For example, the spatial and temporal organization of Ca2+ signaling in the central nervous system is fundamental for neurotransmission, where local changes in cytosolic Ca2+ concentration are needed to transmit information from neuron to neuron, between neurons and glia, and even regulating local blood flow according to the required activity. However, under pathological conditions, Ca2+ homeostasis is altered, with increased cytoplasmic Ca2+ concentrations leading to the activation of proteases, lipases, and nucleases. This review aimed to highlight the role of Ca2+ signaling in neurodegenerative disease-related apoptosis, where the regulation of intracellular Ca2+ homeostasis depends on coordinated interactions between the endoplasmic reticulum, mitochondria, and lysosomes, as well as specific transport mechanisms. In neurodegenerative diseases, alterations-increased oxidative stress, energy metabolism alterations, and protein aggregation have been identified. The aggregation of α-synuclein, β-amyloid peptide (Aβ), and huntingtin all adversely affect Ca2+ homeostasis. Due to the mounting evidence for the relevance of Ca2+ signaling in neuroprotection, we would focus on the expression and function of Ca2+ signaling-related proteins, in terms of the effects on autophagy regulation and the onset and progression of neurodegenerative diseases.Calcium (Ca Signaling pathways utilizing Ca2+ are present during all stages of life, from egg fertilization to death, and have also been implicated in several pathologies and aging. Indeed, Ca2+ plays a central role in muscle contraction, cell proliferation, differentiation, metabolism, and death, as well as autophagy and other processes. However, the connection between Ca2+ signaling and central nervous system (CNS) protection remains elusive.As a key second messenger, Ca2+ mediates essential signaling processes and plays an important role in synaptic plasticity. In fact, the regulation of Ca2+ concentrations in each compartment is a prerequisite for normal neuronal function. The neuronal Ca2+ signaling machinery is able to simultaneously activate different Ca2+-dependent processes, even from a distance. Due to the fact that neurons are susceptible to slight perturbations in intracellular Ca2+ levels, regulating these levels can be viewed as neuroprotective.In neurons, Ca2+ concentrations, in an acute or chronic manner, lead to catastrophic cellular consequences, thus demonstrating the role this key second messenger has in controlling cell fate. In the present review, we discussed the role of Ca2+ signaling in neurodegenerative diseases, highlighting the interplay between the proteins altered in those pathological conditions and the subsequent alterations in Ca2+ regulation. A better understanding of these processes is directly relevant to the onset and progression of neuronal cell death.There are several circumstances where altered Ca2+ homeostasis and subsequent changes in the Ca2+ levels of cellular compartments can promote survival or induce the activation of cell death pathways [Altered Capathways ,2. IncreThe extrinsic apoptotic pathway is induced by the activation of death receptors in the cellular membrane. For example, tumor necrosis factor receptor 1 (TNFR1) promotes the activation of initiator caspases  and activates effector caspases  , as well as other molecules stored in the intermembrane space (IMS) [c binds to apoptotic protease activating factor 1 (APAF1), ATP/dADP, and procaspase 9, forming an apoptosome that subsequently activates effector caspases, with caspase 3 being especially susceptible to activation [During activation of the intrinsic pathway, Bax translocates from the cytosol to the outer mitochondrial membrane (OMM) , where ice (IMS) . In the tivation . The exttivation .2+, nicotinamide adenine dinucleotide (NAD+), proteins, glutathione, and other metabolites into the cytosol  opening. Described by Hunter et al. (1976), the mPTP is a voltage-operated channel, located in the inner mitochondrial membrane (IMM) . These piewed by ). In addiewed by ). These iewed by ,16. As wiewed by ), which Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) are among the most prevalent neurodegenerative diseases. In the elderly population, AD is perhaps the most frequently diagnosed neurodegenerative disorder, progressively impairing the memory and learning processes. Most cases of AD and PD are sporadic and characterized by late-onset, mostly affecting people with 60 years of age or more; however, about 10% corresponds to familial cases, having an early onset and commonly observed in individuals that are around 50 years of age or younger. On the other hand, HD is an inherited monogenic autosomal dominant disease, with symptoms often appearing at 40–50 years of age.2+ signaling include:Components associated with familial cases of neurodegenerative diseases that have been found to interfere with Ca(1) AD: mutations in genes codifying amyloid precursor protein (APP) or Presenilins 1 or 2. Presenilins are part of the catalytic subunit of the γ-secretase complex. The γ- and β-secretase enzymes together cleave APP, consequently generating β-amyloid peptides (Aβ), subsequently forming protein aggregates.(2) PD: the presence of intraneuronal protein aggregates called Lewy bodies, mainly composed of α-synuclein. Mutations in leucine-rich repeat kinase 2 (LRRK2) may stimulate protein activity.(3) HD: mutations, present as an expansion of CAG trinucleotides (polyglutamine repeats) close to the N-terminus, of the protein huntingtin (mHtt), which are prone to aggregation.c), known as PrPSc, resulting in neuroinflammation and neurodegeneration. A more thorough discussion related to these protein aggregation events and Ca2+ signaling would be discussed later in this review.Another inherited neurodegenerative disease involving protein aggregation includes frontotemporal dementia (FTD), which is caused by mutations in either the microtubule-associated protein Tau (MAPT:FTDP - 17MAPT) or the progranulin (PGRN:FTDP - 17PGRN) genes. Additionally, Creutzfeldt–Jakob disease (CJD) is associated with the accumulation and aggregation of a misfolded/unfolded isoform of brain cellular prion protein  stress. For example, PrPSc accumulation activates an adaptive response, known as the unfolded protein response (UPR), to correct improperly folded ER proteins [2+ concentrations are tightly regulated and are essential for neuronal function and survival, with Ca2+ overload being related to excitotoxic neuronal cell death. In the following sections, the mechanisms by which Ca2+ promotes cell death and the effect of neurodegenerative disease-related proteins on ionic buffering would be discussed.Moreover, it is well known that disruptions in Caproteins . Thus, i2+ levels is responsible for the selective activation of Ca2+-mediated signal transduction pathways. Neurons can modulate the activity of pumps, as well as voltage and/or ligand-gated ion channels, to generate Ca2+ fluxes through the plasma membrane. The activation of voltage-gated Ca2+ channels (VGCC) promotes Ca2+ influx with the electrochemical gradient, resulting in increased cytoplasmic Ca2+ concentration-dependent enzyme activation, neurotransmitter release, neurite outgrowth, hormone secretion, and other Ca2+-dependent processes.The regulation of Ca2+ channel subtype. The L-type Ca2+ channels participate in Ca2+-induced long-term potentiation in dendritic spines and postsynaptic dendrites, while P- and Q-type Ca2+ channels, present in the presynapse, generate inward Ca2+ currents and initiate neurotransmitter release and presynaptic plasticity. The opening of T-type Ca2+ channels results in currents that induce the rhythmic action potential production in the sinoatrial node of the heart  and low voltage-activated (T-type channels), with the α1 subunit determining the Caiewed by ).2+ influx through L-type VGCCs in a variety of cellular models , an effiewed by , and Aβ1activity . Interesactivity . In FTD,athology .pars compacta (SNpc) neurons produce action potentials without the need of synaptic input, setting them apart from most other types of neurons [V1.3 channel activity made SNpc cells more susceptible to Ca2+-mediated excitotoxicity, caused by subjacent mitochondrial dysfunction [2+ influx and low levels of intracellular Ca2+-buffering proteins, such as calbindin; thus, slight increases in Ca2+ levels could have potentially damaging results [Substantia Nigra  neurons . A previfunction . Further results .2+ channels, which contributes to the increased susceptibility of SNpc neurons to cell death [2+ channel activity is increased in SNpc neurons. Remarkably, in an in vivo model of PD, the pharmacological inhibition of Cav1.3 L-type Ca2+ channels, with isradipine, restores the normal activity of dopaminergic cells, protecting SNpc neurons from cell death [In the α-synuclein A53T transgenic mouse model for PD, the aggregate-prone mutant protein has been associated with the upregulation and hyperactivation of N-type Call death . During ll death .The VGCCs have also been shown to be sensitive to dihydropyridines, which are commonly used to treat hypertension. Additionally, amlodipine, an L-type VGCC blocker, is being tested in a clinical trial (NCT02913664), as a drug candidate that can reduce the risks for developing AD, and nilvadipine, an L-type VGCC antagonist, has recently completed a phase III trial as a treatment for AD (NCT02017340). In PD, some dihydropyridine compounds have been shown to reduce the overall population risk for developing PD ,31, and c has been associated with VGCC dysfunctions. A synthetic PrP fragment (PrP106–126) has been found to inhibit L-type Ca2+ channel activity in rat cerebellar granule cells, decreasing Ca2+ currents, and leading to apoptosis [Sc has presented N-type Ca2+ channel dysfunction, thus attenuating Ca2+ responses [2+ influx via VGCC, which could potentially impair Ca2+ homeostasis  receptors [3 levels, group II (mGluR2 and mGluR3) and group III (mGluR4 and mGluR6–8) are coupled to Gi protein and results in decreased cyclic AMP (cAMP) levels. Pathologically, Aβ42 oligomers, but not monomers, alter Ca2+ release from ER stores, via mGluR5. It was also previously reported that Aβ42 oligomers reduced Ca2+ release induced by IP3 and increased caffeine-induced ryanodine receptor (RyR) Ca2+ release without altering the total Ca2+ content in the intracellular stores [Physiologically, many stimuli can cause intracellular Caeceptors . Glutamar stores .2+ peak, primarily due to contributions from the AMPA and kainate receptors, which exhibit fast kinetics and rapid (millisecond) desensitization [2+ levels by activating, through the N2B subunit, NMDAR [2+ dysregulation and increases oxidative stress, consequently impairing synaptic plasticity [Glutamate primarily acts on ionotropic membrane receptors, such as N-methyl-D-aspartic acid (NMDA), β-amino-3-hydroxy-5-methylsoxazole (AMPA), and kainate receptors. The NMDA receptors (NMDAR) are composed of NR1 and NR2A-D subunits, and during action potential generation, there is a rapid depolarization resulting in a high, transient Catization . It is wtization . Howevert, NMDAR . Additiot, NMDAR . It has asticity . 