Sustainability in a broad sense: An essential aspect of scientific conferences.

This article reflects on sustainability in the context of scientific conferences with emphasis on environmental, diversity, inclusivity, and intellectual aspects. We argue that it is imperative to embrace sustainability as a broad concept during conference organization. In-person conferences have an obvious environmental impact but mitigating strategies can be implemented, such as incentivizing low-emission travel, offering fellowships to support sustainable traveling, and promoting use of public transport or car-pooling. Utilizing eco-conscious venues, catering, and accommodations, along with minimizing resource wastage, further reduces environmental impact. Additional considerations include facilitating hybrid format conferences that allow both in-person and online attendance. Hybrid conferences enhance global participation whilst reducing resource consumption and environmental impact. Often-overlooked benefits can arise from the simple recording of talks to enable asynchronous viewing for people unable to attend in person, in addition to providing a legacy of knowledge that, for example, could support the training of early career researchers (ECRs) or newcomers in the field. The longevity of a research field, intellectual sustainability, requires an inclusive conference atmosphere, offering optimal opportunities for ECRs, minority groups, and researchers from emerging countries. Diversity and inclusivity not only enrich conference experiences but also enhances creativity and innovation.

Cellular effects of BAPTA: Are they only about Ca2+ chelation?

Intracellular Ca2+ signals play a vital role in a broad range of cell biological and physiological processes in all eukaryotic cell types. Dysregulation of Ca2+ signaling has been implicated in numerous human diseases. Over the past four decades, the understanding of how cells use Ca2+ as a messenger has flourished, largely because of the development of reporters that enable visualization of Ca2+ signals in different cellular compartments, and tools that can modulate cellular Ca2+ signaling. One such tool that is frequently used is BAPTA; a fast, high-affinity Ca2+-chelating molecule. By making use of a cell-permeable acetoxymethyl ester (AM) variant, BAPTA can be readily loaded into the cytosol of cells (referred to as BAPTAi), where it is trapped and able to buffer changes in cytosolic Ca2+. Due to the ease of loading of the AM version of BAPTA, this reagent has been used in hundreds of studies to probe the role of Ca2+ signaling in specific processes. As such, for decades, researchers have almost universally attributed changes in biological processes caused by BAPTAi to the involvement of Ca2+ signaling. However, BAPTAi has often been used without any form of control, and in many cases has neither been shown to be retained in cells for the duration of experiments nor to buffer any Ca2+ signals. Moreover, increasing evidence points to off-target cellular effects of BAPTA that are clearly not related to Ca2+ chelation. Here, we briefly introduce Ca2+ signaling and the history of Ca2+ chelators and fluorescent Ca2+ indicators. We highlight Ca2+-independent effects of BAPTAi on a broad range of molecular targets and describe some of BAPTAi's impacts on cell functions that occur independently of its Ca2+-chelating properties. Finally, we propose strategies for determining whether Ca2+ chelation, the binding of other metal ions, or off-target interactions with cell components are responsible for BAPTAi's effect on a particular process and suggest some future research directions.

The Complex Effects of PKM2 and PKM2:IP3R Disruption on Intracellular Ca2+ Handling and Cellular Functions.

Pyruvate kinase M (PKM) 2 was described to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and suppress its activity. To further investigate the physiological importance of the PKM2:IP3R interaction, we developed and characterized HeLa PKM2 knockout (KO) cells. In the HeLa PKM2 KO cells, the release of Ca2+ to the cytosol appears to be more sensitive to low agonist concentrations than in HeLa wild-type (WT) cells. However, upon an identical IP3-induced Ca2+ release, Ca2+ uptake in the mitochondria is decreased in HeLa PKM2 KO cells, which may be explained by the smaller number of contact sites between the ER and the mitochondria. Furthermore, in HeLa PKM2 KO cells, mitochondria are more numerous, though they are smaller and less branched and have a hyperpolarized membrane potential. TAT-D5SD, a cell-permeable peptide representing a sequence derived from IP3R1 that can disrupt the PKM2:IP3R interaction, induces Ca2+ release into the cytosol and Ca2+ uptake into mitochondria in both HeLa WT and PKM2 KO cells. Moreover, TAT-D5SD induced apoptosis in HeLa WT and PKM2 KO cells but not in HeLa cells completely devoid of IP3Rs. These results indicate that PKM2 separately regulates cytosolic and mitochondrial Ca2+ handling and that the cytotoxic effect of TAT-D5SD depends on IP3R activity but not on PKM2. However, the tyrosine kinase Lck, which also interacts with the D5SD sequence, is expressed neither in HeLa WT nor PKM2 KO cells, and we can also exclude a role for PKM1, which is upregulated in HeLa PKM2 KO cells, indicating that the TAT-D5SD peptide has a more complex mode of action than anticipated.

