Cytoplasmic Calcium Research Articles

Diabetes mellitus exacerbates post-myocardial infarction heart failure by reducing sarcolipin promoter methylation.

AimsSarcolipin (SLN) is a key regulator of sarcoplasmic reticulum calcium‐ATPase (SERCA)2a, which handles intracellular calcium re‐uptake. This study was aimed to investigate the involvement of SLN in post‐myocardial infarction (MI) heart failure (HF) in diabetes.Methods and resultsDiabetes/MI rat models were established. Altered SLN expression in diabetic hearts was screened out by microarray. A myocardiotropic viral vector was used to deliver siRNA to silence SLN. DNA methylation was evaluated by bisulfite sequencing. Cardiac functions were evaluated by invasive haemodynamic examinations. The SERCA2a activity, cytoplasmic calcium concentration ([Ca2+]i), calcium spark, and myocyte contraction were detected. Correlation between HF and diabetes was analysed in a cohort consisted of 101 ST‐segment elevated myocardial infarction (STEMI) patients between 2017 and 2019 [53.54 ± 4.64 years old; 61.4% male gender; HbA1c% 6.15 ± 2.00; and left ventricular ejection fraction (LVEF%) 40.64 ± 3.20%]. SLN expression was evaluated in left ventricular tissue sample from six STEMI patients complicated with diabetes and six STEMI patients without diabetes. Expressions of DNA methyltransferase 1a and DNA methyltransferase 3 were reduced in diabetic hearts, leading to down‐regulation of SLN promoter methylation, resulting in increased SLN expression in rats. Impaired heart systolic functions were found in experimental diabetic MI rats, which were attenuated by SLN silencing. SERCA2a activity reduction and [Ca2+]i elevation were attenuated by SLN silencing in diabetic animal hearts and high‐glucose incubated primary myocytes. SLN silencing suppressed calcium sparks and improved contraction and sarcoplasmic reticulum calcium re‐uptake in high‐glucose incubated primary myocytes. Expression of SLN was up‐regulated in LV sampled from STEMI patients complicated with diabetes compared with non‐diabetic ones (P < 0.05). LVEF% was reduced in STEMI patients complicated with diabetes compared with non‐diabetic ones (P < 0.01). HbA1c% and LVEF% was related (r = −0.218, P = 0.028). Increased HbA1c% was correlated with reduced LVEF% after adjustment for age, sex, body mass index, cigarette smoking, creatinine, UA, low density lipoprotein, K+, Na+, and troponin I (adjusted odds ration = 0.75, 95% confidence interval 0.62–0.90, P = 0.002).ConclusionsDiabetes increases the vulnerability of STEMI patients to post‐MI HF by down‐regulating SLN promoter methylation, which further regulates SERCA2a activity via increasing cardiac SLN expression.

224-LB: Role of the Mitochondrial Calcium Uniporter in the Regulation of Hepatic Mitochondrial Metabolism and Gluconeogenesis

The mitochondrial calcium uniporter (MCU) is a key regulator of mitochondrial calcium, however, the role of MCU in mediating hepatic mitochondrial metabolism and gluconeogenesis has received limited attention. In order to address these questions, we examined rates of mitochondrial oxidation in both liver slices and in awake liver-specific MCU knockout (MCU KO) mice. We found that deletion of MCU significantly increases glucagon-induced intrahepatic lipolysis (p&amp;lt;0.04) and glucose production (p&amp;lt;0.04) in ex vivo liver slices. We also observed significantly reduced liver triglyceride accumulation (p&amp;lt;0.05) and increased hepatic long chain acyl-CoA (p&amp;lt;0.02) and acetyl-CoA (p=0.05) content in MCU KO mice, consistent with increased intrahepatic lipolysis and fat oxidation. This effect was not due to changes in white adipose tissue lipolysis. Deletion of MCU also impaired glucose tolerance in chow-fed mice, indicating dysregulated hepatic glucose production in these mice. These metabolic alterations could be attributed to reduced mitochondrial buffering of cytoplasmic calcium, leading to a 77% increase in cytoplasmic calcium (p&amp;lt;0.0001). This led to activation of CAMKII (p&amp;lt;0.04) and subsequent increase in ATGL phosphorylation (p&amp;lt;0.04) in hepatocytes lacking MCU. In conclusion, these results demonstrate a critical role for MCU in mediating hepatic mitochondrial oxidation and hepatic gluconeogenesis. Disclosure T.E. LaMoia: None. J. Lee: None. M. Guerra: None. R. Goodman: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc. Advisory Panel; Spouse/Partner; Merck &amp; Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. Funding National Institutes of Health (T32GM0007324)

