca2+]MT, using parvalbumin genetically targeted to the mitochondrial matrix (PV-Mito), suppressed the production of ROS and development of metastatic properties. Further analysis of the pathways involved led to the conclusion that increased expression of mitochondrial Ca2+ uniporter and enhanced uptake of Ca2+ to the mitochondrial matrix cause an increase in ROS via changes in NAD/NADH, SIRT3 and superoxidase dismutase 2 (SOD2), as shown schematically in In human HCC tissue, the mitochondrial Cah in HCC . Studies2+ uniporter regulator 1 enhances HCC cell survival via the p53 pathway and also enhances metastasis via the Nrf2 pathway. Expression of mitochondrial Ca2+ uniporter regulator 1, was found to be high in many HCC liver samples compared with that on non-tumor liver tissue MT, increased mitochondrial ROS, decreased apoptosis and increased cell proliferation [2+ uniporter regulator 1 exhibited enhanced growth compared with that of tumors derived for HCC cells exhibiting low mitochondrial Ca2+ uniporter regulator 1 expression [2+ uniporter regulator 1 enhanced the proliferation of HCC cells by increasing [Ca2+]MT and generation of mitochondrial ROS, leading to enhanced degradation of p53, decreased autophagy and increased cell cycle activity and cell proliferation , decreased expression of mesenchymal markers (N- cadherin and vimentin) and decreased expression of the transcription factor snail, a regulator of the epithelial mesenchymal transition [2+ uniporter regulator 1 caused the opposite changes. Measurement of the expression of mitochondrial Ca2+ uniporter regulator 1 in human HCC tissue samples indicated that higher levels of mitochondrial Ca2+ uniporter regulator 1 were associated with more advanced HCC. From these studies it was concluded that increased expression of mitochondrial Ca2+ uniporter regulator 1 increases [Ca2+]MT, ROS production, activates Nrf2 and the Notch pathway which mediates the epithelial mesenchymal transition . In HepG2 cells, erinacine decreased cell proliferation, colony formation, migration and invasion. These changes were associated with decreases in mitochondrial membrane potential, expression of components of the PI3kinase/Akt/GSK-3β pathway, cyclin D, vimentin, β-catenin and Bcl2 and increased expression of E- cadherin. Further, erinacine increased apoptosis, as indicted by increased Bax and caspase-9. Erinacine also enhanced the action of LY294002 on these pathways. In a mouse xenograft liver tumor model, erinacine reduced tumor size [2+]cyt, [Ca2+]MT, the mitochondrial permeability pore transition and the components of the PI3kinase/Akt/GSK-3β pathway in the mechanism of erinacine action. However, further experiments are needed to define the exact roles of [Ca2+]cyt, [Ca2+]MT and the mitochondrial permeability pore transition.Enhanced opening of the mitochondrial permeability transition pore leading to increased apoptosis has been implicated in the mechanism by which the natural product erinacine, obtained from the mushroom CC cells . Expressmor size . These r2+ from the ER to mitochondria, thereby inducing apoptosis ER and increased [Ca2+]MT and cellular ROS. Decreased expression of mitochondrial Ca2+ uptake proteins 1 and 2 was also observed in HepG2 cells overexpressing mitofusin-2. The authors concluded that increasing the expression of mitofusin-2 in HCC cells induces apoptosis by enhancing Ca2+ entry to mitochondria from the ER cyt (assessed as an increase in the percentage of cells positive for elevated [Ca2+]cyt) and increased the phosphorylation of Akt, but not that of ERK or JNK. Other experiments identified miR-671-5p as an important regulator of tuftelin1 expression. On the basis of these and other observations, Dou and colleagues proposed the following pathway for the involvement of tuftelin1 and Ca2+ in hypoxia-induced HCC progression. Hypoxia, via miR671-5p, activates transcription factor HIF-1α which induces increased expression of tuftelin1. This in turn increases [Ca2+]cyt, activates the Ca2+/PI3kinase/Akt pathway leading to an enhancement of the growth, metastasis and epithelial mesenchymal transition of HCC cells [The tuftelin1 (TUFT1) protein is involved in the development and mineralization of tooth tissue and its expression is increased in hypoxia . Noting CC cells .2+/calmodulin-dependent protein kinase IV (CamKIV) as a potential therapeutic target [Experiments directed towards identifying the mechanism by which another micro RNA, miR-129-5p, inhibits HCC growth have identified Cac target . Expressc target .Stephania tetrandra plant. The mechanism by which tetrandrine inhibits HCC involves an increase in ROS and activation of the Akt pathway. In a subsequent study, Huang and colleagues, using structural modeling, identified inhibition of CaMKIIγ as the possible target of tetrandrine action. This was confirmed by measuring the IC50 for the inhibition by tetrandrine of cell proliferation and the level of expression CaMKIIγ expression in each of a panel of eight different HCC cell lines [50 of about 1 µM, which is about 30 times lower than the IC50 for the multikinase inhibitor sorafenib [About ten years ago, tetrandrine, was shown to inhibit the development of HCC in studies employing HCC cell lines and a mouse HCC tumor model . Tetrandll lines . Huh7 cell lines . To try ll lines . These worafenib .Berberis amurensis [Inhibition of CaMKIIγ may also be a target of berbamine, another member of the bisbenzylisoquinoline family, with a chemical structure similar to that of tetrandrine . Berbamimurensis . Studiesmurensis . Howevermurensis . From stmurensis .In further investigation of the role of CaMKIIγ in HCC, Meng and colleagues used bioinformatics to identify protein kinases that regulate gene expression in human HCCs and in a diethylnitrosamine mouse HCC model . One of Calcium-binding protein 39 has been shown to be important in the migration and metastasis of HCC cells, suggesting that it may be a potential pharmacological target for the treatment of late stage HCC [2+-signaling proteins that could be potential targets for therapeutic treatment of HCC were identified. Targets in liver cancer stem cells, which are thought to drive the initiation and progression of HCC and development of resistance to current drugs, are of particular potential value, as are those which could inhibit the proliferation, migration and metastasis in advanced HCC. However, none of these Ca2+-signaling targets has been seriously studied any further than laboratory research experiments. A few of the Ca2+-signaling proteins have been identified as targets for natural products previously known to reduce HCC. Promising Ca2+-signaling targets seem to be voltage-operated Ca2+ channels in liver cancer stem cells, InsP3Rs, store-operated Ca2+ entry, SERCA2b and Ca2+/calmodulin protein kinases. However, for several Ca2+-signaling proteins so far identified as potential drug targets, the situation seems complicated, in that the effects of inhibition or activation may depend on the stage of the HCC. The targets so far identified have, to a large extent, evolved from somewhat serendipitous research investigations and from studies of the mechanisms of the actions of natural products. The application of more systematic studies in the future is expected to reveal new Ca2+-signaling protein targets and to consolidate priorities for those already identified. Such studies may include expanded genomics and gene expression, RNA-seq and improved knowledge of the fundamental biology and pathology of HCC. If one or more pharmacological inhibitors or activators of Ca2+-signaling proteins is shown to be useful in clinical practice, it is likely that such a compound will be most effective in combination with other drugs and will likely be delivered via drug-emitting bead transcatheter arterial chemoembolization.Numerous Ca