2+ signaling in the spine, the coordinated enzymatic activities of Ca2+calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN)—a Ca2+-dependent phosphatase—are involved. In synapses, CaMKII and CaN are expressed at high levels, with slight alterations in Ca2+ concentrations, resulting in reduced CaMKII autophosphorylation and increased CaN activity, thus shifting the balance of these enzyme activities. Synaptic CaMKII is essential for LTP, being centrally involved in memory formation, whereas CaN activation is necessary for LTD processes in the hippocampus. In AD, alterations in Ca2+ signaling have been suggested to shift the synaptic activities of CaMKII and CaN, favoring CaN and disrupting the equilibrium between LTP and LTD mechanisms. Due to the subsequent LTD stimulation and LTP inhibition, synapses are lost in AD brains, resulting in memory impairment  and long-term depression (LTD) . This syiewed by ).2+ influx leads to apoptotic cell death, induced by increased ROS generation, sustained mPTP opening, and the release of pro-apoptotic signals [2+ and an essential component of excitatory synapses [Caenorhabditis elegans expressing a mutant form of Tau, 2N4R-TauA152T (TauAT). In this model, TauAT expression increased cytosolic Ca2+ concentrations, due to release from ER stores, hyperactivated CaN, and initiated necrosis [2+ signaling. Furthermore, in FTD, the involvement of AMPA receptors (AMPAR) has been associated with disease-related presenile onset due to the presence of anti-GluA3 (AMPAR) antibodies in the sera of patients, which promote dendritic spine loss in culture [2+ signaling could contribute to neuronal damage [The hyperactivation of glutamate receptors, mainly NMDAR, can inflict cell injury and, in some cases, result in cell death , a proce signals . It is psynapses . More renecrosis , thus pr culture . Anotherl damage .2+ homeostasis is altered when challenged with this excitatory amino acid [With regards to PD, in an experimental dopaminergic denervation model, increased cortico-striatal glutamatergic activity was observed . In addiino acid .2+ signaling [2+ currents have been associated with fast receptor translocation to the cell membrane [There is evidence that indicates there is a striatal synaptic dysfunction in HD that is related to alterations in Caignaling . For exaignaling . It is lignaling  since inmembrane . As a remembrane ). In extmembrane . Consistmembrane , as wellmembrane ,63. Howemembrane .1-42 [1-42/40 in a transgenic mouse model [1-40 promotes α7 nAChR activation in a neuroblastoma cell line [1-42/42 induced the activation of both α7 and non-α7, and increased Ca2+ influx [Besides glutamatergic signaling, cholinergic neurons are also associated with the cognitive impairments observed in AD patients , where A1-42 ,69. Addise model , and thaell line . Anotherell line . Finally+ influx .Sc initiated a synaptotoxic signaling cascade that activates NMDA and AMPA receptors in hippocampal neurons, increases intracellular Ca2+ concentrations, stimulates p38 mitogen-activated protein kinase (MAPK), and depolymerizes actin filaments in dendritic spines, leading to the collapse of the cytoskeleton [c protected neurons from Ca2+ overload via the modulation of ionotropic glutamatergic receptors function [c was reported to promote axonal growth in a Ca2+-dependent manner [c was shown to form a complex with mGluR5, and this complex could be activated by ligands, such as β-amyloid oligomers and laminin, promoting Ca2+ influx into the neurons [c induced abnormal Ca2+ currents in cultured neurons and cerebellar slices, resulting in glutamate-induced excitotoxicity and neuronal death [In prion disease, Fang et al. (2018) demonstrated that PrPskeleton . Synaptifunction . In addit manner . PrPc wa neurons . In factal death .2+ storage, lipid and protein biosynthesis, secretion, and transport [2+ concentrations in response to increased levels of the ion in the cytosol [The ER is the largest organelle in a cell and is responsible for a variety of functions, ranging from Caransport . Numerou cytosol . In addi cytosol . Notably2+ homeostasis in the ER, extracellular Ca2+ concentrations range from 1–2 mM, cytosolic is about 100 nM, and the ER lumen is 100–800 μM [3 receptor (IP3R), in the ER membrane, promotes Ca2+ release from the ER to cytosol, but other Ca2+ homeostasis mechanisms also exist in the ER (reviewed by [2+-mediated ryanodine receptor (RyR) activation leads to Ca2+-induced Ca2+ release (CICR) [2+ ATPase (SERCA), a pump that mediates the transport of Ca2+ back to the lumen of ER, represents a Ca2+ reuptake mechanism  and plasma membrane protein ORAI1 assemble to form the selective pore of store-operated Ca2+ channel (SOCC) and increase cytoplasmic Ca2+ concentrations when the ER stores are depleted . For exe (CICR) , and actiewed by ). Furtheiewed by ). 2+ signaling in AD. Additionally, mutant presenilins have also been implicated in AD-related Ca2+ dysregulation, exerting similar effects through different mechanisms. In the ER, presenilins have been proposed to function as Ca2+-leak pores, and several familial AD mutations may compromise the pore function, leading to Ca2+ accumulation in the ER [3R activity, consequently augmenting channel-mediated Ca2+ release [2+ due to physical interactions between these proteins and SERCA [2+ influx via SOCC and low levels of STIM1, indicating that mutant presenilins could dysregulate Ca2+ homeostasis [Previously, we discussed the impact of Aβ and Tau alterations on Can the ER ,87. Thes release ,89. Pres release . Previound SERCA . Recentleostasis .2+ release from the ER into the cytoplasm [3ryanodine binding, which is indicative of upregulated RyR protein expression [2+ release is increased [2+ contributions, mediated through RyR and IP3R, ultimately leading to ER stress and apoptosis [There is evidence that supports the role of dysregulated RyR function in AD, caused by excessive Caytoplasm . For exapression . In the ncreased . In priopoptosis .2+ influx, consequently resulting in SH-SY5Y cell degeneration in vitro. Conversely, the overexpression or activation of TRPC1 protected neurons against the effects of the neurotoxin [2+-independent phospholipase A2 group 6 (PLA2g6) protein, have also been associated with altered Ca2+ homeostasis. Interestingly, skin fibroblasts obtained from idiopathic and familial PD patients with the PLA2g6R747W mutation displayed reduced Ca2+ entry via SOCC and depleted Ca2+ stores in the ER. Inhibition of PLA2g6-dependent Ca2+ signaling in a mouse model, using genetic tools, triggered autophagic dysfunction in and death of dopaminergic SNpc neurons [Neurotoxins, such as 1-methyl, 4-phenyl pyridinium (MPP+) and salsolinol, used for pharmacological PD models, were found to downregulate transient receptor potential canonical l (TRPC1) expression and induce thapsigargin-mediated Caurotoxin ,98. Stud neurons .2+ stores are also indirectly influenced by mHtt. For example, mHtt has been shown to bind to IP3R1, increasing the affinity of the receptor for IP3, and leaking Ca2+ ions from the ER [2+ leakage through the receptor and depleting internal ER Ca2+ stores [2+ influx due to sensitization of IP3R to IP3 agonists in the ER, consequently leading to continuous ER Ca2+ store depletion. In an attempt to regulate Ca2+ influx via SOCC, there is an upregulation of STIM2 [2+ depletion.The ER Cam the ER . Additio+ stores . In HD, of STIM2  and TRPCof STIM2  expressi2+ homeostasis, with intense ion exchange occurring through mitochondria-associated ER membranes (MAM). This ER-specific domain is enriched with cholesterol and anionic phospholipids, allowing the ER and OMM to become very close (10–30 nm)  is the primary channel for transporting Ca2+ from the OMM to the IMM, and subsequently taken up into the mitochondrial matrix by mitochondrial Ca2+ uniporter (MCU) [3R activation, Ca2+ stored in the ER is released and travels across the MAM, and taken up by VDAC in the OMM of the mitochondria [2+ homeostasis and cell function. Furthermore, the protein deglycase DJ-1 and parkin play a role in the maintenance of ER-mitochondria contact sites, and parkin may also directly stimulate phospholipase Cγ1 (PLCγ1) in the cell membrane, ultimately leading to increased cytosolic Ca2+ concentrations (reviewed by [The mitochondria and ER play important roles in cellular Caiewed by ), and fuer (MCU) . Followichondria . In termiewed by ).2+ regulation (reviewed by [+), through the coordinated activities of enzyme complexes of the electron transport chain (ETC), located in the IMM . Many oiewed by ).2+ storage and flux in the organelle, which can influence the expression and activity of several enzymes and carriers. In the IMS, FAD-glycerol phosphate dehydrogenase (GPDH) has been shown to be regulated by changes in Ca2+ levels [2+ concentrations stimulate enzymes that participate in the tricarboxylic acid cycle (TCA). In addition, components of the ETC are also modulated in response to mitochondrial Ca2+ levels. In fact, it has been shown that increased Ca2+ levels stimulate, by about two-fold, the activities of ETC complexes I, III, and IV [2+ has also been shown to modulate the activity of F0F1-ATP synthase [Interestingly, many functions of the mitochondria are directly linked to Ca+ levels . While i, and IV . Moreovesynthase .Mitochondria gained notoriety in HD pathogenesis when it was discovered that systemically administering a mitochondrial complex II inhibitor, 3-nitropropionic acid (3-NP), to rats caused preferential striatal degeneration and impaired energy metabolism, resembling characteristics of the neurodegenerative disease . Additio2+ across the OMM is mediated by VDAC1, a channel permeable to ions and small hydrophilic molecules, of which some are related to metabolic functions, such as succinate, malate, pyruvate, NADH, ATP, ADP, and phosphate [2+ concentration changes in the IMS can impact the activity of some metabolic enzymes. Finally, to reach the mitochondrial matrix, Ca2+ must cross the IMM, which is impermeable to the ion. This transport step is achieved using the ΔΨm to create a driving force for Ca2+ accumulation in the matrix. However, any factor that can cause the ΔΨm to collapse will consequently reduce and/or suppress mitochondrial Ca2+ uptake [2+ entry into the mitochondria, the movement of Ca2+ through the IMM is more complex and requires the specialized MCU. This IMM protein modulates mitochondrial Ca2+ uptake when concentrations of this ion are elevated in the cytosol [2+ channel, which strongly binds to the ion and transports it against the electrochemical gradient and into the mitochondrial matrix [2+ levels in the mitochondrial matrix and that the overexpression of this protein caused a notable accumulation of this ion [The transport of Cahosphate ,118. As + uptake . Compare cytosol . The MCUl matrix . In vitrthis ion .It is possible that Aβ inhibits the transport of nuclear-encoded proteins into the mitochondria since interactions with the translocase of the outer mitochondrial membrane 40 (TOM40) complex reduce the import of complex IV subunits. Moreover, Aβ interferes with the function of ETC complexes IV and V, while Aβ and Tau act synergistically to impair complex I function. The accumulation of Aβ in the IMS has also been shown to compromise the permeability of both the IMM and OMM (reviewed by ).2+ homeostasis can contribute to neuronal degeneration in an in vitro model of PD [Neurotoxins, such as MPP+, induce SH-SY5Y cell death by activating the intrinsic apoptosis pathway, which is highly dependent on mitochondria. Additionally, it appears that MCU plays a role in MPP+-induced cell death since MCU overexpression partially attenuates neuronal cell death, and downregulation results in augmented autophagy and cell death due to AMPK activation, thus demonstrating that disruptions in mitochondrial Cael of PD . Furtherel of PD .2+ dysregulation when compared with normal fibroblasts. For example, fibroblasts from AD patients exhibited long-lasting mPTP activation, and pharmacological blockage with Cyclosporine A (CsA) improved mitochondrial and cytosolic Ca2+ dysregulation. Additionally, the AD fibroblasts displayed a pattern of mitochondrial dysfunction that resembled what is observed in AD brains, thus highlighting the potential utility in employing fibroblasts as peripheral biomarkers for early AD detection [Previous studies have shown that Aβ can trigger the opening of the mPTP by interacting with and translocating cyclophilin D (CypD), a key component of the mPTP, adenine nucleotide translocase (ANT), and VDAC to the IMM (reviewed by ). In AD,etection . The opeetection  and, in etection .2+ [c release, even at lower Ca2+ concentrations, and triggers neuronal apoptosis [Several findings suggest that mHtt interacts with the IMM and OMM through its cytosolic N-terminus. This interaction might have a negative impact on protein import  and coul2+ . Additio2+ , and stupoptosis . Althoug2+ is also stored in a variety of acidic organelles, including lysosomes, lysosome-related organelles, endosomes, secretory vesicles, vacuoles, and Golgi complex [2+-stores ranges from pH 4–5, with the stored Ca2+ playing a central role in intracellular Ca2+ signaling processes, hydrolase activity, and autophagy regulation [2+-permeable ion channels, including members of the transient receptor potential channel (TRP) and two-pore channel (TPCs) families [Besides the ER and mitochondria, Ca complex ,134, wit complex . Dependigulation . These ofamilies .2+ can vary considerably among TRP channels. While most display low selectivity for Ca2+ (PCa/PNa 0.3–10), TRPV5 and TRPV6 exhibit high Ca2+ selectivity (PCa/PNa > 100), and TRPM4 and TRPM5 are Ca2+ impermeable [The TRP family is divided into two groups, which are further divided into seven subfamilies of channels. Group 1 encompasses the TRPC , TRPV (vanilloid), TRPM (melastatin), TRPN , and TRPA (ankyrin) subfamilies, while Group 2 contains TRPP (polycystin) and TRPML (mucolipin) (reviewed by ). These ermeable .2+-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) [2+ release from acidic Ca2+ stores that subsequently triggers amplified Ca2+ release from the ER, via IP3R and RyRs [2+ release from acidic organelles, dysfunctions in these channels have been related to PD [2+ handling of acidic stores [2+ signals with BAPTA-AM -ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester), which is a Ca2+ chelator. Additionally, another study demonstrated that TPC-mediated lysosomal Ca2+ release stimulated autophagy via Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ) in cells over-expressing an LRRK2 mutation that causes an autosomal dominant form of PD and activates autophagy [2+ concentrations due to increased TPC-mediated Ca2+ efflux from the acidic organelles  ,141. Iniand RyRs . Due to ed to PD . For exac stores , and alsc stores . Hockey c stores . Interesutophagy . Upreguliewed by ). The ro2+ release can be mediated by the activation of transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1), TRPML3, and transient receptor potential cation channel, melastatin subfamily, member 2 (TRPM2) , a member of the MiT-TFE helix–loop–helix leucine-zipper (bHLH-Zip) family of transcription factors, that, once in the nucleus, binds to the coordinated lysosomal expression and regulation (CLEAR) sequence, transcriptionally regulating the expression of TRPML1, as well as other autophagic and lysosomal genes [2+ release activates CaN, a phosphatase that binds and dephosphorylates TFEB. Furthermore, TRPML1 overexpression was shown to induce autophagy, whereas TRPML1 silencing attenuated autophagy in HeLa cells [Previous work has shown that lysosomal Caiewed by ). Each oiewed by . More spal genes ,149. TRPLa cells . 2+ release is necessary for the activity of mTORC1 [Under nutrient-rich conditions, serines  of TFEB are phosphorylated by mammalian/mechanistic target of rapamycin (mTOR) , and S14f mTORC1 ,153 thatf mTORC1 .Located in early and late endosomes/lysosomes, TRPML3 has also been associated with autophagy regulation. In HeLa cells, it was demonstrated that TRPML3 overexpression potentiated, and channel silencing inhibited autophagy, and expression levels of this channel also influenced endocytosis and membrane trafficking .2+ release is stimulated by adenosine diphosphoribose [2+ release is much higher than the values reported for other channels stimulated by this second messenger. Currently, the physiological relevance of NAADP-mediated TRPM2 activation is still unclear [2+ release [2+ release was shown to be responsible for autophagy activation via CaMKKβ . 2+ release from late endosomes and lysosomes [The physiological importance of the TRPML1-mediated Caysosomes  is perhaysosomes .2+ homeostasis [2+ concentrations were reduced [In cases of early-onset AD, presenilin 1 mutations alter lysosomal activity, impairing V-ATPase-mediated lysosomal acidification, and perturbing Caeostasis . In caseeostasis , and, in reduced . TherefoWith regards to the familial α-synuclein PD models, it was recently demonstrated that TRPML1 agonists promoted lysosomal exocytosis, reduced α-synuclein secretion, and prevented α-synuclein accumulation in a human neuroglioma cell line. Thus, demonstrating the promising therapeutic potential of specific channel activating compounds could contribute to the treatment of PD .2+ release, lysosomal storage, vesicle trafficking, and lysosomal acid homeostasis, and the dysregulation of these processes has been associated with several degenerative disorders [2+ release, Coen et al. (2012) demonstrated that the content of lysosomal Ca2+ stores was significantly reduced in presenilin gene deleted cells and neurons [Previous studies have shown that TRPML1 is an important component of lysosomal Caisorders ,170,171.isorders . Using G neurons . This re neurons . Autophagy has evolved to become a lysosomal catabolic process, allowing cells to degrade and recycle deleterious components or organelles by autophagosomes and preserve cell homeostasis. Under normal physiological conditions, autophagy activation is maintained at basal levels but can be induced in response to nutrient deprivation, hypoxia, ER stress, and DNA. Autophagy supplies the cells with the essential amino acids, nucleotides, and fatty acids, needed for the synthesis of new molecules. However, post-mitotic cells, such as neurons, have a low proliferative rate and depend heavily on autophagy for cellular maintenance, making them extremely vulnerable to changes in this pathway damage (reviewed by ).2+-sensitive manner—which is in contrast to microautophagy or CMA, where cargos are sequestered and internalized with or without the aid of lysosomal membrane receptors  ,177. Maciewed by ).2+ levels and Ca2+ dependent kinases, such as Ca2+/calmodulin-dependent kinase 2 (CaMKK2), and blocks mTORC1, ultimately inducing autophagy [Autophagy is directly related to anabolic and catabolic processes, participating in a variety of regulatory pathways involved in cell growth and proliferation. In fact, the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and MAPK pathways are typically activated by the mechanistic target of rapamycin complex 1 (mTORC1) (reviewed by ), the mautophagy .2+ release leads to ATG1 protein Unc-51 like autophagy activating kinase (ULK1) activation and autophagy induction [The ATG proteins have also been shown to participate in the activation of autophagy, modulating the machinery responsible for autophagosome formation. Previous studies have shown that AMPK-mediated Canduction ,182. Tognduction ). It hasnduction .2+ in autophagy was performed by Gordon et al. (1993) and demonstrated that extracellular and intracellular Ca2+ chelators (BAPTA-AM) reduced the levels of autophagy in isolated rat hepatocytes [2+ mobilizing agents  induced autophagosome accumulation, which was accompanied by mTORC1 inhibition and CAMKK2 and AMPK activation [2+ can exert both inhibitory and stimulatory effects on autophagy [2+-mediated autophagy response depends on both the amplitude of the response and the specific channels involved  demonstrated that deleting Fkbp1b resulted in RyR leakage and mTOR induction . Furthertivation . In C2C1tivation . 2+ levels with a concomitant loss of ER Ca2+ stores, blocked autophagic flux and inhibited autophagosome and lysosome fusion [2+ levels. This compound has been shown to inhibit nutrient starvation-induced autophagy [The role of SERCA in autophagy induction is poorly understood. Studies showed that thapsigargin treatment, which increases cytosolic Cae fusion . Indeed,utophagy . Additioutophagy .2+ uptake increased AMP:ATP ratios, activated AMPK, and induced mTORC1 independent autophagy [With regards to MCU, data suggested that it negatively regulated basal autophagy rates since the pharmacological agent Ru360 and genetic inhibition of the uniporter abrogated mitochondrial Cautophagy ,201,202.2+ channel antagonists  increase autophagy independent of mTORC1, while L-type Ca2+ channel agonist BAYK8644 activates calpains and increases cAMP levels, which, in turn, positively regulates IP3 levels and results in increased cytosolic Ca2+ levels. As a result of continuous calpain activation, a cyclic pathway of autophagy inhibition is created [It is well known that VGCC has a regulatory role in autophagy; however, the specific function of these channels in autophagy is still unclear and likely depends on the type of channel activated or inhibited. For example, L-type Ca created .2+ entry by SKF-96365 was shown to stimulate autophagy through AKT/mTORC1 pathway inhibition [Several intracellular processes, including apoptosis, cellular proliferation, and gene expression, involve SOCC, and the available data in the literature suggests that these channels can differentially influence autophagy depending on cell type and autophagy inducer (reviewed by ). Moreovhibition ,206.2+ entry via SOCC positively regulates autophagy. In endothelial progenitor cells, increased Ca2+ transport through SOCC activated autophagy via the CAMKK2/mTOR pathway, and both Ca2+ chelators (EGTA or BAPTA-AM) and STIM1-silencing inhibited autophagy, thus confirming the involvement of SOCC in this process [On the other hand, studies have also demonstrated that Ca process . Under h process , which i process . Therefo2+ signaling has on autophagy regulation, with evidence suggesting that cellular Ca2+ signals are involved at different stages of the autophagy process, and playing roles in the initiation of phagophore formation and elongation and autophagosome/endosome fusion with the lysosome. Ca2+ signaling at ER-mitochondria contact sites would likely influence autophagy initiation, whereas Ca2+ channel activity at ER-lysosome contact sites would modulate lysosomal function, autophagosome fusion, and cargo degradation. For instance, with respect to AD, the deletion of presenilin-1 leads to a loss of lysosomal vATPase activity, which raises lysosomal pH, perturbs TRPML-mediated lysosomes, and blocks autophagy [The study results presented up to this point clearly demonstrates channel diversity and emphasizes the importance Cautophagy .2+ signaling and Ca2+ sources, the role this ion plays in autophagy is extremely complex and relies on multiple factors, such as cell type, status, including nutritional, along with ion channel type, localization, expression levels, isoforms, and regulatory mechanisms. Indeed, ion channel dysfunctions