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing MCL-1-addicted cancer cells.

Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we discovered that BAPTAi alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTAi provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTAi diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTAi. Notably, these effects were also induced by a BAPTAi analog with low affinity for Ca2+. Consequently, our findings uncover PFKFB3 inhibition as an Ca2+-independent mechanism through which BAPTAi impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

Calcium signalling pathways in prostate cancer initiation and progression.

Cancer cells proliferate, differentiate and migrate by repurposing physiological signalling mechanisms. In particular, altered calcium signalling is emerging as one of the most widespread adaptations in cancer cells. Remodelling of calcium signalling promotes the development of several malignancies, including prostate cancer. Gene expression data from in vitro, in vivo and bioinformatics studies using patient samples and xenografts have shown considerable changes in the expression of various components of the calcium signalling toolkit during the development of prostate cancer. Moreover, preclinical and clinical evidence suggests that altered calcium signalling is a crucial component of the molecular re-programming that drives prostate cancer progression. Evidence points to calcium signalling re-modelling, commonly involving crosstalk between calcium and other cellular signalling pathways, underpinning the onset and temporal progression of this disease. Discrete alterations in calcium signalling have been implicated in hormone-sensitive, castration-resistant and aggressive variant forms of prostate cancer. Hence, modulation of calcium signals and downstream effector molecules is a plausible therapeutic strategy for both early and late stages of prostate cancer. Based on this premise, clinical trials have been undertaken to establish the feasibility of targeting calcium signalling specifically for prostate cancer.

Quantal leaps in understanding Ca2+ signaling: A "Taylored" approach.

On 2 September 2022, about 85 scientists gathered in person at Queens' College in Cambridge, UK, for a scientific meeting to celebrate the career of Colin W. Taylor of Cambridge University upon his retirement. The meeting was organized by the authors, who are all former graduate students in the Taylor laboratory, which has been at the forefront of Ca2+ signaling for more than 30 years.

A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca2+ signalling through IP3 receptors.

Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IP3R)-interacting protein by mass spectrometry. The PKM2:IP3R interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IP3R isoforms. Moreover, fluorescence microscopy indicated that both IP3R and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IP3R at a highly conserved 21-amino acid site (corresponding to amino acids 2078-2098 in mouse type 1 IP3R isoform). Synthetic peptides (denoted 'TAT-D5SD' and 'D5SD'), based on the amino acid sequence at this site, disrupted the PKM2:IP3R interaction and potentiated IP3R-mediated Ca2+ release both in intact cells (TAT-D5SD peptide) and in a unidirectional 45Ca2+ flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IP3R-mediated Ca2+ signalling in intact cells without altering the ER Ca2+ content. These data identify PKM2 as an IP3R-interacting protein that inhibits intracellular Ca2+ signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.

Off-target inhibition of NGLY1 by the polycaspase inhibitor Z-VAD-fmk induces cellular autophagy.

The polycaspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide: N-glycanase (NGLY1), an endoglycosidase which cleaves N-linked glycans from glycoproteins exported from the endoplasmic reticulum (ER) during ER-associated degradation (ERAD). Both pharmacological N-glycanase inhibition by Z-VAD-fmk and siRNA-mediated knockdown (KD) of NGLY1 induce GFP-LC3-positive puncta in HEK 293 cells. The activation of ER stress markers or induction of reactive oxygen species (ROS) is not observed under either condition. Moreover, Ca2+ handling is unaffected when observing release from intracellular stores. Under conditions of pharmacological NGLY1 inhibition or NGLY1 KD, upregulation of autophagosome formation without impairment of autophagic flux is observed. Enrichment of autophagosomes by immunoprecipitation (IP) and mass spectrometry-based proteomic analysis reveals comparable autophagosomal protein content. Gene ontology analysis of proteins enriched in autophagosome IPs shows overrepresentation of factors involved in protein translation, localization and targeting, RNA degradation and protein complex disassembly. Upregulation of autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13-deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan-caspase inhibitor, Q-VD-OPh, does not induce cellular autophagy. Therefore, experiments with Z-VAD-fmk are complicated by the effects of NGLY1 inhibition, including induction of autophagy, and Q-VD-OPh represents an alternative caspase inhibitor free from this limitation. ENZYMES: Peptide:N-glycanase1, Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase [EC:3.5.1.52].

Fundamentals of Cellular Calcium Signaling: A Primer.

Ionized calcium (Ca2+) is the most versatile cellular messenger. All cells use Ca2+ signals to regulate their activities in response to extrinsic and intrinsic stimuli. Alterations in cellular Ca2+ signaling and/or Ca2+ homeostasis can subvert physiological processes into driving pathological outcomes. Imaging of living cells over the past decades has demonstrated that Ca2+ signals encode information in their frequency, kinetics, amplitude, and spatial extent. These parameters alter depending on the type and intensity of stimulation, and cellular context. Moreover, it is evident that different cell types produce widely varying Ca2+ signals, with properties that suit their physiological functions. This primer discusses basic principles and mechanisms underlying cellular Ca2+ signaling and Ca2+ homeostasis. Consequently, we have cited some historical articles in addition to more recent findings. A brief summary of the core features of cellular Ca2+ signaling is provided, with particular focus on Ca2+ stores and Ca2+ transport across cellular membranes, as well as mechanisms by which Ca2+ signals activate downstream effector systems.

Cardiac pathology in neuronal ceroid lipofuscinoses (NCL): More than a mere co-morbidity.

The neuronal ceroid lipofuscinoses (NCLs) are mostly seen as diseases affecting the central nervous system, but there is accumulating evidence that they have co-morbidities outside the brain. One of these co-morbidities is a decline in cardiac function. This is becoming increasingly recognised in teenagers and adolescents with juvenile CLN3, but it may also occur in individuals with other NCLs. The purpose of this review is to summarise the current knowledge of the structural and functional changes found in the hearts of animal models and people diagnosed with NCL. In addition, we present evidence of structural changes that were observed in a systematic comparison of the cardiomyocytes from CLN3Δex7/8 mice.

Creating a New Cancer Therapeutic Agent by Targeting the Interaction between Bcl-2 and IP3 Receptors.

Bcl-2 is a member of a family of proteins that regulate cell survival. Expression of Bcl-2 is aberrantly elevated in many types of cancer. Within cells of the immune system, Bcl-2 has a physiological role in regulating immune responses. However, in cancers arising from cells of the immune system Bcl-2 promotes cell survival and proliferation. This review summarizes discoveries over the past 30 years that have elucidated Bcl-2's role in the normal immune system, including its actions in regulating calcium (Ca2+) signals necessary for the immune response, and for Ca2+-mediated apoptosis at the end of an immune response. How Bcl-2 modulates the release of Ca2+ from intracellular stores via inositol 1,4,5-trisphosphate receptors (IP3R) is discussed, and in particular, the role of Bcl-2/IP3R interactions in promoting the survival of cancer cells by preventing Ca2+-mediated cell death. The development and usage of a peptide, referred to as TAT-Pep8, or more recently, BIRD-2, that induces death of cancer cells by inhibiting Bcl-2's control over IP3R-mediated Ca2+ elevation is discussed. Studies aimed at discovering a small molecule that mimics BIRD-2's anticancer mechanism of action are summarized, along with the prospect of such a compound becoming a novel therapeutic option for cancer.