A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pHi), voltage and [Ca2+]i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion.Intracellular calcium ([Ca Fertilisation in marine invertebrates, such as the sea urchin, occurs during broadcast spawning events in which males and females of co-localised species ejaculate sperm and spawn eggs synchronously. During these events, spermatozoa have to find and fertilise conspecific eggs in the midst of all the other ones, which is a remarkable navigation and mating choice achievement. Sperm cells do this by navigating towards the source of species-specific peptides released by the egg, steered by spatial and temporally orchestrated peaks in intracellular calcium concentration that trigger sudden reorientations. How these calcium spikes are regulated and timed remains elusive. Different calcium channels have been implicated by indirect experimental evidence giving rise to a complex network of putative interacting components. We gained insight into the structure and function of this network by modelling it as a set of candidate modules that could be studied separately. By using this ‘divide and conquer’ approach to the complexity of the network, we could characterise the potential dynamics of each module and confront these dynamics with specific quantitative data. Our results indicate that the channel mediating calcium signals in sea urchin sperm is likely CatSper, a calcium channel necessary for human male fertility. Its elevation above resting levels conveys diverse physiological signals and may trigger cellular processes such as programmed cell death and egg activation. Both versatility and specificity of this ion as a physiological signal lie in how it is temporally and spatially organised within a cell i-spike train, the temporal organisation of which could be modulated by Ca2+-activated Cl- and K+ channels. Another hypothesis i oscillations. The pHi rise would promote the opening of CatSper that drives an upsurge in [Ca2+]i and membrane depolarisation, which in consequence would inactivate respectively CatSper and the exchanger. The cycle would reinitiate once the membrane hyperpolarises again.The signalling network that mediates calcium-steered chemotaxis in sea urchin sperm has been an object of intense study, and several decades of experimental research have implicated multiple channels and molecular components , 8. It iwnstream . The risthesised , 11 thatpothesis  is that 2+]i-spike trains is summarised in The evidence for the two types of calcium channels proposed to participate in SAP-activated i-spike trains. Our aim is to contrast the different hypotheses by their quantitative implications in order to identify critical properties that can be used to experimentally disentangle them.In the present work, we explore which putative calcium channels may mediate the CaV generated by the cGMP response, and two alternative downstream modules that can generate Ca2+ oscillatory responses. The CaV+BK module includes voltage-dependent Ca2+-channels and Ca2+-dependent K+-channel BK; while the CatSper+NHE module is composed of the voltage-, pHi- and Ca2+-dependent channel CatSper, the voltage-dependent sodium-proton exchanger and the intraflagellar proton concentration. The variables, parameters and differential equations used to model the components of this network are described in Sec. 4.1 and Sec. 4.2.The potential signalling network illustrated in 2+-channels were constrained such that given the input of the Upstream module, they generated an output that was consistent with the observed Ca2+-spike trains, i.e. these two putative modules were constrained by independent data sets at the both input and output levels. Hence, by coupling the downstream CaV+BK and CatSper+NHE modules to this Upstream module, we show in Sec. 2.3 that either module can independently account for the observed Ca2+-spike trains patterns.The modular structure facilitated modelling and analysis. As a strategic simplification, we hypothesised that most of the components of the Upstream module were independent of the variables downstream and studied it isolated from the remaining dynamics. We tested the validity of this simplifying assumption by showing that this Upstream module accounted quantitatively for experimental time series of cGMP and membrane potential in response to SAP (Sec. 2.2). Once the values of the parameters of this module were obtained, they remained fixed in the subsequent analyses of the dynamics of the downstream components. In these analyses, the modules featuring CaA. punctulata sperm stimulated with the SAP Resact exhibit a rapid cGMP response that peaks before ∼400 ms and rapidly drops to a pseudo plateau that slowly decays over several seconds, as observed by stopped-flow kinetics [i or [Ca2+]i [V [cGMP is the core component of the Upstream module . cGMP lekinetics . This bi [Ca2+]i . In orde [Ca2+]i . The gooa2+]i i spike trains observed in individual sperm cells responding to the SAP stimulus are noisy but show some conspicuous trends, as illustrated by those of S. purpuratus sperm . The rationale for the first selection criterium is twofold. First, it is the most representative pattern of the experimental spike trains analysed here as well as of those reported in other species (e.g. see fig. 3 in reference i (C). The nadirs of the [Ca2+]i oscillations are well above the basal concentration of this cation and this minimal plateau of about 500 nM is maintained until the Ca2+ activity vanishes at about 17 s. A similar oscillatory behaviour with similar period but different phases is observed on the fractions of open channels (second row) and the respective currents (third row), as well as the membrane potential (bottom).The numerical solution of the variables of the model including the Ca2+]i fluctuations predicted by this module can be easily understood qualitatively. The hyperpolarisation induced by KCNG enables the transition from inactive to closed forms of the CaV and the spHCN-mediated increase in membrane potential opens these closed channels. The open CaV channels will overcome the hyperpolarising current driven by KCNG and thus depolarise the membrane as Ca2+ ions flow in. The depolarisation of the membrane leads to the inactivation of the CaV channels with the hyperpolarisation currents predominating again. At the hyperpolarised membrane potentials the inactive channels will again transit to closed form, which eventually will open as described above, reinitiating the cycle. Intraflagellar calcium, by activating the BK channels, adds a strong hyperpolarising current to that of KCNG. This couples the net hyperpolarising currents to the amplitude of the previous Ca2+-spike, in such a way the higher the [Ca2+]i peak, the higher the BK-driven hyperpolarising current will be.The temporal dynamics of the [Ca2+]i spikes follows closely that of the cGMP levels upstream. This led us to speculate that the time course of cGMP might offer a way to understand the fluctuations envelope. To gain a better quantitative insight, a variant of the model was made wherein cGMP concentration, G, instead of a variable became a constant parameter, thus ignoring the upstream variables and reducing the state space (Sec. 4.3). In this simplified model, the qualitative changes of the [Ca2 +]i and its period at the stationary states were analysed as a function of the G parameter. This is known as bifurcation analysis in dynamical systems theory, which is useful for characterising the global properties of a system of differential equations, in particular the dependence of asymptotic behaviours on a given parameter of interest (bifurcation parameter). These behaviours can correspond to attractors of fixed point (invariant in time) or limit cycles (stationary periodic), which in turn can be stable or unstable, among others. Wherever there is a qualitative alteration of these behaviours by changing the parameter of interest, it is said that a bifurcation has occurred.The envelope of the [Ca2+]i stable stationary state to a limit cycle. When it reaches II, the limit cycle disappears giving rise again to a stable stationary equilibrium by means of an inverse Hopf-type bifurcation. Further on in III, the system undergoes an inverse saddle-node bifurcation, defined over cGMP concentration increments, in which a stable equilibrium state coalesces with an unstable one. In this way, in traversing the branch over the stable equilibrium state, the system goes to an unstable state. Now, when advancing on this last unstable equilibrium, this turns to a stable one by means of the direct saddle-node bifurcation (IV). The presence of points III and IV suggest the possible existence of a cusp bifurcation defined in a space of higher dimension, which is corroborated in panel B of the same figure. Finally, the last stable equilibrium state, again by means of a direct Hopf-type bifurcation, gives rise to another limit cycle.When cGMP concentration is varied in this subsystem, the bifurcation diagram shows 5 bifurcation points , the saddle-node bifurcation would occur at a lower cGMP concentration value and these low amplitude fluctuations would not be revealed . This explains the abrupt i. As a first approach to investigate how the coupling of the modules would affect the dynamics, we considered a scenario in which the actual composition of the system would be a weighted combination of the two modules by means of weight parameters given by θcs = θ and θcs = (1 − θ) (implementation details in Sec. 4.5). For θ = 0, one recovers the submodel with CatSper module alone, for θ = 1 one recovers the submodel featuring CaV+BK module only, and when θ = 0.5 the total conductance of the components in each module are half of their respective reference values. Taking the CatSper+NHE module we titrated in the components of the CaV+BK