and altered autophagy regulation have been implicated in various diseases, suggesting that Ca2+ channel expression and/or activity could be exploited as potential therapeutic targets for the modulation of autophagic processes.Given the diversity of cellular Ca2+ release involving activated TPC channels. Following the release of Ca2+ from the lysosome, Ca2+ is also released from the ER, resulting in Ca2+/CaMKK/AMPK pathway activation and autophagy induction. The increase in LRRK2 activity and Ca2+ release leads to a pathological increase in autophagy induction and defective lysosomal acidification [2+-induced cell death, representing yet another potential cause of PD pathogenesis [In PD, the presence of Lewy bodies formed by aggregated α-synuclein is probably the result of protein aggregates that are resistant to autophagic degradation . Accordification . The ovefication ,216. Besogenesis .2+ homeostasis; however, the exact role that Ca2+ plays in apoptosis and autophagy regulation, especially in neurodegenerative diseases, remains unknown. In the CNS, Ca2+ controls cell fate and metabolism, accounting for most of the energy expenditure of the body. Altered Ca2+ homeostasis has been linked to numerous pathological processes and can lead to the activation of death pathways. Therefore, Ca2+ levels must be well regulated under these conditions since neurons are particularly susceptible to damage. The ER, mitochondria, and lysosomes mainly control Ca2+ buffering, and the interplay between these organelles, along with Ca2+ transport mechanisms involved in shuttling this ion across membranes, are essential to cellular homeostasis, as well as the modulation of autophagy. This catabolic process emerges as a neuroprotective strategy since the basis of neurodegenerative diseases lies in the accumulation of unfolded proteins that consequently impair cell function, alter metabolism, and upregulate the unfolded protein response, to the demise of the cytoskeleton. In addition, it has been reported that during aging and in age-related neurodegenerative pathologies, there is an impairment of autophagy, accompanied by elevated oxidative stress and unbalanced energy. Understanding the role of Ca2+ regulation buffering in apoptosis and autophagy could reveal novel therapeutic targets for combating neurodegenerative diseases.Based on the results from a variety of experimental models, it is clear that specific AD, PD, and HD-related proteins can disrupt Ca


Yet, considerable information is lacking distinguishing functional differences between mammalian α-parvalbumin (PVALB) and oncomodulin (OCM), a branded β-parvalbumin. Herein, we provide an overview detailing the current body of work centered around OCM as an EF-Hand Ca2+-binding protein and describe potential mechanisms of OCM function within the inner ear and immune cells. Additionally, we posit that OCM is evolutionarily distinct from PVALB and most other β-parvalbumins. This review summarizes recent studies pertaining to the function of OCM and emphasizes OCM as a parvalbumin possessing a unique cell and tissue distribution, Ca2+ buffering capacity and phylogenetic origin.EF-hand Ca As shown in Sri), in the inner ear  is a small EF-hand Ca2+ buffers do not?The term “oncomodulin” is derived from the initial discovery of OCM in cancerous tissue as an oncoprotein and from its similarity to calmodulin as a CaBP , birds (n = 4), amphibians (n = 17), and mammals (n = 11) in order to better elucidate how OCM fits within parvalbumins Although OCM shares many of the essential features of β-parvalbumins (described below), we show that mammalian OCM does not share a common phylogenetic lineage with lower vertebrate β-parvalbumins of the parvalbumin family . While tn = 7). Contrarily, the α-parvalbumin branch in Xenopus subsp. shared a higher degree of phylogeny and grouped with the β-parvalbumins from lower vertebrates rather than with mammalian OCM. On this basis, it seems that OCM in Amphibia was mislabeled and needs to be reclassified as another type of β-parvalbumin instead of being termed OCM. Indeed, Ictalurus punctatus and parvalbumin thymic-like protein from Callorhinchus milii, based on their shared phylogeny. It is worth mentioning that there are different thymic parvalbumins which are not extremely closely related despite the shared name, e.g., CPV3 and thymic parvalbumin in chicken  in  chicken . While a chicken  could be2+ and selective interactions with effector proteins  relative to apical cuticular plate (3.4 mM). In a separate study, OCM concentration was observed as high as 2.4 mg/g dry weight cochlea  organized into AB, CD and EF domains. The CD and EF domains are adjacent domains and contain the two Cang sites . OCM difng sites . Henzl ang sites  determin2+-bound OCM allows for potential effector protein interactions. Unlike calmodulin, which is known to interact with several proteins in a Ca2+-specific manner -based electromotility mechanism. Yang et al.  mice on the C57BL/6 genetic background exhibit progressive hearing loss starting at 1–2 months and are essentially deaf by 3–4 months. In the Ocm KO, progressive hearing loss at 2 months occurs prior to the loss of hair cells, suggesting that OCM may be protective against hearing loss  showed only minor impacts on hearing  regulation of Ca2+ transients that modulate Rho-dependent actin polymerization and hair cell elongation  receptors or L-type Ca2+ channels lead to Ca2+ transients amplified by Ca2+-induced Ca2+ release (CICR) from nearby cisternae, which increases intracellular levels of Ca2+. In addition to the amount of CICR, the magnitude and duration of these Ca2+ transients would be a function of cytosolic buffers and uptake into organelles  through Ca2+ transporters. Importantly, the effects of OCM buffering would depend on its binding kinetics. If OCM is a very fast buffer, then it would decrease the amplitude of the Ca2+ transient and slow its decay. However, as a slow buffer, OCM would bind Ca2+ more slowly, having less of an impact on the amplitude of the Ca2+ transient, but a larger effect on its decay. Such is the case for PVALB in skeletal muscle, which binds Ca2+ slowly and therefore has minimum impact on the amplitude of Ca2+ transients, but significantly increases the speed of its decay contributing to the increased rate of relaxation  and Slc26a5 as the top three genes that define the OHC transcriptome. Similar to OCM, Sorcin is an EF-hand CaBP localized throughout the body and lateral membrane of OHCs and is speculated to function in CICR from the OHC cisternae. If OCM has a similar role in OHCs as PVALB does in skeletal muscle, then it makes sense that OCM would work synergistically with Sorcin and other CICR Ca2+ regulators, or through regulating the Ca2+ signals induced by ACh.In the adult mammalian ear, OCM serves as the dominate EF-hand CaBP in OHCs and its absence leads to OHC dysfunction as inferred from diminished DPOAEs (Hackney et al., m et al.  identifivia an endocytic mechanism, but only when intracellular cAMP is elevated above basal levels. The mechanism detailing how elevated intracellular cAMP causes OCM binding is unknown.A general hypothesis of OCM-induced regeneration was laid out by a series of experiments from Kurimoto et al. . Brieflyde novo synthesis of OCM just as loss of PVALB can lead to upregulation of OCM in certain brain regions. All of these proposed roles for OCM beg the question whether it is possible for a single small molecule to function in such widely disparate ways. Our proposed models of OCM function are based on data from cochlear hair cells and retinal macrophages that present very divergent mechanisms of OCM function. Fully delineating the molecular details of how OCM protects adult auditory function and augments nerve regeneration will be essential next steps.In this review, we show that OCM is evolutionarily distinguished from the majority of lower vertebrate β-parvalbumins. Experiments to date suggest that OCM may have an ambiguous function that depends upon the cell type in which it is expressed, and this ambiguity further distinguishes it from other EF-hand CaBPs. In sensory cells, recent studies suggest that OCM plays an essential role in maintaining auditory function, most likely affecting OHC motility mechanisms. In immune cells, OCM may be secreted in response to inflammatory signals and facilitates axon regeneration. Although originally defined as a potential oncoprotein, whether it plays a major role in this regard remains inconclusive. As such, the term “oncomodulin” may be a misnomer for mammalian β-parvalbumin. Independent of any oncogenic function, the process of transforming certain cells can lead to the LC, AC and DS wrote the manuscript. AC and TR performed the phylogenetic analysis. All authors contributed to commenting and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aimed to elucidate the underlying mechanisms of DCF-induced relaxations. The effects of DCF on airway smooth muscle (ASM) cells were explored using multiple biophysiological techniques, such as isometric tension measurement and patch-clamping experiments. Both high K+- and ACh-evoked contraction of TRs in mice were relaxed by DCF in a dose-dependent manner. The results of isometric tension and patch-clamping experiments demonstrated that DCF-induced relaxation in ASM cells was mediated by cytosolic free Ca2+, which was decreased via inhibition of voltage-dependent L-type Ca2+ channels (VDLCCs), nonselective cation channels (NSCCs), and Na+/Ca2+ exchange. Meanwhile, DCF also enhanced large conductance Ca2+ activated K+ (BK) channels, which led to the relaxation of ASMs. Our data demonstrated that DCF relaxed ASMs by decreasing the intracellular Ca2+ concentration via inhibition of Ca2+ influx and Na+/Ca2+ exchange. Meanwhile, the enhanced BK channels also played a role in DCF-induced relaxation in ASMs. These results suggest that DCF is a potential candidate for antibronchospasmic drugs used in treating respiratory diseases such as asthma and chronic obstructive pulmonary disease.Diclofenac sodium (DCF) is a nonsteroidal anti-inflammatory drug (NSAID) and is widely used as an analgesic and anti-inflammatory agent. Herein, we found that DCF could relax high K Approximately 300 million individuals suffer from asthma worldwide . Asthma 2+ plays important roles in regulating the tone of airway smooth muscles (ASMs) (2+ ([Ca2+]i) in ASM cells depends on many factors, including intracellular Ca2+ release, extracellular Ca2+ influx, and Na+/Ca2+ exchange , nonselective cation channels (NSCCs), and Na+/Ca2+ exchange  . The conexchange . Among texchange . In the  of ASMs .2+ homeostasis via regulating Na+/Ca2+ exchange (Diclofenac sodium (DCF) is a nonsteroidal anti-inflammatory drug (NSAID) widely used in the treatment of inflammatory diseases and pain . NSAIDs exchange  and NSCCexchange . Therefo+ (80 mM K+) or ACh to mimic the physical state of airway hyperactiveness to evaluate the potential relaxant ability in TRs and elucidate the underlying mechanisms. Our results demonstrate that DCF effectively relaxed ACh/high K+-precontracted TRs by inhibiting VDLCC-mediated extracellular Ca2+ influx and enhancing the activity of BK channels. These data suggest that DCF is potential new anti-asthma drug candidate.Our lab is focusing on developing new drugs for bronchospasm via two methods: screening new ingredients from natural plants and old drugs. DCF is screened as a potential candidate for relaxing precontracted ASMs in an “old drug, new function” way. In the