2094-P: Effects of Irisin on Human Pancreatic Islets from Type 2 Diabetic Subjects

Irisin is a hormone secreted by skeletal muscle following physical activity and excess of saturated fatty acids, able to improve metabolic homeostasis and promote energy expenditure. In a previous study, we have demonstrated that, in beta-cells, irisin promotes proliferation and glucose-stimulated insulin secretion (GSIS), increases insulin content levels, and reduces lipotoxicity-induced apoptosis. The aim of this study was to evaluate the effects of irisin on the function and survival of pancreatic islets isolated from patients with type 2 diabetes mellitus (T2DM), characterized by reduced ability to secrete insulin and high levels of apoptosis, and to investigate the molecular mechanisms underlying this action. Pancreatic islets isolated from T2DM patients (n=7) and nondiabetic subjects (ND) (n=3), as well as INS-1E cells, were exposed to 100 nM irisin for different times. GSIS, insulin content, apoptosis and cytoplasmic calcium levels were measured by ELISA assays; the activation of the main mediators of beta-cell intracellular signalling (AKT, CREB and PKA) was evaluated by immunoblotting. Irisin increased GSIS approximately 2-fold (p&amp;lt;0.05) in both ND and T2DM islets. Notably, in T2DM islets, characterized by reduced GSIS, this was restored to the levels of ND islets. In addition, irisin increased insulin content (1.6-fold in ND, 1.9-fold in T2DM, p&amp;lt;0.05) and tendentially decreased apoptosis levels only in T2DM islets. Moreover, irisin was able to activate AKT and CREB under conditions of high glucose in the ND islets, without changing cytoplasmic calcium levels. In contrast, in the T2DM islets, irisin did not induce AKT or CREB activation. In conclusion, the myokine irisin is able to increase the secretory function and survival of human pancreatic islets and to improve the functional defects typical of the T2DM islets. The mechanisms underlying the secretagogue effects of irisin in the T2DM islets do not apparently involve the known signaling machinery of this novel hormone. Disclosure N. Marrano: None. A. Natalicchio: Consultant; Self; AstraZeneca, Novo Nordisk Inc., Sanofi-Aventis. G. Biondi: None. A. Cignarelli: None. L. Vincenti: None. L.G. Lupo: None. S. Perrini: None. L. Laviola: Advisory Panel; Self; Abbott, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi-Aventis. Speaker’s Bureau; Self; Abbott, Medtronic. F. Giorgino: Advisory Panel; Self; AstraZeneca, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi. Consultant; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., LifeScan, Inc., MedImmune, Merck Sharp &amp; Dohme Corp., Roche Diabetes Care, Sanofi. Research Support; Self; Eli Lilly and Company, LifeScan, Inc., Takeda Development Centre Europe Ltd. Funding European Union (AIM1810057)

Role of AMP-activated protein kinase during postovulatory aging of mouse oocytes†

Studies suggested that postovulatory oocyte aging might be prevented by maintaining a high maturation-promoting factor (MPF) activity. Whether AMP-activated protein kinase (AMPK) plays any role in postovulatory oocyte aging is unknown. Furthermore, while activation of AMPK stimulates meiotic resumption in mouse oocytes, it inhibits meiotic resumption in pig and bovine oocytes. Thus, the species difference in AMPK regulation of oocyte MPF activities is worth in-depth studies. This study showed that AMPK activation with metformin or 5-aminoimidazole- 4-carboxamide- 1-beta-d- ribofuranoside and inactivation with compound C significantly increased and decreased, respectively, the activation susceptibility (AS) and other aging parameters in aging mouse oocytes. While AMPK activity increased, MPF activity and cyclic adenosine monophosphate (cAMP) decreased significantly with time post ovulation. In vitro activation and inactivation of AMPK significantly decreased and increased the MPF activity, respectively. MPF upregulation with MG132 or downregulation with roscovitine completely abolished the effects of AMPK activation or inactivation on AS of aging oocytes, respectively. AMPK facilitated oocyte aging with increased reactive oxygen species (ROS) and cytoplasmic calcium. Furthermore, treatment with Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitors significantly decreased AS and AMPK activation. Taken together, the results suggested that AMPK facilitated oocyte aging through inhibiting MPF activities, and postovulatory oocyte aging activated AMPK with decreased cAMP by activating CaMKs via increasing ROS and cytoplasmic calcium.