module under several combination schemes. First, we explored the addition of CaV and BK simultaneously maintaining their conductance ratio as in the original CaV+BK module (θ ∼1 or θ ∼0) the mixed models predict the characteristic decay of the amplitude and the increase interspike interval. BK alone added to the CatSper+NHE module will cancel the oscillations at values as small as 10% of its reference value. At even lower densities it is functionally negligible, modulating the average interspike interval or the duration of the whole spike train without modifying the characteristics of the oscillations. These results are not entirely unexpected considering that in each oscillation of the CaV+BK module the system spends most of the time hyperpolarised, while in the CatSper+NHE it spends a significant fraction of the cycle depolarised, which is an absolute prerequisite for pHi to return to lower levels closing CatSper gate.In the previous sections, we have shown that both alternative modules, once coupled with the Upstream module, can account for the observed data patterns. These two modules share some features and their components could potentially coexist in the flagellum being coupled through the membrane potential, pHK module , then weK module . This siV+BK and CatSper+NHE modules were systematically explored in broad ranges, between negligible and excessive conductances. The generation of parameter vector variants ensured a random exploration of the parameter space centred on the realistic estimates of the other parameters, to avoid diversions into spurious parameter regimes. The final ensemble of parameter vectors produced by the algorithm contains both a population that was selected for being coherent with experimental data and an unselected population derived from and closely located to the former  shows 6 clusters, labelled a-f, depicted in V+BK module, are more spread both in parameter space  and in the plane  summarising the predicted output. To understand the mixed scenarios we asked whether the presence of all the channels CaV, BK or CatSper was essential to produce the predicted dynamics. To this end, we compared the numerical solution obtained with a given parameter set with the solutions obtained by specifically cancelling the conductance of CaV and BK on the one hand, or that of CatSper, on the other hand . Ou. Ou26]).i oscillations. The parameter vectors in the other clusters display very transient depolarisation times, with sharp Ca-spikes  show in each oscillation cycle the typical preponderance of depolarisation, with blunt Ca-spikes and marked pHa-spikes  and pHi K module . As we wi dynamics. The NHE and [H+]i can be coupled to the CaV+BK module in the extended model by making θcv = 1 and θcs = 0 in Eqs i does not feedback on any of the processes that determine Ca2+ fluctuations (proton concentration H does not appear in any of the equations that govern the variables of that model variant). Therefore, this module would predict that changes in the pHi should not affect the Ca2+-spike trains. In contrast, in the CatSper+NHE module, the proton dynamics is tightly coupled to the Ca2+ oscillations and is an essential part of the mechanisms underlying the oscillatory behaviour. In our analysis of the parameter dependence of the CatSper+NHE module, we systematically found oscillations in [Ca2+]i that were concomitant with pHi oscillations having the same period, albeit phase differences. Parameters that lead to slower proton dynamics such that the pHi oscillations vanish, resulted in cancellation of the oscillation in [Ca2+]i.The two candidate modules for the signalling network activated by SAPs are, by construction, differently coupled to the pHi inside the flagellum. In this situation, the CaV+BK module would predict that the Ca2+-fluctuations would be unaffected, while the CatSper+NHE module predicts that sustained pHi increase would cancel [Ca2+]i fluctuations and this cation would be sustained at higher concentration. These effects are illustrated in 2+ dynamics in the presence of ammonium chloride (NH4Cl), an agent that arguably raises the pHi without affecting any of the other channels modelled here. As observed in the experimental trace, the exogenous alkalinisation gives rise to a dynamics that is qualitatively similar to the one predicted by the pHi-dependent CatSper+NHE module. The conspicuous effect of alkalinisation on [Ca2+]i dynamics is not predicted by any of the mixed scenarios that were otherwise coherent with the experimental time series studied the previous Sec. 2.8  , insteaV channels or shifting to higher opening rate of the voltage-gate of CatSper) but not too strong to prevent the oscillatory [Ca2+]i dynamics. This property of the signalling network is amenable to experimental corroboration by assessing whether the membrane potential recovery is only partial under a setting in which all calcium entry is prevented by using zero external concentrations of this cation.The Upstream module alone was able to explain the early hyperpolarisation dynamics based on the joint action of KCNG and spHCN channels , bottom.2+]i fluctuations by the dynamics of the network. The key question was: what components of the downstream modules correctly predict this link? Our analysis indicates that either one of two modules featuring voltage-dependent channels could be responsible for the dynamics of [Ca2+]i fluctuations. The first module calls into action CaV channels that undergo voltage-dependent sequential transitions from inactivated to closed, to open and back to inactivated states. These irreversible transitions bring forth a limit cycle with constant period with an amplitude that is controlled by cGMP activity. These CaV channels, which are predominantly inactivated at the resting potential, are released from inactivation upon a transient hyperpolarisation and subsequently opened when spHCN channels activity raises the membrane potential. In this module, the oscillations are strongly modulated in amplitude and tempo by the Ca2+-dependent channel BK to produce the experimentally observed progressive increase in the intervals between [Ca2+]i spikes. The oscillations predicted by this module vanish when cGMP falls below a critical value involving a Hopf-type bifurcation . Conversely, the progressive decrease in the amplitude observed experimentally is better accounted for by the CaV+BK module than by the CatSper+NHE module under all the parameters studied here.It is noteworthy that, under the reference parameters used in this article, the overall temporal scale of the calcium response produced by the CatSper+NHE module is slower compared to the experimental tracings, in terms of real time units; however, if the relative progressive increase of interspike intervals is contrasted instead, this property becomes more marked in this model than in the one featuring the Caobserved . In the V + BK channels pair. This envelope has a shape similar to that of the [Ca2+]i spikes. In contrast, the CatSper+NHE module predicts that the amplitude in the membrane potential peaks should increase until it suddenly ceases  or too high intracellular GMP concentrations, the model with CatSper predicts a sustained rise i as observed experimentally following the addition of NH4Cl to S. purpuratus spermatozoa  or, alternatively, it might mean that intraflagellar pH is not oscillating periodically. In the latter case, the hypothesis that CatSper is the main responsible for fluctuating Ca2+ currents, at least in the way we modelled it here, is questionable and must be reexamined opening a new avenue for modelling research. In this context, it might be relevant to note that oscillatory fluxes of Ca2+ and protons have been reported with the same period as growth oscillations of pollen tubes .i precedes the first Ca2+ spike in sea urchin sperm stimulated by SAP has been interpreted as an indication that Ca2+ currents are a consequence of this early alkalinisation [2+ fluctuations. It is worth calling attention to the early time courses of pHi and [Ca2+]i obtained by the numerical solution of the model featuring the CaV+BK module . In res2+]i spikes. It is important to bear in mind that the present analysis involves an asymptotic approximation, i.e. in our case G was set to a fixed value as a way to transform this variable into a parameter and quantify the amplitude and period of the stable limit cycles. The experimental corroboration of such analysis would require development of a cGMP-clamping technique, analogously to classic voltage-clamping experiments or synthetic biology approaches that seem presently unfeasible in sea urchin sperm.We used bifurcation analysis of a subset of the differential equations of the full system as a heuristic tool to understand how the temporal evolution of cGMP activity affects the envelope and intervals between i