Constitutive IP3 signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP3 receptor disruptor BIRD-2.

Anti-apoptotic Bcl-2 proteins are upregulated in different cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), enabling survival by inhibiting pro-apoptotic Bcl-2-family members and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated Ca2+-signaling. A peptide tool (Bcl-2/IP3R Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IP3Rs by targeting Bcl-2's BH4 domain. BIRD-2 triggers cell death in primary CLL cells and in DLBCL cell lines. Particularly, DLBCL cells with high levels of IP3R2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IP3R2-expression levels, but also on constitutive IP3 signaling, downstream of the tonically active B-cell receptor. The basal Ca2+ level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP3 production. This constitutive IP3 signaling fulfilled a pro-survival role, since inhibition of phospholipase C (PLC) using U73122 (2.5 µM) caused cell death in SU-DHL-4 cells. Milder inhibition of IP3 signaling using a lower U73122 concentration (1 µM) or expression of an IP3 sponge suppressed both BIRD-2-induced Ca2+ elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP3 signaling also fulfilled a pro-survival role in other DLBCL cell lines, including Karpas 422, RI-1 and SU-DHL-6 cells, whereas PLC inhibition protected these cells against BIRD-2-evoked apoptosis. Finally, U73122 treatment also suppressed BIRD-2-induced cell death in primary CLL, both in unsupported systems and in co-cultures with CD40L-expressing fibroblasts. Thus, constitutive IP3 signaling in lymphoma and leukemia cells is not only important for cancer cell survival, but also represents a vulnerability, rendering cancer cells dependent on Bcl-2 to limit IP3R activity. BIRD-2 seems to switch constitutive IP3 signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy.

Examining Cardiomyocyte Dysfunction Using Acute Chemical Induction of an Ageing Phenotype.

Much effort is focussed on understanding the structural and functional changes in the heart that underlie age-dependent deterioration of cardiac performance. Longitudinal studies, using aged animals, have pinpointed changes occurring to the contractile myocytes within the heart. However, whilst longitudinal studies are important, other experimental approaches are being advanced that can recapitulate the phenotypic changes seen during ageing. This study investigated the induction of an ageing cardiomyocyte phenotypic change by incubation of cells with hydroxyurea for several days ex vivo. Hydroxyurea incubation has been demonstrated to phenocopy age- and senescence-induced changes in neurons, but its utility for ageing studies with cardiac cells has not been examined. Incubation of neonatal rat ventricular myocytes with hydroxyurea for up to 7 days replicated specific aspects of cardiac ageing including reduced systolic calcium responses, increased alternans and a lesser ability of the cells to follow electrical pacing. Additional functional and structural changes were observed within the myocytes that pointed to ageing-like remodelling, including lipofuscin granule accumulation, reduced mitochondrial membrane potential, increased production of reactive oxygen species, and altered ultrastructure, such as mitochondria with disrupted cristae and disorganised myofibres. These data highlight the utility of alternative approaches for exploring cellular ageing whilst avoiding the costs and co-morbid factors that can affect longitudinal studies.

Oncogenic KRAS suppresses store-operated Ca2+ entry and ICRAC through ERK pathway-dependent remodelling of STIM expression in colorectal cancer cell lines.