present study, mouse tracheal rings (TRs) and ASM cells were isolated. The tissues/cells were stimulated using high KUnless otherwise stated, all chemicals were purchased from Sigma-Aldrich Corporation.DCF  was purchased from Yuanye Biotechnology Co., Ltd. . Nifedipine was dissolved in dimethyl sulfoxide (DMSO) as a stock solution.2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.4). To generate the high K+ solution (80 mM K+), the NaCl in the PSS was replaced with an equimolar concentration of KCl. All experiments were conducted at room temperature (22–25°C).The physiological saline solution (PSS) solution comprised the following components (in mM): 135 NaCl, 5 KCl, 1 MgClad libitum access to food and water. All protocols and experiments involving animals were conducted in strict accordance with the guidelines of the Institutional Animal Care and Use Committee of the South-Central University for Nationalities. This study was approved by the Animal Ethical Committee of South-Central University for Nationalities .Male SPF BALB/c mice (6–8 weeks) were purchased from the Hubei Provincial Center for Disease Control and Prevention . Mice were housed in a 12-h light/dark cycle with Muscle force of TRs was measured as we previously reported elsewhere . Briefly2+ PSS (LCPSS) composed of 135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM glucose, 10 mM HEPES, and 0.1 mM CaCl2 (pH 7.4). After removal of the epithelial cells using a fine forceps, the ASM trips were cut off and incubated in LCPSS containing 1 mg/ml papain, 0.5 mg/ml dithioerythritol, and 1 mg/ml BSA for 20 min at 37°C. Then, the muscle trips were transferred into LCPSS containing 1 mg/ml collagenase H, 1 mg/ml dithiothreitol, and 1 mg/ml BSA for 20 min at 37°C. After three washes in LCPSS, the tissues were triturated using a fire-polished glass pipette to obtain single ASM cells. Single ASM cells were stored in 1.5-ml Eppendorf tubes on ice and used within 4 h.Single mouse ASM cells were enzymatically isolated as previously reported . Briefly2+ as the charge carrier with an EPC-10 patch-clamp amplifier . The composition of the pipette solution was (in mM): 130 CsCl, 10 EGTA, 4 MgCl2, 4 Mg-ATP, 10 HEPES, and 10 tetraethyl ammonium chloride (pH 7.2). Meanwhile, the bath solution contained (in mM) 105 NaCl, 6 CsCl, BaCl2, 11 glucose, 10 HEPES, and 0.1 niflumic acid (NA) (pH 7.4). The holding voltage was −70 mV. Currents were recorded following depolarization for 300 ms from −70 to +40 mV in 10-mV increments every 1 s.VDLCC currents were recorded as previously reported with some modifications . Briefly+ channels, the whole-cell recording method was used as described previously . The bath solution contained (in mM) 150 NaCl, 5.4 KCl, 0.8 MgCl2, 5.4 CaCl2, and 10 HEPES (adjusted to pH 7.2 with KOH).To measure the currents of Keviously . Briefly+ ion concentrations were used to record BK currents. The intracellular solution composition was as follows (in mM): 140 KCl, 1 MgCl2, 5 EGTA, 4.37 CaCl2, and 10 HEPES (adjusted to pH 7.2 with KOH). The bath solution contained (in mM) 140 KCl, 1 MgCl2, 4.9 CaCl2, 1 EGTA, and 10 HEPES (adjusted to pH 7.2 with KOH). The digitization rate was 4 kHz and filtered at 1 kHz. All-point amplitude histogram and single channel open probability (Po) were acquired using Clampfit 9 software .Single BK currents were measured as described previously . Briefly2O. The mice were exposed to aerosol ACh at different concentrations . Respiratory system resistance (Rrs) was acquired for 120 s for ACh/DCF at each concentration and calculated using flexiVent software.Airway reactivity to DCF was measured using a forced oscillation technique as previously described with some modifications . BrieflyP < 0.05 was considered statistically significant.Values are expressed as the means ± SEM. Statistical analysis and significance were measured with Student’s t-test or a one-way analysis of variance (ANOVA) using Origin 9.0 software . +-induced contraction in a dose-dependent manner. The maximal relaxation produced by DCF was 80.5% (IC75 = 298 μM). Meanwhile, high K+-elicited contraction was completely inhibited by a specific inhibitor of voltage-dependent L-type Ca2+ channels, nifedipine . The addition of 100 μM ACh elicited a small contraction, which was significantly smaller than that in the PSS group (P < 0.05) and completely relaxed by 230 μM DCF .CFC, YY, SS, and SH performed the experiments. MY wrote the manuscript. CLC and QL analyzed the data. JS designed the experiments and revised the manuscript.This work was supported by the National Natural Science Foundation of China (Grant No. 31771274), “the Fundamental Research Funds for the Central Universities,” South-Central University for Nationalities (Grant No. CZY19020), and fund for Key Laboratory Construction of Hubei Province (Grant No. 2018BFC360).CL was employed by Wuhan Youzhiyou Biopharmaceutical Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Both RyR2 and SERCA2a/PLN are highly regulated by post-translational modifications and/or different partners’ proteins. These control mechanisms guarantee a precise equilibrium between SR Ca2+ reuptake and release. The review then discusses how disruption of this balance alters SR Ca2+ handling and may constitute a first step toward cardiac damage and malignant arrhythmias. In the last part of the review, this concept is exemplified in different cardiac diseases, like prediabetic and diabetic cardiomyopathy, digitalis intoxication and ischemia-reperfusion injury.The present review focusses on the regulation and interplay of cardiac SR Ca Indeed, alterations that constitute the hallmark of these diseases, like contractile dysfunction, cardiac arrhythmias or cell death are in great part a reflection of the impairment in Ca2+ handling and the altered function of the SR, a pivotal responsible of Ca2+ cycling within cardiac myocytes.Cardiovascular diseases are the leading cause of morbidity and mortality worldwide, being Ca2+ handling proteins involved in SR Ca2+ uptake and release, i.e., SERCA2a/PLN and RyR2. Both proteins are highly regulated by additional partners’ proteins and/or PTMs that may increase or decrease their activity. (2) To describe how the disruption of the interplay among these proteins may constitute a key determinant of Ca2+ triggered arrhythmias and cardiac damage. (3) To recapitulate experimental evidence in which alterations of the balance between these key players contribute to SR Ca2+ mishandling with the ensuing production of Ca2+ triggered arrhythmias and cell death in different cardiac diseases.The purpose of this review is: (1) To summarize the regulation of the main cardiac SR Ca2+ ions are the link between the two processes (Cardiomyocyte excitation-contraction coupling (ECC) is the process that links membrane depolarization, at the surface cell level, with myofilament interaction that drives contraction, inside the cell. Carocesses .2+ ions the main players. Ryanodine Receptors 2 (RyR2) and SERCA2a are highly and precisely regulated by several proteins and kinases, which allow a fine-tuned synchronization of Ca2+ cycling and therefore of cardiac contraction and relaxation processes. We will mention here several key modulators and PTMs of these two main proteins. For a further review see for instance , by AGEs (glycation) and by acetylation/deacetylation processes. SERCA2a sumoylation appears to prolong the lifetime of SERCA2a as well as to increase its intrinsic activity by SUMO1 binding to Lys480 and Lys585 residues. Indeed, increasing SUMO1 expression restores SERCA2a levels, improves hemodynamic performance, and reduces mortality in heart failure   . Phosphoactility .16 site and simultaneously inhibits PP1 through the PKA dependent phosphorylation of two additional proteins, inhibitor-1 (I-1) and the small heat shock protein 20 (Hsp20)  . Under βibit PP1 .2+ uptake  associated protein X-1 (HAX1), a ∼35 kDa protein, which was identified forming a complex with HS-1 in lymphocytes (2+ binding protein (HRC), which has been shown to interact with both SERCA2a and triadin on the SR luminal side  may also influence SR Ca+ uptake . Among t+ uptake . PLN pla+ uptake . Moreovetivation . Severalphocytes , the intand load . As will release .2+ handling proteins. RyR forms a homotetrameric assembly comprising four monomers of 565 kDa each   ; the Ca2n region . This con region . This al2+ on the RyR2 without having the inherent ability to open or close the channel per se  was first described by Witcher and collaborators as a CaMKII site  site as the primary phosphorylation site responsible for SR Ca2+ leak and arrhythmogenic events in HF  were protected from arrhythmias and cardiac dysfunction induced by several diseases  was characterized as a PKA phosphorylation site using classical phospho-epitope mapping , results in structural changes in the RyR2 phosphorylation domain that enhance the interaction with PKAc. These findings strongly suggest that phosphorylation of Ser2814 site may affect the activity of PKA and impact on Ser2808, i.e., nearby phosphorylation sites might influence one each other , to avoid the simultaneous phosphorylation of Ser2814 residue , showing Serch other . This po residue , i.e., p2+ leak as well as cellular arrhythmias without significant changes in SR Ca2+ load and contractility  is the predominant isoform expressed in T-tubules and SR membranes of adult cardiomyocytes. Therefore, it is strategically positioned to modulate the activity of the RyR2s. ROS produced by NOX2 stimulates SR Ca2+ release via at least two pathways: direct oxidation or S-glutathionylation of RyR2s or indirectly through CaMKII activation -triggered activity of RyR2s. Moreover, it decreased the amplitude of the Ca2+ transient without affecting the amplitude or kinetics of ICa, reducing the “gain” of ECC mechanism. Sorcin seems to be a key RyR2-associated protein under stress conditions since its ablation displayed a significantly higher incidence of cardiac arrhythmias and sudden death in sorcin-KO mice when subjected to acute or chronic stress challenge (2+ uptake (2+ channels (LTCC)  . All the3H-ryanodine ([3H]Ry) binding to RyR2 at free Ca2+ concentrations of about 150 nM, while a significantly increased [3H]Ry binding occurred at Ca2+ concentrations greater than 300 nM. Hypothesizing a reduced RyR2 P0 at diastolic cytoplasmic Ca2+ levels, S100A1 would reduce SR Ca2+ leak in quiescent cardiomyocytes  process ; channel activity is maximal at 10–100 μM cytosolic Ca2+, while elevating cytosolic Ca2+ beyond this point leads to a reduction in P0  which eliminates Ca2+ regulation by luminal but not by cytosolic Ca2+ was recently identified , have received great attention from physiologists and clinicians. We will describe below two main consequences of the unbalance of SR Ca2+ uptake and release, i.e., Ca2+ triggered arrhythmias and cellular apoptosis and necroptosis, with a main focus on those produced by PTM of Ca2+ handling proteins.The normal interplay among the different proteins responsible for the release and reuptake of Ca2+ triggered arrhythmias, i.e., arrhythmias that are originated due to abnormal Ca2+ handling.As stated above, RyR2s are highly regulated molecules. Genetic or PTM of RyR2 are a main cause of CaCa (+ current (INa), has been recognized as an important player to set up the conditions for EADs, by producing SR Ca2+ overload, via the reduction of repolarization reserve and the increase in intracellular Na+ concentration. In addition, experimental evidence and modeling studies indicate that