Metoprolol alleviates arginine vasopressin-induced cardiomyocyte hypertrophy by upregulating the AKT1\u2013SERCA2 cascade in H9C2 cells

BackgroundArginine vasopressin (AVP) is elevated in patients with heart failure, and the increase in the AVP concentration in plasma is positively correlated with disease severity and mortality. Metoprolol (Met) is a beta blocker that is widely used in the clinic to treat pathological cardiac hypertrophy and to improve heart function. However, the specific mechanism by which Met alleviates AVP-induced pathological cardiac hypertrophy is still unknown. Our current study aimed to evaluate the inhibitory effects of Met on AVP-induced cardiomyocyte hypertrophy and the underlying mechanisms.MethodsAVP alone or AVP plus Met was added to the wild type or AKT1-overexpressing rat cardiac H9C2 cell line. The cell surface areas and ANP/BNP/β-MHC expressions were used to evaluate the levels of hypertrophy. Western bolting was used to analyze AKT1/P-AKT1, AKT2/P-AKT2, total AKT, SERCA2, and Phospholamban (PLN) expression. Fluo3-AM was used to measure the intracellular Ca2+ stores.ResultsIn the current study, we found that AKT1 but not AKT2 mediated the pathogenesis of AVP-induced cardiomyocyte hypertrophy. Sustained stimulation (48 h) with AVP led to hypertrophy in the H9C2 rat cardiomyocytes, resulting in the downregulation of AKT1 (0.48 fold compared to control) and SERCA2 (0.62 fold), the upregulation of PLN (1.32 fold), and the increase in the cytoplasmic calcium concentration (1.52 fold). In addition, AKT1 overexpression increased the expression of SERCA2 (1.34 fold) and decreased the expression of PLN (0.48 fold) in the H9C2 cells. Moreover, we found that Met could attenuate the AVP-induced changes in AKT1, SERCA2 and PLN expression and decreased the cytoplasmic calcium concentration in the H9C2 cells.ConclusionsOur results demonstrated that the AKT1–SERCA2 cascade served as an important regulatory pathway in AVP-induced pathological cardiac hypertrophy.

SIX2 Regulates Human β Cell Differentiation from Stem Cells and Functional Maturation In Vitro

SUMMARYGeneration of insulin-secreting β cells in vitro is a promising approach for diabetes cell therapy. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are differentiated to β cells (SC-β cells) and mature to undergo glucose-stimulated insulin secretion, but molecular regulation of this defining β cell phenotype is unknown. Here, we show that maturation of SC-β cells is regulated by the transcription factor SIX2. Knockdown (KD) or knockout (KO) of SIX2 in SC-β cells drastically limits glucose-stimulated insulin secretion in both static and dynamic assays, along with the upstream processes of cytoplasmic calcium flux and mitochondrial respiration. Furthermore, SIX2 regulates the expression of genes associated with these key β cell processes, and its expression is restricted to endocrine cells. Our results demonstrate that expression of SIX2 influences the generation of human SC-β cells in vitro.

Mitochondrial Dysfunction and Calcium Dysregulation in Leigh Syndrome Induced Pluripotent Stem Cell Derived Neurons.

Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characterized by neurodegeneration and astrogliosis in the basal ganglia or the brain stem. At present, there is no cure or treatment for this disease, partly due to scarcity of LS models. Current models generally fail to recapitulate important traits of the disease. Therefore, there is an urgent need to develop new human in vitro models. Establishment of induced pluripotent stem cells (iPSCs) followed by differentiation into neurons is a powerful tool to obtain an in vitro model for LS. Here, we describe the generation and characterization of iPSCs, neural stem cells (NSCs) and iPSC-derived neurons harboring the mtDNA mutation m.13513G>A in heteroplasmy. We have performed mitochondrial characterization, analysis of electrophysiological properties and calcium imaging of LS neurons. Here, we show a clearly compromised oxidative phosphorylation (OXPHOS) function in LS patient neurons. This is also the first report of electrophysiological studies performed on iPSC-derived neurons harboring an mtDNA mutation, which revealed that, in spite of having identical electrical properties, diseased neurons manifested mitochondrial dysfunction together with a diminished calcium buffering capacity. This could lead to an overload of cytoplasmic calcium concentration and the consequent cell death observed in patients. Importantly, our results highlight the importance of calcium homeostasis in LS pathology.

Autophagic Burden on Calcium Signaling in Sympathetic Activation

Signaling via calcium plays essential roles in all aspects of cell functions. Macroautophagy is a catabolic process that regulates important aspects of cell metabolism. While many studies have indicated that calcium is involved at various steps in the regulation of autophagic flux, the impact of autophagic flux on calcium signaling is unknown. We conceptually hypothesized that an “autophagic burden” exists in which enhanced requirement of the cell to process autophagic cargos would exert negative impact on calcium signaling. To test this hypothesis, we utilized an “non‐stimulation” approach to increase the “autophagic burden” by heterologously expressing in cells the wildtype microtubule‐associated proteins 1A/1B light chain 3B (LC3) or a mutant version in which the C‐terminal glycine responsible for ATG4‐mediated cleavage for LC3 lipidation has been removed (LC3DG) and examined the impact on adrenergic receptor‐mediated calcium signaling. HEK293 cells produced oscillatory intracellular calcium signals in response to agonists of b adrenoceptors (isoproterenol), α1A adrenoceptor (A61603), or a combination thereof (10 mM norepinephrine, (NE)). Cells expressing a fusion between the RFP mKate2 and wildtype LC3 (mKate2‐LC3) responded to 10 mM NE with substantial reductions in the number of intracellular calcium oscillations in the physiological ranges of 300 – 600 nM, 600 – 900 nM, or 900 – 1200 nM, as compared to the non‐expressing cells examined in the same microscopic fields, recognized by absence of mKate2 fluorescence. In contrast, cells expressing the mKate2‐LC3DG produced the same number of oscillatory calcium signals compared to non‐expressing cells in the same microscopic fields across the same ranges of cytoplasmic calcium stimulated by NE. The data suggest that an “autophagic burden” exists that may dampen the effects of acute sympathetic stimulation significantly via reduction in adrenoceptor‐mediated calcium signals.Support or Funding InformationIOER Grant 091707 to Q‐KT

Empagliflozin Reduces Arrhythmic Events and Improves Ca2+ Transient in Hypoxia‐induced Injury Rat Cardiomyocytes