The KRAS GTPase plays a fundamental role in transducing signals from plasma membrane growth factor receptors to downstream signalling pathways controlling cell proliferation, survival and migration. Activating KRAS mutations are found in 20% of all cancers and in up to 40% of colorectal cancers, where they contribute to dysregulation of cell processes underlying oncogenic transformation. Multiple KRAS-regulated cell functions are also influenced by changes in intracellular Ca2+ levels that are concurrently modified by receptor signalling pathways. Suppression of intracellular Ca2+ release mechanisms can confer a survival advantage in cancer cells, and changes in Ca2+ entry across the plasma membrane modulate cell migration and proliferation. However, inconsistent remodelling of Ca2+ influx and its signalling role has been reported in studies of transformed cells. To isolate the interaction between altered Ca2+ handling and mutated KRAS in colorectal cancer, we have previously employed isogenic cell line pairs, differing by the presence of an oncogenic KRAS allele (encoding KRASG13D), and have shown that reduced Ca2+ release from the ER and mitochondrial Ca2+ uptake contributes to the survival advantage conferred by oncogenic KRAS. Here we show in the same cell lines, that Store-Operated Ca2+ Entry (SOCE) and its underlying current, ICRAC are under the influence of KRASG13D. Specifically, deletion of the oncogenic KRAS allele resulted in enhanced STIM1 expression and greater Ca2+ influx. Consistent with the role of KRAS in the activation of the ERK pathway, MEK inhibition in cells with KRASG13D resulted in increased STIM1 expression. Further, ectopic expression of STIM1 in HCT 116 cells (which express KRASG13D) rescued SOCE, demonstrating a fundamental role of STIM1 in suppression of Ca2+ entry downstream of KRASG13D. These results add to the understanding of how ERK controls cancer cell physiology and highlight STIM1 as an important biomarker in cancerogenesis.

Deleterious effects of calcium indicators within cells; an inconvenient truth.

The study of cellular Ca2+ signalling is indebted to Roger Tsien for the invention of fluorescent indicators that can be readily loaded into living cells and provide the means to measure cellular Ca2+ changes over long periods of time with sub-second resolution and microscopic precision. However, a recent study [1] reminds us that as useful as these tools are they need to be employed with caution as there can be off-target effects. This article summarises these recent findings within the wider context of confounding issues that can be encountered when using chemical and genetically-encoded Ca2+ indicators, and briefly discusses some approaches that may mitigate against misleading outcomes.

Contractile responses to endothelin-1 are regulated by PKC phosphorylation of cardiac myosin binding protein-C in rat ventricular myocytes.

The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca2+]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ETA- or ETB-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initial transient negative and sustained positive inotropy) and increases the peak amplitude of systolic Ca2+ transients in adult rat ventricular myocytes. The negative inotropic phase was ETB receptor-dependent, whereas the positive inotropic response and increase in peak amplitude of systolic Ca2+ transients required ETA receptor engagement. Both effects of ET-1 required phospholipase C (PLC)-activity, although distinct signalling pathways downstream of PLC elicited the effects of each ET receptor. The negative inotropic response involved inositol 1,4,5-trisphosphate (InsP3) signalling and protein kinase C epsilon (PKCε). The positive inotropic action and the enhancement in Ca2+ transient amplitude induced by ET-1 were independent of InsP3 signalling, but suppressed by PKCε. Serine 302 in cardiac myosin binding protein-C was identified as a PKCε substrate that when phosphorylated contributed to the suppression of contraction and Ca2+ transients by PKCε following ET-1 stimulation. Thus, our data provide a new role and mechanism of action for InsP3 and PKCε in mediating the negative inotropic response and in restraining the positive inotropy and enhancement in Ca2+ transients following ET-1 stimulation.

The regulation of autophagy by calcium signals: Do we have a consensus?