EADs may directly arise from Na+ channel reactivation  of an AP. Afterdepolarizations are divided into early and delayed afterdepolarizations, EAD, and DAD, respectively. EADs are defined as a slowing or reversal of normal repolarization that occurs before completion of AP, usually in phases 2 and 3 of human AP, whereas DADs occur after AP completion . These mCa . HoweverCa . The lattivation .2+ releases from the SR . This promotes a transient Na+ current (Iti), that depolarizes cell membrane and may eventually trigger a spontaneous AP ”. In post-infarction myocytes the post-refilling refractory period was reduced, an effect attributed to CaMKII phosphorylation and redox modifications of RyR2. In the context of Caractions . Diaz et content . Once sphreshold”. Due to  (SOICR)”. It has R occurs”. More re results , it was It is generally accepted that mitochondria are at the central stage of cell death . Indeed,2+ cycling, contraction, and relaxation  leads to mitochondrial membrane depolarization, failure to produce ATP and release of mitochondrial proteins such as cytochrome c, which initiate cell death pathways  due to RyR2 mutation only suffer arrhythmias after β-ARS. Experiments performed in S2814D myocytes, in which Ser2814 was mutated to aspartic acid and behaves as pseudo constitutively phosphorylated (0 of RyR2 produced by CaMKII phosphorylation was not able to evoke an SR Ca2+ leak higher than the one observed in wild type (WT) myocytes unless they are challenged by increasing extracellular Ca2+ or β-ARS , which have a fully loaded SR, have not proven to be prone to arrhythmias under basal conditions   . In the KO cells . Similarprotocol . Unexpeclar Ca2+  or I/R , which lacks leptin receptors  and the consequent decrease in SR Ca2+ content and Ca2+ transient. Moreover, a major mechanism for the ischemia-induced increase in diastolic Ca2+ is an increase in the frequency of Ca2+ sparks which may switch to arrhythmogenic Ca2+ waves during reperfusion , and exacerbating the effect of the preceding ischemia. Indeed, it was shown that reperfusion may trigger life-threatening arrhythmias, accounting for up to half of the total I/R-induced infarcts . Althougc damage . The inca2+ load , which ierfusion .2+ overload and subsequently ROS production trigger mitochondrial permeability transition pore and ROS production via ROS-induced ROS release mechanisms  and oxidized-CaMKII (ox-CaMKII) at the onset of reperfusion , alleviated post-ischemic cardiac injury  beneficial, by opposing to SR Ca2+ leak and rescuing at least in part SR Ca2+ load and contractility; (b) detrimental, because the increase in SR Ca2+ load would favor SR Ca2+ leak, arrhythmias, and cardiac damage. Interestingly, a greatly exacerbated increase in SR Ca2+ uptake, as that produced by PLN ablation, does contribute to increasing SR Ca2+ leak and cardiac damage by incrementing SR Ca2+ load and leak. Paradoxically, and in spite of the exacerbated SR Ca2+ leak, this increase may prevent Ca2+ triggered arrhythmias, by a different mechanism, i.e., diminishing cytosolic Ca2+ and avoiding Ca2+ wave propagation. Post-translational activation of RyR2 activity would produce a deleterious effect by increasing SR Ca2+ leak and predisposing to cardiac damage and arrhythmias. When this modification occurs only at the level of SR Ca2+ release/leak, the short-lived enhancement of SR Ca2+ leak may produce a decrease in contractility due to the decrease in SR Ca2+ load, without any further detrimental effect. Moreover, the increased activity of RyR2 during systole tends to preserve contractility, even at lower SR Ca2+ loads. The detrimental effect of RyR2 activation  can only take place when it occurs associated to an increase in SR Ca2+ uptake, able to maintain SR Ca2+ leak. This is the typical case of Ser2814D myocytes, with constitutive pseudo-phosphorylation of RyR2 at the CaMKII site.The normal interplay among the proteins involved in SR CaJP and AM generated the idea. JP, AM, MF, and CV wrote the manuscript, designed, and edited the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therefore, the identification of new therapeutic targets requires the analysis of the calcium-regulated elements at transcriptional levels. In this review, we focus onto the direct regulation of gene expression by KCNIP proteins (KCNIP1–4). These proteins constitute the class E of Ca2+ sensor family with four EF-hand Ca2+-binding motifs and control gene transcription directly by binding, via a Ca2+-dependent mechanism, to specific DNA sites on target genes, called downstream regulatory element (DRE). The presence of putative DRE sites on genes associated with unfavorable outcome for GBM patients suggests that KCNIP proteins may contribute to the alteration of the expression of these prognosis genes. Indeed, in GBM, KCNIP2 expression appears to be significantly linked to the overall survival of patients. In this review, we summarize the current knowledge regarding the quiescent GSLCs with respect to Ca2+ signaling and discuss how Ca2+via KCNIP proteins may affect prognosis genes expression in GBM. This original mechanism may constitute the basis of the development of new therapeutic strategies.Glioblastomas (GBMs) are the most aggressive and lethal primary astrocytic tumors in adults, with very poor prognosis. Recurrence in GBM is attributed to glioblastoma stem-like cells (GSLCs). The behavior of the tumor, including proliferation, progression, invasion, and significant resistance to therapies, is a consequence of the self-renewing properties of the GSLCs, and their high resistance to chemotherapies have been attributed to their capacity to enter quiescence. Thus, targeting GSLCs may constitute one of the possible therapeutic challenges to significantly improve anti-cancer treatment regimens for GBM. Ca More thl of GBM  and in hl of GBM . Thus, t2+ is a crucial second messenger (2+ concentration ([Ca2+]i) is tightly regulated and involves Ca2+ channels, pumps, and exchangers both at the plasma membrane and at the membrane of endoplasmic reticulum, mitochondria, or Golgi apparatus  and by its spatial  and time-dependent  components   . These dCCNA2, CCNB1, and CCNE2), or upregulated and classified as tumor suppressors, including the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the G0/G1 switch gene 2 (G0S2)  . Quiesce2 (G0S2)  and acid2 (G0S2) .2+ is an important regulator of the balance between quiescence and proliferation in hematopoietic stem cell (HSC)  and mitochondrial Ca2+-uptake  are downregulated in quiescence while others are upregulated . A functional analysis through a bioluminescent Ca2+ imaging approach showed that quiescence in GSLCs does not involve Cav1 channels like in HSCs, but is rather due to the modification of the kinetics of the store-operated Ca2+ entry (SOCE), mediated by plasma membrane ORAI channels associated with the ER membrane protein STIM1. The inhibition of store-operated channels (SOC) by SKF96365 triggers quiescence, further supporting the crucial role of SOC in quiescence in GSLCs. Interestingly, the use of bioluminescent Ca2+ reporter targeted to mitochondria revealed that this change in SOCE kinetics is due to an increased capacity of quiescent GSLCs’ mitochondria to capture Ca2+ and not to the modification of the SOCE mechanism itself  . In HSCsnt state . A RNAseit genes , establim itself . These d2+ homeostasis such as Ca2+ channels, pumps, and exchangers and established that some of these proteins contribute to tumorigenesis through regulation of proliferation, migration, or apoptosis   . The ide2+ sensor family, are globular proteins of 217–270 amino acids in size, with variable N- and C-termini and a conserved core domain containing four EF-hand Ca2+-binding motifs . Among the four EF hands, EF-1 is not able to bind Ca2+  and several alternatively spliced variants , which constitute the class E of Caind Ca2+ . Drosophvariants . In mamm tissues . KCNIP3, tissues  and in t tissues . KCNIP2  tissues , 2010. Dat E10.5  and Kcnifore E13 . In the ogenesis  and in tritories .via a Ca2+-dependent mechanism, to specific DNA sites, called DRE, of target genes. DRE sites are localized in the proximal 5′ sequence of the gene, downstream of the TATA box and upstream of the start codon, with the sequence GTCA forming the central core of the DRE site  results in a significant increase in the expression of these KCNIP3-dependent target genes . A recent study incidentally provides additional information on KCNIP expression in BT189 GSLC  and of transient receptor potential cation channels . Of note, close examination of this list revealed that KCNIP1–4 are indeed expressed in the BT189 GSLCs, although with different expression levels, and that KCNIP2 is upregulated by ING5 in this GLSC ; KCNN4, a potassium Ca2+-activated channel (KCa3.1); KCNB1, a voltage-gated potassium channel (Kv2.1); KCNJ10, a potassium Ca2+-activated channel (Kir4.1); and classified as Ca2+-binding protein; PRKCG, Ca2+-dependent serine/threonine protein kinase Cγ (PKCγ); PRKCB, Ca2+-dependent serine/threonine protein kinase Cβ (PKCβ); and CAMK2A, the Ca2+-calmodulin-dependent protein kinase IIα. KCNIP proteins are known to control gene transcription directly by binding to DRE sites. To test whether KCNIP proteins may be involved in the regulation of the expression of these selected prognosis genes, we searched for the presence of DRE sites within their proximal promoters. The CACNA1H and PRKCB genes present both one DRE potential site in their proximal promoter and KCNB1 presents two DRE-binding sites , their DRE sites respect orientation and repartition, even for tandem organization. This promising observation has to be tested for KCNIP binding efficiency. Recent evidence argues for the existence of functional DRE sites within the CAMK2A proximal promoter. KCNIP3 mutants with two amino acids substitution in the EF-hands two, three, and four are unable to respond to Ca2+ and function as a constitutively dominant active (daDREAM) transcriptional repressor (CAMK2A mRNA level is reduced by 1.7-fold compared to wild type (KCNN4 is conserved (79%), but in a lesser extend concerning DRE sequences. These sequence alignments for proximal promoters let guess a putative regulatory role of KNCIPs in the expression of some prognosis genes in GBM. Of note, not all of these prognosis genes exhibit DRE-like sites, as no DRE putative sequence was detected for KCNJ10 or PRKCG  and the TCGA (The Cancer Genome Atlas) identified genes belonging to the Ca outcome , 2018. Tannel Cav.2; KCNN4epressor . In tranild type . Mouse pannel Cav.2; KCNN4CG Table  interfac2+-binding proteins and according to their interaction partners and subcellular localization one can discriminate at least three main functions: (1) regulation of cellular excitability, (2) regulation of intracellular signaling, and (3) control of transcription.Although no specific data are available for KCNIPs’ function in GBM or even cancers, one can speculate taking into account published functions of KCNIP in other cell types. KCNIPs are in fact multifunctional EF hand Ca2+ channel Cav3  by interacting with the ryanodine modulator, presenilin  can also interact with other transcription factors such as CREB and therefore affects transcription of genes that do not contain DRE sites (review in f KCNIP3 . As CAMKIn conclusion, since no experimental data exists for the moment in the literature concerning the function of KCNIP family in GBM, this opens a new field of research. In other models, KCNIPs have pleiotropic effects. Their well-known role as transcriptional repressors, and the presence of DRE sites in the promoter region of some GBM prognosis genes argue for a transcriptional function of KCNIPs in GBM. However, non-transcriptional roles have also to be considered more closely in the future.IN, JH, M-CK, FA, MM, and CL designed the experiments. IN, FA, and CL performed and analyzed the experiments. IN, JH, M-CK, MM, and CL wrote the manuscript. JH, MM, and CL analyzed the data, provided financial support, and the final approval of manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