Sodium glucose cotransporter 2 inhibitors (SGLT2i) emerged as promising antidiabetic drugs that decreasing glucose reabsorption as well as effectively reduce heart failure hospitalization and cardiovascular death in type 2 diabetes patients. Recently, it has been shown that the SGLT2i also exert beneficial cardiovascular effects in non‐diabetic heart failure patients. However, the cardioprotective mechanisms of these drugs remain unknow. The present study aimed to test the hypothesis that the cardioprotective effects of SGLT2i empagliflozin (EMPA) may be associated with changes of cardiac electrical activity and Ca2+ homeostasis in hypoxia‐induced injury ventricular myocytes. Cardiomyocytes isolated from neonatal rats submitted to chemical hypoxia (200 μM CoCl2) or normoxia were incubated with EMPA (1 μM) or vehicle during 24 hours. Electrophysiological recordings were obtained using multielectrode array (MEA) system and intracellular microelectrode. Cytoplasmic calcium transients were investigated via fura‐2 fluorescence. Compared with vehicle, MEA analysis showed a significant decrease in field potential duration in EMPA‐group in both conditions [hypoxia: 83 ± 4 (N=19) vs. 37 ± 3 (N=44); normoxia: 213 ± 5 (N=53) vs. 62 ± 4 (N=36) ms, p&lt;0.001]. EMPA reduced action potential duration at 30%, 50% and 90% repolarization (APD30, APD50 and APD90, respectively) in hypoxia [APD30: 109 ± 2 (N=41) vs. 98 ± 1 (N=30); APD50: 150 ± 3 (N=41) vs. 134 ± 1 (N=30); APD90: 292 ± 4 (N=41) vs. 271 ± 5 (N=30) ms, p&lt;0.05] and normoxia [APD30: 94 ± 2 (N=30) vs. 81 ± 1 (N=39); APD50: 129 ± 2 (N=30) vs. 111 ± 1 (N=39); APD90: 259 ± 4 (N=30) vs. 244 ± 4 (N=39) ms, p&lt;0.05]. No differences were observed in amplitude and resting membrane potential. Action potential recordings also revealed that the percentage of arrhythmic cells was lower in EMPA‐group in both conditions [hypoxia: 19 (N=41) vs. 10 (N=30) %; normoxia: 7 (N=30) vs. 3 (N=39) %]. In addition, EMPA significantly increased in Ca2+ transient amplitude in hypoxia [F/F0: 0.16 ± 0.01 (N=21) vs. 0.21 ± 0.01 (N=19), p&lt;0.01] and normoxia [F/F0: 0.17 ± 0.01 (N=19) vs. 0.23 ± 0.01 (N=20) μM, p&lt;0.01]. These results suggest that EMPA modulates electrical activity, reduces arrhythmic events and improves Ca2+ transient, which may contribute to cardioprotective effects of SGLT2i.Support or Funding InformationFAPESP and CNPq

Development of erianin-loaded dendritic mesoporous silica nanospheres with pro-apoptotic effects and enhanced topical delivery

BackgroundPsoriasis is a malignant skin disease characterized as keratinocyte hyperproliferation and aberrant differentiation. Our previous work reported that a bibenzyl compound, erianin, has a potent inhibitory effect on keratinocyte proliferation. To improve its poor water-solubility, increase anti- proliferation activity, and enhance the skin delivery, erianin loaded dendritic mesoporous silica nanospheres (E/DMSNs) were employed.ResultsIn this work, DMSNs with pore size of 3.5 nm (DMSN1) and 4.6 nm (DMSN2) were fabricated and E/DMSNs showed pore-size-dependent, significantly stronger anti-proliferative and pro-apoptotic effect than free erianin on human immortalized keratinocyte (HaCaT) cells, resulting from higher cellular uptake efficiency. In addition, compared to free erianin, treatment with E/DMSNs was more effective in reducing mitochondrial membrane potential and increasing cytoplasmic calcium levels, which were accompanied by regulation of mitochondria and endoplasmic reticulum stress (ERS) pathway. Porcine skin was utilized in the ex vivo accumulation and permeation studies, and the results indicated higher drug retention and less drug penetration in the skin when administered as the E/DMSNs-loaded hydrogel compared to the erianin-loaded hydrogel.ConlusionsThis work not only illustrated the further mechanisms of erianin in anti-proliferation of HaCaT cells but also offer a strategy to enhance the efficiency of erianin and the capacity of skin delivery through the DMSNs drug delivery systems.

Lanthanum chloride impairs spatial learning and memory by inducing [Ca2+]m overload, mitochondrial fission-fusion disorder and excessive mitophagy in hippocampal nerve cells of rats.

Lanthanum (La) is a kind of rare earth element (REE) widely found in nature. La has neurotoxicity and can impair learning and memory, but the underlying mechanism is still not completely clear. The mitochondrial calcium uniporter (MCU) complex can cause the uptake of cytoplasmic calcium ([Ca2+]c) into mitochondria and thereby resist [Ca2+]c overload. However, the abnormal increase of calcium in the mitochondrial matrix ([Ca2+]m) can also disturb the mitochondrial fission-fusion balance, and then induce excessive mitophagy, and disrupt mitochondrial quality control (MQC). It is unclear whether La can interfere with the function of nerve cells through the above-mentioned mechanism and thus impair learning and memory. In this study, four groups of Wistar rats were treated with 0%, 0.25%, 0.5% and 1.0% (w/v) lanthanum chloride (LaCl3) from the embryonic phase to 1 month after weaning. The results showed that La could impair the spatial learning and memory of rats, promote the uptake of [Ca2+]c by MCU, induce the abnormal increase of [Ca2+]m, up-regulate p-Drp1 Ser616 expression and inhibit Mfn1/2 expression, enhance mitochondrial fission and lead to mitochondrial fission-fusion disturbance in hippocampal nerve cells. Meanwhile, La could also activate the PINK1-Parkin signaling pathway, up-regulate LC3B-II expression and decrease p62 expression, and thereby induce excessive mitophagy. These results suggested that learning and memory impairment caused by La may be related to MQC disturbance. The present data provide some novel clues for elucidating the neurotoxic effect mechanism of La.