Macroautophagy (hereafter called 'autophagy') is a cellular process for degrading and recycling cellular constituents, and for maintenance of cell function. Autophagy initiates via vesicular engulfment of cellular materials and culminates in their degradation via lysosomal hydrolases, with the whole process often being termed 'autophagic flux'. Autophagy is a multi-step pathway requiring the interplay of numerous scaffolding and signalling molecules. In particular, orthologs of the family of ∼30 autophagy-regulating (Atg) proteins that were first characterised in yeast play essential roles in the initiation and processing of autophagic vesicles in mammalian cells. The serine/threonine kinase mTOR (mechanistic target of rapamycin) is a master regulator of the canonical autophagic response of cells to nutrient starvation. In addition, AMP-activated protein kinase (AMPK), which is a key sensor of cellular energy status, can trigger autophagy by inhibiting mTOR, or by phosphorylating other downstream targets. Calcium (Ca2+) has been implicated in autophagic signalling pathways encompassing both mTOR and AMPK, as well as in autophagy seemingly not involving these kinases. Numerous studies have shown that cytosolic Ca2+ signals can trigger autophagy. Moreover, introduction of an exogenous chelator to prevent cytosolic Ca2+ signals inhibits autophagy in response to many different stimuli, with suggestions that buffering Ca2+ affects not only the triggering of autophagy, but also proximal and distal steps during autophagic flux. Observations such as these indicate that Ca2+ plays an essential role as a pro-autophagic signal. However, cellular Ca2+ signals can exert anti-autophagic actions too. For example, Ca2+ channel blockers induce autophagy due to the loss of autophagy-suppressing Ca2+ signals. In addition, the sequestration of Ca2+ by mitochondria during physiological signalling appears necessary to maintain cellular bio-energetics, thereby suppressing AMPK-dependent autophagy. This article attempts to provide an integrated overview of the evidence for the proposed roles of various Ca2+ signals, Ca2+ channels and Ca2+ sources in controlling autophagic flux.

Calcium phosphate particles stimulate interleukin-1ß release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release.

AIMS: Calcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1ß (IL-1ß). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs. METHODS AND RESULTS: Using ELISA to measure IL-1ß release from VSMCs, we demonstrated that CaP particles stimulated IL-1ß release from proliferating and senescent human VSMCs, but with substantially greater IL-1ß release from senescent cells; this required caspase-1 activity but not LPS-priming of cells. Potential inflammasome agonists including ATP, nigericin and monosodium urate crystals did not stimulate IL-1ß release from VSMCs. Western blot analysis demonstrated that CaP particles induced rapid activation of spleen tyrosine kinase (SYK) (increased phospho-Y525/526). The SYK inhibitor R406 reduced IL-1ß release and caspase-1 activation in CaP particle-treated VSMCs, indicating that SYK activation occurs upstream of and is required for caspase-1 activation. In addition, IL-1ß and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1ß in exosomes isolated from VSMC media. Furthermore, CaP particle treatment stimulated exosome secretion by VSMCs in a SYK-dependent manner, while the exosome-release inhibitor spiroepoxide reduced IL-1ß release. CONCLUSIONS: CaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1ß, at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro-calcific potential of microcalcification.

Tissue Specificity: Store-Operated Ca2+ Entry in Cardiac Myocytes.

Calcium (Ca2+) is a key regulator of cardiomyocyte contraction. The Ca2+ channels, pumps, and exchangers responsible for the cyclical cytosolic Ca2+ signals that underlie contraction are well known. In addition to those Ca2+ signaling components responsible for contraction, it has been proposed that cardiomyocytes express channels that promote the influx of Ca2+ from the extracellular milieu to the cytosol in response to depletion of intracellular Ca2+ stores. With non-excitable cells, this store-operated Ca2+ entry (SOCE) is usually easily demonstrated and is essential for prolonging cellular Ca2+ signaling and for refilling depleted Ca2+ stores. The role of SOCE in cardiomyocytes, however, is rather more elusive. While there is published evidence for increased Ca2+ influx into cardiomyocytes following Ca2+ store depletion, it has not been universally observed. Moreover, SOCE appears to be prominent in embryonic cardiomyocytes but declines with postnatal development. In contrast, there is overwhelming evidence that the molecular components of SOCE (e.g., STIM, Orai, and TRPC proteins) are expressed in cardiomyocytes from embryo to adult. Moreover, these proteins have been shown to contribute to disease conditions such as pathological hypertrophy, and reducing their expression can attenuate hypertrophic growth. It is plausible that SOCE might underlie Ca2+ influx into cardiomyocytes and may have important signaling functions perhaps by activating local Ca2+-sensitive processes. However, the STIM, Orai, and TRPC proteins appear to cooperate with multiple protein partners in signaling complexes. It is therefore possible that some of their signaling activities are not mediated by Ca2+ influx signals, but by protein-protein interactions.