I highlight the critical factors for dynamic modulation of transmitter release and presynaptic short-term plasticity.Small size of an axon and presynaptic structures have hindered direct functional analysis of axonal signaling and transmitter release at presynaptic boutons in the central nervous system. However, recent technical advances in subcellular patch-clamp recordings and in fluorescent imagings are shedding light on the dynamic nature of axonal and presynaptic mechanisms. Here I summarize the functional design of an axon and presynaptic boutons, such as diversity and activity-dependent changes of action potential (AP) waveforms, Ca In this article, I like to overview these recently clarified presynaptic mechanisms for transmitter release, particularly focusing on small synapses.Direct patch-clamp recording from an axonal compartment tells a lot about the axon physiology and mechanisms of synaptic transmission. Unfortunately an axon and presynaptic terminals are usually too small to apply patch-clamp technique, except for unusually large structures, such as synapses of calyx of Held in the auditory brainstem and presynaptic boutons of hippocampal mossy fiber . Most co2+ influx to a presynaptic bouton and the amount of exocytosis of synaptic vesicles (2+ influx as ∼2 nA upon a presynaptic depolarization pulse to 0 mV (2+ currents at mossy fiber boutons in hippocampus (∼5 μm) and those in the cerebellum (∼6 μm)  . Recentl ∼300 pA . Thus, gon pulse . Thus, eon pulse . It shou2+ current amplitude likely depend on the first to second power of presynaptic diameter. Thus, we are tempted to assume that the RRP size estimated by Cm measurement simply reflects the surface area size of presynaptic structure, implying that the release machinery downstream of activation by Ca2+ may operate in a similar manner at various neuronal boutons. However, it should be noted that the vesicular release estimated by Cm increases upon depolarization for tens of ms sometimes reflects the fusion of multiple states of synaptic vesicles, including those already primed at and those loosely coupled to the release sites  than in a PC bouton (∼300 μs), in spite that once started the release kinetics are similar  in hippocampal mossy fiber boutons  as ∼-50 mV in a terminal of the calyx of Held synapse (GABA, presynaptic GABAAR activation depolarizes presynaptic boutons, facilitating Ca2+ influx upon the subsequent AP arrival and hence synaptic transmission. In contrast, axonal GABAARs also exert inhibitory effects in different neurons (The presence of receptors for neurotransmitters in an axon has been demonstrated at various brain region and influences axonal excitability and neurotransmitter release . Becauserebellum . Axonal  synapse  and -45  synapse . Because neurons .2+ concentration and downstream processes of transmitter release mediate short-term plasticity. Residual Ca2+ hypothesis is the simplest candidate mechanism for facilitation (2+ remaining in the cytoplasm after the first AP was suggested to facilitate the following AP-triggered transmitter release based on the ∼third power Ca2+-dependence of release (2+ increase is too small compared with the local Ca2+ to explain synaptic facilitation in most cases.Activity-dependent short-term plasticity of synaptic transmission lasting for milliseconds to minutes is an important element in the neuronal computation . A lot olitation . Tempora release . However2+ channels, which predominantly mediates transmitter release at many synapses, exhibit remarkable facilitation of its current in a Ca2+-dependent manner, resulting in facilitation of transmitter release (2+ to C-lobe of calmodulin, which is basically bound with the P/Q-type channels, is thought to rapidly augment the Ca2+ current by increasing the probability of channel opening. Exogenous expression of mutant Ca2+ channels in superior cervical ganglion neurons in culture demonstrated a tight correlation between facilitation of Ca2+ current and that of synaptic transmission (2+ current facilitation (2+-dependent facilitation of presynaptic Ca2+ influx takes place at glutamatergic boutons in the calyx of Held, which partly contributes to facilitation of synaptic transmission (2+-facilitation of P-type channels still exhibit short-term facilitation at many synapses in a physiological condition, suggesting just a minor contribution of Ca2+-current facilitation to short-term synaptic facilitation (P/Q-type of Ca release . The asssmission . Paired litation . Similarsmission . Howeverlitation .2+ buffer saturation hypothesis. If Ca2+ buffer molecules remain occupied by Ca2+ that entered into a bouton upon the first AP, cytoplasmic free Ca2+ becomes more abundant upon the following APs, and leading to larger transmitter release. Indeed, calbindin contributes to the facilitation at hippocampal mossy fiber-CA3 excitatory synapses and inhibitory synapses in the cerebral cortex (2+ and release machinery (2+-release coupling is indeed loose there, and facilitation depends on the presence of low concentration of fast Ca2+ buffer in the terminal (2+ buffer saturation model.Another candidate mechanism for synaptic facilitation is the Cal cortex . The bufachinery . Recentlterminal , in line2+-dependent facilitation of release sensors (The next hypothesis for short-term facilitation is the Ca sensors . Recent  sensors . The mec2+ current inactivation and AP conduction failure at axonal branches (2+ channels activated by a smaller AP is responsible for the depression of transmitter release at high-frequency activation in a PC bouton. Importantly, the AP conduction faithfulness seems to increase in PC axons along with the development, resulting in smaller frequency-dependent depression at this synapse in mature animals (In addition to facilitation, short-term depression has also been studied, and the RRP depletion is thought to be the major mechanism . Other mbranches  and inacbranches , could abranches . Recentlbranches . Smaller animals .2+ channel activation, and Ca2+-triggered release processes based on fine molecular organizations of release sites and vesicular replenishment systems. Different combinations of these dynamic elements would define the neuronal type-specific functional design of synapses and their short- and/or long-term plasticity. Rapid expansion of techniques for direct recording of axonal signaling with subcellular patch-clamp methods and/or fluorescent imagings is going to clarify neuron-specific and common functional designs of a variety of CNS synapses in the near future, which enriches our understanding of neuronal circuitry.As overviewed here, recent technical advances to study axon physiology are shedding light on rich computational ability of an axon affecting synaptic outputs, rather than the classical view of an axon as a simple reliable digital signal conductor. Transmitter release from a presynaptic bouton is dynamically controlled by a variety of activity-dependent modulations of critical factors, such as AP waveforms, CaS-yK wrote the manuscript and prepared the figures.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Reduced cardiac output in HF leads to compensatory mechanisms via activation of the adrenergic nervous system. In turn, chronic adrenergic overstimulation induces pro-arrhythmic events, increasing the rate of sudden death in failing patients. Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is an innate immune modulator that plays a key role in HF progression. NOD1 deficiency in mice prevents Ca2+ mishandling in HF under basal conditions, but its role during β-adrenergic stimulation remains unknown. Here, we evaluated whether NOD1 regulates the β-adrenergic modulation of Ca2+ signaling in HF. Ca2+ dynamics were examined before and after isoproterenol perfusion in cardiomyocytes isolated from healthy and from post-myocardial infarction (PMI) wild-type (WT) and Nod1-/- mice. Isoproterenol administration induced similar effects on intracellular [Ca2+]i transients, cell contraction, and sarcoplasmic reticulum (SR)-Ca2+ load in healthy WT and Nod1-/- cells. However, compared with WT-PMI cells, isoproterenol exposure induced a significant increase in the [Ca2+]i transients and cell contraction parameters in Nod1-/--PMI cells, which mainly due to an increase in SR-Ca2+ load. NOD1 deficiency also prevented the increase in diastolic Ca2+ leak (Ca2+ waves) induced by isoproterenol in PMI cells. mRNA levels of β1 and β2 adrenergic receptors were significantly higher in Nod1-/--PMI hearts vs WT-PMI hearts. Healthy cardiomyocytes pre-treated with the selective agonist of NOD1, iE-DAP, and perfused with isoproterenol showed diminished [Ca2+]i transients amplitude, cell contraction, and SR-Ca2+ load compared with vehicle-treated cells. iE-DAP-treated cells also presented increased diastolic Ca2+ leak under β-adrenergic stimulation. The selectivity of iE-DAP on Ca2+ handling was validated by pre-treatment with the inactive analog of NOD1, iE-Lys. Overall, our data establish that NOD1 deficiency improves the β-adrenergic modulation of Ca2+ handling in failing hearts.Heart failure (HF) is a complex syndrome characterized by cardiac dysfunction, Ca Heart failure (HF) is a complex clinical disorder characterized by the inability of the heart to deliver blood and nutrients to metabolic tissues. Chronic HF is a progressive disease, with high morbidity and mortality, and poses a significant economic burden on the healthcare system. Given its poor prognosis, the identification of molecular pathways contributing to HF is a major research goal.Compensatory activation of the adrenergic nervous system is a pathophysiological response to HF progression, which functions to maintain cardiac homeostasis through activation of neural hormones, mainly catecholamines . Accordi2+ mishandling , and this is amplified by a greater release of Ca2+ from the sarcoplasmic reticulum (SR) by ryanodine receptors (RyR2), increasing the intracellular Ca2+ concentration ([Ca2+]i) – a process known as Ca2+-induced Ca2+ release. This elevation in [Ca2+]i results in myofilament activation and cell contraction. During relaxation, Ca2+ is removed from the cytosol primarily by two mechanisms: Ca2+ re-uptake by the SR-ATPase (SERCA2a) and Na+/Ca2+ exchanger activation.Ca2+ transporters such as ICaL, RyR2, and phospholamban, among others  background were used in this study  and xylazine . Unconscious mice were shaved in the anterior region of the neck and the chest, intubated by tracheostomy, and connected to a small animal ventilator  for artificial ventilation at 150 strokes/minute and 230 μL stroke volume. A 2–3 mm incision parallel to the lower costal edge was made and the left pectoralis major muscle was dissociated until the ribs were exposed. Left thoracotomy was performed between the third and fourth ribs to visualize the anterior surface of the heart and left lung. A 1-mm-thick piece of gelatin sponge (Spongostan®), slightly moistened with saline, was inserted through the hollow thoracotomy to protect the lung. The ribs were separated with an eyelid-retractor and a branch of the left coronary artery was ligated with a blue polypropylene monofilament surgical non-absorbable suture 6/0. The Spongostan® sponge was then removed and the chest was closed with braided silk non-absorbable suture 4/0. Respiratory stimulation was performed to return the heart to spontaneous breathing. For postoperative analgesia, buprenorphine  was applied subcutaneously. Mice were kept on a warm electric blanket until spontaneous recovery. Sham-operated mice were used as the control group and underwent the same procedure, but without coronary ligation.Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine  and type II collagenase . Successful digestion was assumed when the flux rate increased and the heart color changed from red to pallor. The heart was then taken off the apparatus, and the ventricles were removed, minced into small pieces, and mechanically dissociated in a thermostatic bath at 37°C in the enzymatic solution. The cardiomyocyte cell suspension was filtered through a nylon mesh (250 μm) and centrifuged at 300 rpm for 3 min. The cell pellet was suspended in Tyrode solution containing CaCl2 (0.5 mM) and centrifuged as before. Finally, the cells were suspended in Tyrode solution containing CaCl2 (1 mM). The Tyrode solution contained (in mM): 130 NaCl, 5.4 KCl, 0.5 MgCl2, 25 HEPES, 0.4 NaH2PO4, and 22 glucose, which was adjusted to pH 7.4 with NaOH.Adult male mice (2 months of age) were anesthetized with sodium pentobarbital (100 mg/kg i.p.) and heparinized (4 UI/g i.p.). Hearts were rapidly removed and cannulated via the ascending aorta for Langendorff perfusion. Retrograde perfusion was initiated with a free calcium Tyrode solution containing 0.2 mM EGTA over 2–3 min at room temperature, and subsequently with Tyrode solution containing CaCl2+ concentration, [Ca2+]i transients, and Ca2+ waves were analyzed in intact isolated cardiomyocytes loaded with the fluorescent Ca2+ dye Fluo-3AM . [Ca2+]i transients were recorded in cells electrically excited at 2 Hz by field stimulation using two parallel platinum electrodes. Ca2+ waves were acquired in quiescent myocytes. SR Ca2+ load was assessed by rapid caffeine application (10 mM) in cells previously excited at 2 Hz.Local increases in intracellular CaF) were normalized by the basal fluorescence (F0) to obtain the fluorescence ratio (F/F0).Images were acquired by confocal microscopy , by scanning the cell with an argon laser every 1.54 ms. Fluo-3AM was excited at 488 nm and emitted fluorescence was collected at >505 nm. Data analysis was performed with homemade routines using software written on the IDL platform , designed by Dr. AM Gómez . Images were corrected for background fluorescence. The fluorescence values  for 1–5 min and intracellular Ca2+ dynamics were recorded. In some experiments, cells were pretreated with the selective NOD1 agonist C12-iE-DAP , which has significant cell membrane permeability and high potency  with a Polytron system (MWR). RNA (250 ng) was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems #4368813). This template cDNA was used in the qPCR reaction with Power SYBR Green PCR Master mix (Applied Biosystems #4367659) and specific primers in a 7900HT Fast Real Time PCR system (Applied Biosystems). 18s-RNA was used as a housekeeping gene. The primer sequences (5′-3′) for β1 and β2 adrenergic receptors and 18s-RNA were as follows:Frozen heart tissue was pulverized to powder and then homogenized in TRIZOL Reagentβ1-F CGCTGATCTGGTCATGGGATβ1-R GAAGAAGGAGCCGTACTCCCβ2-F AATAGCAACGGCAGAACGGAβ2-R TCACAAAGCCTTCCATGCCT18s-F CCAGTAAGTGCGGGTCATAAGC18s-R CCTCACTAAACCATCCAATCGG2 test, as appropriate. All statistical analyses were performed with Origin 8.0 software . Significance was set at P < 0.05.Results are reported as mean ± SEM. Statistical analysis was performed using two-way analysis of variance, paired (two-sided) Student’s t test or χTable 1). By contrast, Nod1-/--PMI mice showed similar heart weight, heart weight/body weight, and heart weight/tibia length values to Nod1-/- mice (Table 1). These data indicate that deficiency of NOD1 attenuates cardiac hypertrophy in our PMI mouse model.Compared with healthy WT mice, post-myocardial infarction (PMI)-mice 6 weeks after surgery showed significantly increased heart weight, heart weight/body weight ratio, and heart weight/tibia length ratio, consistent with the development of cardiac hypertrophy , cell contraction , and SR-Ca2+ load  in WT and Nod1-/- cardiomyocytes.We first analyzed whether deficiency of NOD1 determined the β-adrenergic response to Ca2+ dynamics plays a key role in HF-related cardiac dysfunction, we next questioned whether NOD1 deficiency impacted isoproterenol-induced modulation of Ca2+ handling in cardiomyocytes after PMI. Figure 1D shows representative line-scan Ca2+ fluorescence images obtained after electric field stimulation at 2 Hz in all groups. Under β-adrenergic stimulation, both WT-PMI and Nod1-/--PMI cardiomyocytes exhibited increased [Ca2+]i transients , augmented cell contraction , and faster time decay of [Ca2+]i transients , but all these changes were significantly greater in Nod1-/--PMI cells. These results reveal that, compared with WT-PMI cells, Nod1-/--PMI myocytes show a significant improvement in Ca2+ mishandling, which was mostly due to the increased amplitude of [Ca2+]i transients  and also higher cell contraction parameters . In this line, mean values of the amplitude of [Ca2+]i transients (F/F0) in cardiomyctes obtained under isoproterenol administration were 5.31 ± 0.52 (n/N = 13/5) in WT, 5.18 ± 0.32 (n/N = 14/4) in Nod1-/-, 4.18 ± 0.22 (n/N = 12/4) in WT-PMI, and 5.41 ± 0.21 (n/N = 13/4) in Nod1-/--PMI . Thus, [Ca2+]i transients obtained in the Nod1-/--PMI group during isoproterenol perfusion were similar to those of equivalent healthy WT and Nod1-/- cardiomyocytes. Consistent with this finding, treatment of WT-PMI mice with the selective NOD1 inhibitor nodinitib-1 resulted in higher amplitude isoproterenol-induced [Ca2+]i transients in isolated cardiomyocytes compared with cells isolated from vehicle-treated mice  in cardiomyocytes during isoproterenol exposure was 7.07 ± 0.69 (n/N = 9/3) in WT, 7.34 ± 0.93 (n/N = 10/4) in Nod1-/- , 5.77 ± 0.46 (n/N = 13/4) in WT-PMI, and 7.40 ± 0.50 (n/N = 12/4) in Nod1-/--PMI . Thus, the amplitude of [Ca2+]i transients triggered by caffeine was significantly higher in Nod1-/--PMI mice than in WT-PMI mice, both in the absence or the presence of isoproterenol . Additionally, the high values of SR-Ca2+ load in the Nod1-/--PMI group treated with isoproterenol were similar to those found in healthy WT and Nod1-/- cells treated with isoproterenol.Next, to question whether changes in i transients , cell contraction , and SR-Ca2+ load  compared with vehicle-treated cardiomyocytes. Treatment of cells with the inactive analog of IE-DAP, iE-Lys (20 μg/mL), resulted in a similar isoproterenol response for Ca2+ handling to vehicle-treated cells, demonstrating the selective effect of iE-DAP on Ca2+ handling under β-adrenergic stimulation .We next addressed whether the selective activation of NOD1 impairs the β-adrenergic regulation of Ca2+ release under β-adrenergic stimulation. As shown in Figure 5E, isoproterenol perfusion promoted a significant increase in Ca2+ waves in WT cells pretreated with iE-DAP as compared with vehicle- or iE-Lys-treated cells. Importantly, the percentage of Ca2+ waves in cells that were pretreated with iE-DAP and under isoproterenol perfusion was very similar to that found in WT-PMI cardiomyocytes under the same conditions . Supporting these data, iE-DAP administration failed to increase the percentage of Ca2+-wave-positive Nod1-/- cardiomyocytes (data not shown).Finally, we investigated whether the selective activation of NOD1 impairs diastolic Ca2+ handling due to impairment of systolic Ca2+ release ([Ca2+]i transients) and SR-Ca2+ load, which are highly conditioned by the increased diastolic Ca2+ leak.Overall, these results indicate that NOD1 activation limits the β-adrenergic regulation of CaOver the last decade, a large number of studies have shown that compensatory upregulation of the adrenergic nervous system response is a key factor in disease progression in chronic failing hearts. During the early stages of HF, there is a steady activation of the sympathetic nervous system to maintain cardiac output , whereas2+ release and by maintaining Ca2+ SR load, which are both compromised in HF , and compromising the SR-Ca2+ load (Several groups have shown that other pro-inflammatory mediators including TNFα, CXR4 or IL-1β impair the β-adrenergic response and reduce cardiac contractility , and shoa2+ load .2+ release is the cause of depressed SR-Ca2+ load in HF. Our data show that during β-adrenergic stimulation, diastolic Ca2+ release is significantly increased in failing WT cells, whereas deficiency of NOD1 prevents the increase in the HF-related diastolic Ca2+ waves. These data go some way to explain the improvement in the SR-Ca2+ load and the resulting improved systolic Ca2+ release and cell contractility in Nod1-/--PMI cells under isoproterenol stimulation.In many cases, increased diastolic CaNod1-/--PMI hearts showed physiological levels of phospho-RyR2 (2+ diastolic leak in Nod1-/--PMI cardiomyocytes. Aberrant diastolic Ca2+ leak in many cases such as HF leads to arrhythmogenesis and cardiac dysfunction (2+ leak can cause arrhythmias by Ca2+ waves that trigger delays after depolarizations (2+ mishandling in HF.pho-RyR2 , thus cofunction . RyR-derizations . Pro-arrNod1-/--PMI mice have higher mRNA levels of β1 and β2 adrenergic receptors than WT-PMI hearts, suggesting a possible mechanism for the larger systolic Ca2+ release and SR Ca2+ reuptake in by Nod1-/--PMI mice. β-adrenergic receptors are activated through heterotrimeric G proteins and subsequent activation of the adenylyl cyclase, which modulates the activity of several proteins essential for EC Ca2+ coupling, such as ICaL, RyR2, and phospholamban (Nod1-/--PMI cells may increase the efficiency of cardiac EC coupling through the augmented function of ICaL, RyR, and SERCA, improving both systolic Ca2+ release and SR Ca2+ loading in failing hearts.In line with our results, elevated levels of the inflammatory cytokines TNFα and IL-1β have been reported in both tissue and plasma from HF patients with arrhythmias . Moreoveholamban . Therefo2+ handling by blocking NOD1 can be a new tool to improve the cardiac function in failing hearts.One of the most common treatments to manage β-adrenergic impairment in HF patients is the use of β-blockers. It has been demonstrated that β-blocker treatment reduces the detrimental effects of catecholamine stimulation, such as toxicity, pathological elevated heart rate, and adverse remodeling and arrhythmia development in failing patients. Improving β-adrenergic regulation of Ca2+ handling in isolated cells from failing mice. Therefore, NOD1 emerges as a new potential target in the treatment of cardiac dysfunction and ventricular arrhythmias associated with HF.Our study demonstrates that deficiency of NOD1 improves the β-adrenergic modulation of CaMF-V conceived, designed, and discussed the experiments and wrote the paper. AV-B, JN-G, MT, PP, CZ, MP, LR-R, and MG-F performed the experiments. MF-V, AV-B, JN-G, CD, GR-H, and PP analyzed the data. MF-V, LB, GR-H, and CD contributed reagents, materials, and analysis tools.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.