fluctuations are controlled by different molecular components. A system of ordinary differential equations (ODE) describes the dynamics of the membrane potential and of the intraflagellar concentrations of cGMP, protons and Ca2+. These four essential variables depend on the activities of different flagellar channels and enzymes, the dynamics of which are also described by ODE (Sec. 4.2). The four variables are the main observables in the model and offer the means to assess its predictions by comparison with experimental data.Our strategy is to describe the dynamics of the SAP-activated signalling pathway in order to understand how the i via calmodolin, as documented in other cellular systems and species [i and [Ca2+]i on phosphodiesterases, which is a simplifying assumption that proved to be reasonable by comparison with quantitative data.In this equation, it is assumed that the activities of general guanylate cyclases and phosphodiasterases lead to a constitutive turnover of cGMP, with a constant source a urchin . Althougty by pH , and thi species . Eq 4 neG was compared with measurements of total cGMP by radioimmunoassay in bulk cell experiments previously reported o and [Na+]i are the extracellular and the intraflagellar concentrations of Na+, respectively; pHo is the extracellular pH; and KNa is the dissociation equilibrium constant for Na+.Proton flux as a consequence of exchange activity of NHE, designated kinetics :JH=fnhaVi, closed CaVc and open CaVo:There has been suggestive evidence for the involvement of classical voltage-gated calcium channels in the SAP-activated response, i.e. Low-Voltage-Activated , 10 and The gating rates are themselves voltage-dependent exponential functions similar to those of Hodgkin-Huxley (HH) type models:v2 = −55 mV), inactivation (o to i) takes place under marked depolarisation (v8 = −18 mV) and opening of closed channels (c to o) is favoured from potentials slightly hyperpolarised (v5 = −39 mV).Each transition rate is promoted at a different voltage range: recovery from inactivation (i to c) is favoured under hyperpolarisation  are activated by i dynamics in the model the same procedure was applied to the numerical solutions of C. The structure of the spike trains in the model was considered to be coherent with those experimentally observed according to the following selection criteria: the average spike interval is in the range 0.37 s-1.38 s, the predicted  point falls within the convex hull defined by experimental values in the bottom right quadrant, i.e. the spike amplitude decrease whereas the interspike interval increase in time bA < 0 and bT > 0. As mentioned in the Sec. 2.3, this pattern of the spike trains is the most representative of the experimental time series and the most challenging for the models. In addition, to filter out spontaneous oscillations and alternative equilibria in multistable regimes, we imposed that the resting steady state value of [Ca2+]i, S = 0, is about 100nM. It is important to notice that the fluorescent probe intensities were not calibrated and therefore mean flagellar intensities are not accurate [Ca2+]i measurements. We assumed that the fluorescence intensity was proportional to the actual concentration of [Ca2+]i, focusing our analyses on relative differences and values. This assumption underlies the co-plot time series and model solutions presented in the figures and also the direct comparison of the theoretical and experimental bA values. We believe this assumption is reasonable, considering that all Ca-fluorescence intensity spikes analysed were below the control intensities elicited by ionomycin.The  (V by setting θcv = θ and θcs = (1 − θ) while forcing gbk = 0  addi gbk = 0 ; and c)  θcs = 1 .θcv, θcs ∈ {0.0001, 0.1, 0.2, 0.3, …, 2.0}. Each vector in this population was used randomly as a seed to generate a daughter parameter vector obtained by multiplying each parameter value by an independent lognormal distributed random variable . Each daughter parameter vector was appended to the evolving population, irrespective of the output behaviour, serving as random seed to generate new daughters. The method to generate parameter vector variants favoured values close to the reference ones . The dynamics predicted by each parameter vector gcv, gcs), started to visibly coalesce. At this stage, several parameter vectors satisfied the selection criteria for coherence with experimental observations and were selected for further exploration in the second phase of the algorithm. In each iteration, a vector in the selected population was randomly picked to generate a daughter vector by the same procedure as above. This daughter vector was analysed and added to the evolving selected population, serving as seed for further exploration, if it met the selection criteria; otherwise it was stored in a list of unselected parameter vectors. The latter vectors are by construction closely located in the parameter space to those of the evolving selected population from which they derived but, being incoherent with observations, they were not further explored. The selected and unselected vector populations were used to build clusters based on the proximity in the projection plane . The iterative generation of daughter parameters from the selected population was repeated until the clustering in the plane  was stable (i.e. no new clusters were observed after a large number of iterations). Within each final cluster, the fraction of number of selected parameter vectors over the total number of vectors is a kind of measure of the parameter sensitivity. The smaller this fraction the more sensitive the model is within the cluster.A more systematic analysis of mixed scenarios contemplated the conductance of channels that compose the two modules but also variants of the values of all the other parameters, except for those representing the Upstream Module . The values of RT, r2, r3, δG, kL and kH were fitted while fixing the remaining parameters . For each SAP concentration (encoded in grey shades) the dots represent experimental values read from the figures in  and the rameters . Becausede 14000 , 22, 3 ×14000 [5  and 1 × 00 [5 [6  molecule(TIF)Click here for additional data file.S2 Figτ). In the lower pannel, the set of estimated characteristic times was fitted to a Gaussian function, which is our proposed form for the voltage-dependent characteristic time of spHCN gating . Data extracted from figure 4a of  (gray liting Eqs  and 21.(TIF)Click here for additional data file.S3 Figi-dependent gate variable in equilibrium, 2+) produced by taking the holding voltage from 0 mV to −100 mV, under different pHi values, and measured by whole-cell patch-clamp in mouse sperm, . C and D correspond to the G/V (conductance/voltage) curves of mouse and sea urchin CatSper, respectively, using the s2, as an initial guess. This last parameter has not been estimated in sea urchin due to the lack of patch-clamp measurements.In A and B, experimental data on mouse sperm (circles) are shown, along with model fittings (lines) related to the voltage- and pHg. 4c of ). Mouse (TIF)Click here for additional data file.S4 Figθ, which controls the percentage of module CaV + BK that is being added. The parameters are set according to Eqs θcv = θ and θcs = (1 − θ). For reference, the original CatSper-only scenario is shown first, i.e. θ = 0, and the subsequent rows correspond to the gradual increase of θ. The effect of this parameter on the channel densities is reported in the coefficient rg, which measures the total conductance ratio CatSper/CaV, as well as the corresponding gbk value. The greater the value of rg, the greater the predominance of CatSper on the total calcium conductance with respect to CaV.Starting from the model that includes the CatSper module, numerical solutions for calcium are shown under different values of the weighting parameter (TIFF)Click here for additional data file.S5 Figθ, with either BK or CaV conductance density set to 0, respectively. As reference, the original scenario of only CatSper, i.e. θ = 0, is shown in the top row, while the subsequent rows correspond to the gradual increase of θ. In A, the reference value of the CaV conductance density, gcv, is multiplied directly by θ, whereas the reference value of CatSper conductance density is multiplied by (1—θ); The resulting ratio of CatSper/CaV conductance densities, rg, is shown for each θ value. In B, θ sets the fraction of the reference value of gbk by multiplying the conductance density of BK; unlike scenario A, the conductance density of CatSper is not weighted by θ. For each titration, the corresponding modified gbk value is shown.In A and B, we show numerical solutions for calcium under different values of the weighting parameter (TIFF)Click here for additional data file.S1 Algorithm(PDF)Click here for additional data file.


However, the response of cardiomyocytes to simulated microgravity and hypergravity and the effects on Ca2+ signalling remain unknown. Here, we elucidate the mechanisms underlying the proliferation and remodelling of HL‐1 cardiomyocytes subjected to rotation‐simulated microgravity and 4G hypergravity.Cardiac CaThe cardiomyocyte cell line HL‐1 was used in this study. A clinostat and centrifuge were used to study the effects of microgravity and hypergravity, respectively, on cells. Calcium signalling was detected with laser scanning confocal microscopy. Protein and mRNA levels were detected by Western blotting and real‐time PCR, respectively. Wheat germ agglutinin (WGA) staining was used to analyse cell size.ANP and BNP, indicating cardiac remodelling. WGA staining indicated that cell size was decreased following rotation‐simulated microgravity and increased following 4G hypergravity. Moreover, HL‐1 cell proliferation was increased significantly under hypergravity but not rotation‐simulated microgravity.Our data showed that spontaneous calcium oscillations and cytosolic calcium concentration are both increased in HL‐1 cells after simulated microgravity and 4G hypergravity. Increased cytosolic calcium leads to activation of calmodulin‐dependent protein kinase II/histone deacetylase 4 (CaMKII/HDAC4) signalling and upregulation of the foetal genes 2+/CaMKII/HDAC4 signalling plays a pivotal role in myocardial remodelling under rotation‐simulated microgravity and hypergravity.Our study demonstrates for the first time that Ca Ca2+ functions through the Ca2+ binding protein calmodulin (CaM) to activate CaMKII, which is activated by different pathological processes in the heart. This Ca2+‐CaMKII‐dependent gene regulation during cardiac remodelling suggests novel strategies for Ca2+‐CaMKII‐dependent “transcriptional therapies” to control cardiac gene expression and function.Intracellular Ca2+ is a highly versatile intracellular signal that regulates many different cellular processes.2+ signalling plays an essential role in regulating cardiac functions, including cardiac contraction, relaxation and remodelling.2+ handling in cardiomyocytes. HU of rats impaired the function of L‐type Ca2+ channels and decreased intracellular Ca2+ ([Ca2+]i) transients, resulting in reduced responsiveness to β‐adrenoceptor stimulation, which may be partially responsible for the decline in cardiac function. These studies demonstrated that microgravity‐induced changes in Ca2+ signalling play an important role in cardiac remodelling and reduced function. Numerous studies on mammalian organisms have demonstrated that the absence of gravity has severe effects not only on a systemic level but also on a cellular level.Ca2+/CaMKII/HDAC4 signalling pathway.In vitro studies have demonstrated that space flight and simulated microgravity induce significant changes in gene expression patterns,22.1l‐glutamine (Sigma‐Aldrich) in a 5% CO2 atmosphere. Cells were seeded in T‐75 culture plates precoated with 25 µg/mL fibronectin solution.The HL‐1 cardiomyocyte cell line was cultured with Claycomb medium (Sigma‐Aldrich), 10% foetal bovine serum (Gibco), 1% norepinephrine, 1% penicillin‐streptomycin and 1% 2.22 cell culture flasks or plated on 25‐mm glass coverslips and filled with culture medium  clinostat, which was developed and provided by the China Astronaut Research and Training Center Figure A. HL‐1 c2.3A hypergravity centrifuge was used to detect the effects of hypergravity on cells. The details of the procedure were the same as described above for rotation‐simulated microgravity. The hypergravity centrifuge was continuously rotated under the 4G hypergravity condition at 37°C for 48 hours. The hypergravity centrifuge has been described previously.2.42+ levels and dynamic calcium signalling, as described previously.2·6H2O, 1.2 mmol/L NaH2‐PO4·2H2O, 5.4 mmol/L KCl and 1.8 mmol/L CaCl2, pH 7.35 [adjusted with NaOH]). Cells were then rinsed twice with Tyrode solution and mounted on the inverted stage of a confocal microscope. Confocal imaging was performed with an LSM710 microscope (Zeiss) with a 40×, 1.3 NA oil immersion objective, and linescan speed of 2.55 μs/line; the pinhole was nominally set for a 1‐μm optical section. Fluo‐4 AM was excited at 488 nm, and fluorescence emission was measured at 490‐550 nm. Images were acquired every 3 seconds to observe calcium oscillations and analysed using Interactive Data Language (IDL: Research Systems) software. The fluorescence intensity was quantified, and the calcium oscillation trace was acquired. We took the number of oscillations in 1 minute to be the calcium oscillation frequency. To determine resting [Ca2+]i and [Ca2+]i released from the endoplasmic reticulum (ER), cells were scanned for 20‐30 seconds to obtain Fresting; then, the solution was replaced with 0 Ca2+ Tyrode solution containing 4 mmol/L EGTA (Invitrogen), 5 μmol/L thapsigargin (Molecular Probes) and 10 μmol/L A23187 (Sigma‐Aldrich). Stored calcium was immediately released to the cytoplasm, followed by a gradual decline; we defined the peak value as FER release. When stabilized, 100 μmol/L BAPTA‐AM (Molecular Probes) was added to further chelate cytosolic Ca2+, to measure the minimum fluorescence level (Fmin). Then, the solution was replaced with Tyrode solution containing 10 mmol/L Ca2+, 5 μmol/L thapsigargin, 12 μmol/L A23187, 3 μmol/L FCCP (Sigma‐Aldrich) and 20 mmol/L 2‐DG (Sigma‐Aldrich). The stable value was Fmax. The trace is shown in Figure 2+]i was calibrated using a modified version of the equation of Grynkiewicz et alA calcium indicator was used to measure intracellular Ca2.5g at 4°C for 30 minutes. Protein samples were separated by 10% SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin and incubated with specific antibodies overnight. Antibodies used were as follows: CaMKII , p‐CaMKII , HDAC4 , p‐HDAC4 , mTOR , p‐mTOR , PCNA , α‐MHC  and GAPDH .HL‐1cells were lysed in lysis buffer  on ice for 30 minutes. Protein fractions were collected by centrifugation at 15 000 2.6® Premix Ex TaqTMII Kit (TaKaRa). Primers used in this study were as follows:Total RNA from HL‐1 cells was extracted with TRIzol Reagent (Invitrogen) as the manufacturer's instructions. RNA (0.5 μg) was reverse transcribed with PrimeScript RT reagent Kit (TaKaRa) according to the manufacturer's instructions. cDNA was used for detecting mRNA expression by quantitative PCR using SYBR2.7Cells were plated on 25‐mm glass coverslips and transfected with CaMKII small interfering RNA (siRNA) or negative control (NC) at 70% confluence, using Lipofectamine RNAiMAX in OptiMEM according to the manufacturer's instructions (Invitrogen). The NC siRNA sequence was 5′‐UUCUCCGAACGUGUCACGUTT‐3′, and the CaMKII siRNA sequence was 5′‐UCUAGAAUCUGUUGUAUACAA‐3′.2.8HL‐1 cells were fixed with 4% paraformaldehyde for 15 minutes, washed twice with phosphate‐buffered saline (PBS) and incubated with wheat germ agglutinin  at room temperature for 30 minutes. The cells were then washed with PBS and incubated with Hoechst (Invitrogen) for 3 minutes. Images were acquired using a confocal microscope . Cell size analysis was performed with ImageJ software (NIH).2.9t test or one‐way ANOVA for multiple samples. Differences were considered significant at *P < .05, **P < .01, and ***P < .001.All quantitative data are presented as the mean ± standard error of the mean. Data were generated from three independent replicates. Statistical differences among groups were analysed by one‐way analysis of variance (ANOVA) with a post hoc test applied. All statistical analyses were performed with Prism software . Statistical significance was evaluated using unpaired Student's 33.12+‐sensitive fluorescent indicator Fluo‐4 AM, after 48 hours of rotation‐simulated microgravity with the 2D clinostat  and Ca2+ released from the ER ([Ca2+]ER release), which indicated the levels of calcium in the cytoplasm and ER, respectively  and HDAC4 Ser632) was increased significantly after hypergravity 2+‐dependent signalling circuit involved in cardiac hypertrophy and heart failure.Well‐characterized signalling molecules that regulate cardiac remodelling include CaMKII and HDAC4.2+ serves as a central intracellular messenger in the heart. And intracellular Ca2+ handling also takes part in cardiac remodelling regulation.2+ functions through Ca2+ binding protein calmodulin (CaM) to activate CaMKII.2+ signals regulate contraction and also a host of other cellular processes including gene regulation, cellular growth and death.2+ signals is critical, as is the cellular localization of these signals.2+ channels, the voltage‐gated L‐type Ca2+ channels, which control Ca2+ influx elicited by action potentials, and ryanodine receptors (RyRs), which mediate Ca2+ release from intracellular stores, reside on the surface of cell membranes and sarcoplasmic reticulum (SR) membranes, respectively.2+. The frequency of oscillations in [Ca2+]i corresponds to the speed of cyclic release of Ca2+ from the SR and re‐uptake of Ca2+ by SERCA, a calcium uptake channel on the SR membranes.2+]i and calcium oscillation in HL‐1. The difference is that hypergravity induced a stronger increase of calcium signalling, and [Ca2+]ER release increased in the group of hypergravity but not rotation‐simulated microgravity. Our understanding of this issue is incomplete, but it may be the cause of different follow‐up downstream phenotypes.CaPCAN and C‐fos increased in hypergravity, and the level of p‐mTOR/mTOR increased too. This study indicated that hypergravity increased the proliferation and protein synthesis of HL‐1.Besides, cell proliferation plays a major role in maintaining cardiomyocyte homoeostasis.We have reported for the first time that gravity induced changes in calcium signalling in HL‐1 cardiomyocytes Figure ; furtherThe authors declare no commercial or financial conflict of interest.Caizhi Liu, Guohui Zhong, Yuezhang Zhou and Yuchen Yang performed the majority of the experiments, analysed data and prepared the paper. Yingjun Tan provided us technical support. Yuheng Li, Xingcheng Gao, Weijia Sun, Jianwei Li, Xiaoyan Jin, Dengchao Cao and Xinxin Yuan helped with simulated microgravity and hypergravity experiments. Zizhong Liu, Shuai Liang, Youyou Li, Ruikai Du, Yinlong Zhao, Jianqi Xue, Dingsheng Zhao and Jinping Song provided suggestions for the project and critically reviewed the paper. Shukuan Ling and Yingxian Li supervised the project and the paper. Click here for additional data file.


SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated  RUNX1, CEBPA, GATA2), DNA methylation , and genome organization . Acute myeloid leukemia (AML) is a malignant hematopoietic disease derived from myeloid-primed stem cells resulting in accumulation of myeloid blasts. AML patients have a poor prognosis and the only known efficient therapy is bone marrow transplantation combined with chemotherapy. Next-generation sequencing revealed that despite similar cytology and cellular features, the mutational profile of AML clones can be very heterogenic. Leukemogenesis involves multiple types of genomic alterations from single nucleotide variants to large chromosomal abnormalities . Targets of mutagenesis are often genes encoding regulators of gene transcription  belongs to important enzymes of the Swi2/Snf2 family with remodeling activity that is required for successful hematopoietic development in mammals ,2,3. In Drosophila but later they were discovered also in humans, namely, NURF (ATPase motor of the nucleosome remodeling factor), ACF (ATP-utilizing chromatin assembly and remodeling factor), and CHRAC (chromatin assembly complex). Later, additional human complexes were found, such as RSF, NoRC, WICH, CERF, and finally, SNF2H-cohesin )gulation ,16,17,18gulation . SMARCA5U.1 gene . We hereSMARCA5 levels are significantly elevated at the time of diagnosis and decreased after the patients achieved complete hematologic remission   embryos into lethally irradiated adult C57Bl/6J Ly5.1 recipients. Flow cytometric analyses of bone marrow and spleen at several weeks after transplantation revealed that repopulation was detected only in animals transplanted with cells in which the Smarca5 gene was preserved. Thus, homeostatic expression of Smarca5 is very important for hematopoietic reconstitution  cells. In addition, we noted a higher number of cells with polyploid nuclei  as well as the members of the CTCF/cohesin complex across human AML samples. This implicates, albeit indirectly, a role of SMARCA5 in CTCF/cohesin function in AML that also coincides with karyotype abnormalities imposed by a SMARCA5 loss.SMARCA5 gene in K562 cells. Compared with previously published data documenting an inverse relationship between SMARCA5 and hematopoietic transcription factors PU.1 or GATA-1, we observed that upon SMARCA5 deletion in K562 cells the level of SPI1/PU.1 and some of its targets (CSF1R) became downregulated while other transcription factors  were upregulated. The dysregulation of mRNA pattern of SMARCA5 targets upon SMARCA5 deletion can be attributed to the heterogeneity of the AML cell lines and also possibly to multiple genetic/cytogenetic abnormalities imposed by the SMARCA5 loss.We recently showed that SMARCA5 (together with the CTCF/cohesin complex) represses PU.1-mediated myeloid differentiation  and simiSMARCA5 gene, only 186 variants with an amino acid residue substitution exist in nearly ~20 thousand oncologic patients (<1%). There also exist infrequently the variants in ISWI-interacting BAZ proteins detected in cancer, however, the significance of these variants remains also unknown. Importantly, among the AML-associated variants, only the SMARCA5-interacting proteins, CTCF and members of the cohesin complex, were shown consistently mutated in AML [We herein studied how ISWI ATPase SMARCA5/SNF2H controls in AML the proliferation and gene expression of myeloblasts as SMARCA5 appeared to be an interesting target for anti-AML therapy. Our previous work demonstrated a pattern of SMARCA5 upregulation at AML diagnosis followed by its normalization upon achieving the hematologic remission. Importantly, additional work has not identified recurrent mutations of SMARCA5 in AML or any malignant disease . For example, for the d in AML . Based oSMARCA5 gene in AML cell lines with a CRISPR/Cas9-mediated deletion strategy, we could observe that AML cells lacking SMARCA5 markedly slowed the proliferation rate and became dysplastic with multiple karyotypic abnormalities. Inhibiting SMARCA5 to achieve suppression of AML growth may be thus a very efficient strategy as AML cells that are likely addicted to SMARCA5 in order to overcome various chromatin obstacles such as complex karyotype or also polyploidy often seen during progression of AML. Other data further implicated that SMARCA5 is very important also at the stem cell level to regulate their innate function: to repopulate the progeny. Indeed  the AML cells are highly proliferating compared to their normal counterparts, and ii) SMARCA5 being expressed in stem cells implicates that antiSMARCA5 therapy would preferentially target the leukemia stem and progenitor cells. Upon targeting of the Trp53 deletion in murine thymocytes [While SMARCA5 expression represents a potential target for AML therapy, it may also serve as a factor of therapeutic resistance in AML. It is likely that additional factors will be involved in modulating therapy efficacy using SMARCA5 inhibitors in the future. As the Smarca5 loss was sensed in a mouse model by a) increased p53 levels and b) associated with DNA damage response (DDR), and c) activation of the p53 targets , very liSMARCA5 targeting is at least in part caused by karyotype abnormalities, especially cohesion defects, and possibly also by a putative replication defect due to defective chromatin compaction as well as dysregulation of gene expression pattern of the key hematopoietic lineage restricted transcription factors. Interestingly, the nuclear changes after S5 deletion such as polyploidy were also described in other cell lines of hematologic origin [Smarca5 deletion of developing brain or eye lens [SMARCA5 exon5 in AML cells by CRISPR/Cas9 as evidenced by the herein presented data. Data from global CRISPR/Cas9 screen identified that SMARCA5 targeting was very efficient and caused cell growth inhibition in several additional AML cell lines  and also in lymphoma and carcinoma cell lines [Our herein presented data indicate that AML growth is dependent on the expression of chromatin remodeling protein SMARCA5 that is a known partner of AML-associated targets: cohesin complex and CTCF . Data prc origin ,3 but noeye lens ,5. Simileye lens . Importaeye lens . Others eye lens . It seemeye lens . Our worll lines . TogethepX330-Venus (kindly provided by Dr. Bjoern Schuster) produces CRISPR/Cas9 enzyme that cleaves at a specific location based on sequence guide sgRNA defined target sequences in SMARCA5 intron4 (5′-TTCTTACGTTACCCATATACTGG-3′) and SMARCA5 intron5 (5′-ATTTATCATATTTTCAGCGATGG-3′). CRISPR/Cas9 enzyme is also fused with fluorescent protein mVenus, that enables selection of successfully transfected clones by FACS sorting. The DNA sequences for the sgRNA SMARCA5 intron4 and sgRNA SMARCA5 intron5 were synthesized by Sigma-Aldrich as four oligonucleotides with modifications at position 1 (to encode a Guanine due to the transcription initiation requirement of the human U6 promoter). These two pairs of complementary oligos were mixed together, boiled at 95 °C for 10 min, and allowed to cool down to RT to hybridize. Double-stranded oligos also designed with complementary BbsI overhangs on 3′ and 5′ ends were ligated into BbsI linearized pX330-Venus vector using T4 Ligase enzyme . Ligation mixtures were transformed into Subcloning Efficiency DH5α Competent Cells  following the manufacturer’s protocol. pX330-Venus sgRNA hSMARCA5 intron4 and pX330-Venus sgRNA hSMARCA5 intron5 were isolated and purified by GenElute HP Plasmid Midiprep kit  and correct oligo insertion verified by Sanger sequencing. 6 K562 cells using Amaxa Cell Line Nucleofector kit  and 1 × 106 K562 cells using Neon Transfection System 10 μL Kit (Invitrogen). Cells were cultivated for 48 h, Venus-positive cells sorted on BD FACS Aria Fusion and divided to form single cell clones on 96-well plates. DNA from growing clones was used as a template for PCR with the following primers: forward 5′-GAGATGGAGGGCTACACTGTG-3′and reverse 5′-GACATTCCCAAAGTCATCTAGCAG-3′. The resulting amplification produced 1175 bp fragment from wild-type and approximately 632 bp long fragment from CRISPR/Cas9 edited allele of the SMARCA5 gene. Cell smears (0.5–1 × 105 cells) were fixed with methanol and stained with May–Grünwald solution  for 5 min and Giemsa–Romanowski solution  for 12 min. Cell Proliferation Reagent WST-1  was used following manufacturer’s protocol starting from day 0 with seeding 0.5 × 104 cell/100 μL/well in triplicates and continued by daily measurement of absorbance at 430 nm on microplate reader Infinite 200 PRO . Cells were simultaneously counted by Luna Automated Cell Counter . K562 cells  were cultured in 90% Iscove’s Modified Dulbecco’s Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. NB4, SKM-1, and MOLM-13 were cultured in 90% RPMI-1640 medium (Sigma-Aldrich), OCI-M2 in 80% Iscove’s Modified Dulbecco’s medium  at 37 °C and 5% CO2. The media were supplemented with 10-20% fetal bovine serum (Biosera) and 1% penicillin/streptomycin (Biosera). Cell lines were purchased from DSMZ. Both pX330-Venus sgRNA SMARCA5 intron4 (1 μg) and px330-Venus sgRNA SMARCA5 intron5 (1 μg) were transfected into 2.5 × 10www.gencodegenes.org). Reads were counted using FeatureCounts version 1.6.5, normalized to transcripts per million (TPM) and log2 transformed. Log-rank test was performed in survival analysis, Wilcoxon test was used to assess differences in gene expression.RNA-Seq data sets from AML patient samples were previously described including the informed consent and ethical issues ,30,31. Rn = 10) and knockout (n = 5) K562 clones was isolated by TRIzol Reagent (Invitrogen) and reverse-transcribed by High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). Quantitative PCR was run in triplicates on LightCycler 480 (Roche) using LightCycler 480 SYBR Green I Master (Roche) and specific primers for human SMARCA5  and GADPH  gene. Ct values served for fold-change calculation using 2-ΔΔCt equation. Student’s t-test was used for statistical analysis.Total RNA from wild-type (Wild-type and S5KO K562 clones (1 × 10E7) were lysed in RIPA Buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors (Roche). Denatured cell lysates were run on 1 mm thick 10% SDS-PAGE gel (40 μg/lane) in Mini-Protean Electrophoresis system  and semi-dry-blotted onto PVDF membrane (Bio-Rad) using Trans-Blot Turbo transfer system (Bio-Rad). PVDF membrane was blocked for 1 h in 5% nonfat milk in 1x TBS/0.1% Tween-20 and incubated with primary antibodies: Snf2h/ISWI  and β-actin  overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies  visualized bands using Pierce ECL Western Blotting substrate (Thermo Fisher Scientific). Standard cytogenetic methods published previously ,11 were 6 fetal liver cells isolated from E13.5 control (Smarca5fl/+ Rosa26eYFP/+ Vav1-iCRE) and Smarca5-deficient (Smarca5fl/− Rosa26eYFP/+ Vav1-iCRE) with C57Bl/6J Ly5.2 background were transplanted into lethally irradiated (7.5 Gy) adult (8 weeks) C57Bl/6J Ly5.1 recipients. After 12 days, the recipients were euthanized, and their bone marrow and spleen were tested for the presence of donor-derived eYFP+ hematopoietic cells using flow cytometry. The antibody panel included CD45.1, CD45.2, c-Kit, Sca1, and lineage cocktail .For hematopoietic reconstitution experiments, 2.5 × 10n = 3) were isolated from E14.5 embryos, in which the Smarca5 gene contained the LoxP1 sites upstream and downstream of exon5 and also expressed Cre Recombinase-Estrogen receptor fusion protein that translocated into the nucleus upon addition of 4OHT into the cultures for 6 h. Deletion of Smarca5-exon5 represents a null allele [Tp53 gene [flox/flox Cre-Esr1 cells upon 4OHT addition was confirmed by previously published detection methods [S5KO MEF cells (l allele . Productp53 gene . Gene ta methods . Analysi


Infant acute myeloid leukemia (AML) is a rare subgroup of AML of pediatric patients under two years of age. For a long time, they have been included in pediatric forms of AML. However, infant AML demonstrates unusual clinical and biological characteristics, and its prognosis differs from AML in older children. Most treatment protocols do not distinguish age subgroups in pediatric AML, when in reality these unusual forms of infancy might benefit from specific targeted therapeutics. Herein we summarize the various specificities of infant AML. Infant acute myeloid leukemia (AML) is a rare subgroup of AML of children <2 years of age. It is as frequent as infant acute lymphoblastic leukemia (ALL) but not clearly distinguished by study groups. However, infant AML demonstrates peculiar clinical and biological characteristics, and its prognosis differs from AML in older children. Acute megakaryoblastic leukemia (AMKL) is very frequent in this age group and has raised growing interest. Thus, AMKL is a dominant topic in this review. Recent genomic sequencing has contributed to our understanding of infant AML. These data demonstrated striking features of infant AML: fusion genes are able to induce AML transformation without additional cooperation, and unlike AML in older age groups there is a paucity of associated mutations. Mice modeling of these fusions showed the essential role of ontogeny in the infant leukemia phenotype compared to older children and adults. Understanding leukemogenesis may help in developing new targeted treatments to improve outcomes that are often very poor in this age group. A specific diagnostic and therapeutic approach for this age group should be investigated.  Infant acute myeloid leukemia (AML) is a rare disease, historically defined by AML occurring under 1 year of age. This definition has been extended to AML in children up to 2 years of age as patients aged from 1 to 2 years old demonstrate similar clinical and biological pattern of disease . . 4]. HOX cluster genes, demonstrating that about 50% of pediatric non-DS-AMKL patients carry a HOX gene expression program [HOXA but not HOXB genes [KMT2A-R and NUP98-KDM5Asubtypes showed upregulation of  program ,6,50. Cr program . Coenen XB genes .These data demonstrated striking features of infant AML: (i) fusion genes are able to induce AML transformation without additional cooperation, (ii) there is a paucity of associated mutations and (iii) gene expression profile clusters in defined groups ,5,29.The discovery of specific molecular lesions in AML has shed light on both leukemogenesis and hematopoietic development. To understand the mechanistic role of these different molecular lesions, mouse models have been developed using gene editing to create transgenic mice. In parallel, patients derived samples have been used to generate xenotransplants in immunocompromised mice (PDX) ,6,7. TheUsing a transgenic mouse model Okeyo-Owuor et al. explored transformation efficiency of the MLL-ENL (KMT2A-MLLT1) fusion . KMT2A fUsing an inducible transgenic mouse model, Lopez et al., developed a model that phenocopied the human AML CBFA2T3-GLIS2 disease: they demonstrated striking differences in latency, phenotype, and molecular wiring, depending on the ontogenic stage at which the driver fusion oncogene was activated . DevelopThese studies have helped to understand the role of ontogeny in infant leukemogenesis and shed light on the specificities of infant leukemia. It also opens some insight on how to target specific genetic lesion.Infant with AML are currently treated with the same protocols used for older children and adolescents. Some collaborative groups have defined AML<1 year old as a high-risk factor and thus intensify the treatment  and have adapted dosage for chemotherapy to take in account the unique pharmacokinetic and pharmacodynamic profiles due to the young age. However, no protocol has been designed for this specific subgroup even though we demonstrated here that they exhibit unique clinical and biological characteristics.As shown above, some infant AMLs and more specifically some specific genetic subgroups have dismal outcome . FurtherTherefore, knowledge of the molecular and genetic background is of most relevance in order to detect novel leukemia and patient-specific treatment targets . The expCardin et al. were able to recapitulate phenotypic, genomic and transcriptomic characteristics of NUP98-KDM5A AMKL in xenografted mice . They coKrivtsov et al. demonstrated in vitro and in vivo efficacy of Menin inhibitor in KMT2A-driven AML . This smMenin inhibitors could also be of interest in NUP98-R AML, knowing the role of chromatin remodeling in leukemogenesis driven by NUP98-R oncoproteins . By identifying specific phenotype characteristics of CBFA2T3-GLIS2 AMKL, Smith et al. evaluated CD56 as a potential target for therapy . They usThese various studies demonstrate that understanding these rare forms of AML at a molecular and functional level could lead to targeted treatments that are applicable in infant AML resulting in greater treatment tolerance and efficacy. Infants with AML represent a cohort of patients with specific clinical and biological features. They present with some remarkable differences in clinical presentation, cytology, cytogenetic and molecular lesions when compared to older children. Some of these features are unique to AML in children less than 2 years of age. To date, infant AMLs are not differentiated from childhood AMLs in terms of therapeutic approach as they are treated on the same protocols. Understanding their differences could enable the creation of new international protocols designed to better characterize these leukemias and offer new therapeutic approaches.


A balanced intracellular Ca2+ homeostasis is maintained by highly coordinated actions of the intracellular Ca2+ cycling machinery including Ca2+ channels, Ca2+ pumps/transporters located on the plasma membrane, endo/sarcoplasmic reticulum (ER/SR) and mitochondrion in myocytes and fibroblasts [Special Issue, we compiled a coherent set of review and original research articles to introduce the major advances regarding our current understanding of the cardiac Ca2+ signaling via ion channels including voltage-gated Ca2+ channels, ligand-gated Ca2+ channels, ryanodine receptors (RyR2s), inositol 1,4,5-trisphosphate receptors (IP3Rs), Ca2+ pumps/transporters, and transient receptor potential (TRP) channels  [2+ cycling acts as a key contributing factor to an elevated predisposition for arrhythmias and/or impaired cardiac contractility in pathologically altered hearts. Under certain conditions such as exercise, aging, excessive alcohol exposure, obesity, diabetes, inherited mutational burdens, ischemia, systemic inflammation, these extrinsic/intrinsic stress stimuli trigger adaptive remodeling of Ca2+ handling machinery and regulatory signaling pathways in cardiac cells that are necessary to deliver an adequate acute response but also ensures long-term survival. Unfortunately, sustained stimulation of these stress stimuli ultimately disrupts the normal dynamics of cardiac Ca2+ handling, which results in myocardial maladaptive remodeling toward cardiac dysfunction. In addition, disrupted intracellular Ca2+ cycling and SR Ca2+ mishandling critically contribute to the development of fatal ventricular fibrillation (a major cause of sudden cardiac death) and atrial fibrillation . Moreover, detailed discussions by Lahiri et al., McKee et al., Hamilton et al., Wang et al., Rosenberg et al., [2+ dynamics via altered channel function of RyR2, IP3R2, and STIM1-Orai1, which lead to changed SR Ca2+ load and significantly increased diastolic SR Ca2+ leak promoting the initiation or progression of arrhythmias and heart failure. Altered enzyme activity and/or changed interactions between kinases/protein phosphatases and ion channels critically contribute to these stress-evoked changes in the Ca2+ homeostasis and cardiac electrical and mechanical dysfunction. While hyperactivation of CaMKIIδ is well-known to promote maladaptive cardiac remodeling during disease onset and progression, recent studies have uncovered crucial roles of several important kinases and protein phosphatases including stress-response kinase JNK2, striated muscle preferentially expressed protein kinase (SPEG), protein phosphatases PP1, PP2A, and endogenous PP1 inhibitor protein (I-1) in intracellular Ca2+ cycling and SR Ca2+ dynamics as well as enhanced arrhythmogenicity in the stressed heart. These newly identified channel modifiers within the macro-molecular complex of the Ca2+ handling machinery suggest the complexity of the regulation of the ion channel activities and intercellular Ca2+ homeostasis in cardiac myocytes.In this  et al.) , 17–19 h et al., , 17, 18 2+ signaling event occurs in both atrial and ventricular cells but with some notable functionally relevant and structurally based differences. Blatter et al. [2+ release involving the less studied cardiac IP3R type 2 (InsP3R-2) Ca2+ release channel for atrial ECC and atrial alternans in arrhythmogenesis were also presented. Moreover, accumulating studies suggest that mitochondrial Ca2+ and the mitochondrial Ca2+ handling units such as mitochondrial Ca2+ uniporters (MCU) are also involved in physiological Ca2+ homeostasis as well as pathological intracellular Ca2+ mishandling in diseased hearts. Ernst et al. [2+ and cytosolic Ca2+ dynamics using genetically-encoded Ca2+ indicators (GECIs) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and adult cardiomyocytes (ACMs). It should be noted that cardiac maladaptive function during the process of pathological myocardial remodeling  is well known to result in structural and biochemical changes related to the complex alterations of ion channel function, protein abundance, and protein post-translational modifications. Biochemical assays and proteomic analyses have thus been commonly employed. Rennison et al. [2+ mishandling and cardiac dysfunction are clearly worthy of future investigation. A much better understanding of the complex genetic regulatory networks involved in the cardiac pathogenesis will be critical for developing novel personalized therapies for patients.The beat-to-beat Cat et al.  reportedn et al.  reportedn et al.  also rep2+-activated K+ channels and TRP channels also contribute to the intracellular Ca2+ homeostasis. Ca2+-activated small conductance K+ channels, SK, or apamin-sensitive SK (IKAS) are differentially expressed in the heart. Zhang et al. [KAS channels are regulated and are involved in arrhythmias and proposed that sexual dimorphism in autonomic control of IKAS plays a role in J wave syndrome. In contrast to the SK channel, the Ca2+-activated TRPM isoform 4 (TRPM4) is a non-selective monovalent cation channel impermeable to Ca2+ ions. Dysfunction of TRPM4 contributions to electrophysiological abnormalities under a variety of inherited cardiac conduction block conditions and Brugada syndrome and is also associated with various forms of acquired heart defects such as arrhythmias and heart failure. Hu et al. [2+ homeostasis. Feng et al. [+ influx to ER/SR to counter-balance Ca2+ release was thoroughly reviewed by Zhou et al. [+ and Ca2+ can differentially modulate conduction during hyperkalemia and how enhancing determinants of ephaptic coupling may attenuate conduction changes in various physiologic conditions.In addition to the critical regulation of ECC, excitation-transcriptional coupling, and various other intracellular signaling cascades in cardiac contractile dysfunction and cardiac arrhythmia development, other ion channels including Cag et al.  presenteg et al.  conciselu et al.  elegantlg et al.  providedg et al.  knocked u et al. . Not onlu et al.  nicely d2+ cycling mediates various types of cardiac functional abnormalities such has heart failure and arrhythmias. This Special Issue highlights some of the major progresses that have been made regarding changes of intracellular Ca2+ homeostasis via ion channels regulated by either Ca2+-induced SR Ca2+ release or a number of ion channel modifiers including kinases and protein phosphatases in response to various intrinsic or extrinsic stimuli. All of these exciting new perspectives lay the groundwork for future investigation such as the inter-relationship between kinases and protein phosphatases as well as between organelles in calcium handling and homeostasis in both normal function and in the pathological state. The complexity of the genetic regulatory networks in the dysfunction of intracellular cycling and maladaptive cardiac remodeling and disease onset and progression are also worthy of further investigation. Moreover, the interactions between SR, mitochondrion, and nucleus in orchestrating normal intracellular Ca2+ homeostasis as well as aberrant Ca2+ handling under pathological conditions are an interesting research direction deserving more attention. While the functional role of fibroblasts in cardiac electrophysiology and structural remodeling has recently emerged as a research point of interest, the interaction between myocytes and fibroblasts is clearly another new research direction in need of attention. These novel discoveries and new directions in future research will hopefully lead to targeted and effective therapies to prevent the maladaptive remodeling and disease progression in the heart.Over the past few decades, significant progress has been made in understanding the underlying mechanisms by which intracellular Ca