Chronic Methadone Use Alters the CD8+ T Cell Phenotype In Vivo and Modulates Its Responsiveness Ex Vivo to Opioid Receptor and TCR Stimuli.

Endogenous opioid peptides are released at sites of injury, and their cognate G protein-coupled opioid receptors (ORs) are expressed on immune cells. Although drugs of misuse appropriate ORs, conflicting reports indicate immunostimulatory and immunosuppressive activity, in that opioid users have elevated infection risk, opioids activate innate immune cells, and opioids attenuate inflammation in murine T cell-mediated autoimmunity models. The i.v. use of drugs transmits bloodborne pathogens, particularly viruses, making the study of CD8+ T cells timely. From a cohort of nonuser controls and methadone users, we demonstrate, via t-Stochastic Neighbor Embedding and k-means cluster analysis of surface marker expression, that chronic opioid use alters human CD8+ T cell subset balance, with notable decreases in T effector memory RA+ cells. Studying global CD8+ T cell populations, there were no differences in expression of OR and several markers of functionality, demonstrating the need for finer analysis. Purified CD8+ T cells from controls respond to opioids ex vivo by increasing cytoplasmic calcium, a novel finding for OR signal transduction, likely because of cell lineage. CD8+ T cells from controls exposed to μ-OR agonists ex vivo decrease expression of activation markers CD69 and CD25, although the same markers are elevated in μ-OR-treated cells from methadone users. In contrast to control cells, T cell subsets from methadone users show decreased expression of CD69 and CD25 in response to TCR stimulus. Overall, these results indicate a direct, selective role for opioids in CD8+ T cell immune regulation via their ability to modulate cell responses through the opioid receptors and TCRs.

Melatonin fine-tunes intracellular calcium signals and eliminates myocardial damage through the IP3R/MCU pathways in cardiorenal syndrome type 3

Cardiorenal syndrome type-3 (CRS-3) is characterized by acute cardiac injury induced by acute kidney injury. Here, we investigated the causes of CRS-3 by analyzing cardiac function after renal ischemia–reperfusion injury (IRI) using echocardiography and evaluation of pro-inflammatory markers, calcium balance, mitochondrial function, and cardiomyocyte death. Our results show that renal IRI reduces cardiac diastolic function associated with cardiomyocyte death and inflammatory responses. Renal IRI also disrupts cardiomyocyte energy metabolism, induces calcium overload, and impairs mitochondrial function, as evidenced by reduced mitochondrial membrane potential and increased mitochondrial fission. Further, renal IRI induces phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and expression of mitochondrial calcium uniporter (MCU), resulting in cytoplasmic calcium overload and mitochondrial calcium accumulation. Pretreatment with melatonin attenuates renal IRI-mediated cardiac damage by maintaining myocardial diastolic function and reducing cardiomyocyte death. Melatonin also inhibits IP3R phosphorylation and MCU expression, thereby alleviating cytoplasmic and mitochondrial calcium overload. Blockade of IP3R has similar cardioprotective effects, whereas MCU activation abrogates the melatonin-mediated cardioprotection. These results show that the negative effects of renal IRI on myocardial viability and cardiac function are caused by induced IP3R phosphorylation, MCU upregulation, and calcium overload. Melatonin protects cardiac function against CRS-3 by suppressing IP3R-MCU signaling.

Not Fade Away: Mechanisms of Neuronal ATP Homeostasis

In this issue of Neuron, Ashrafi etal. (2020) identify a feedforward signaling mechanism that couples neuronal activity to the homeostatic maintenance of axonal and synaptic ATP production. This mechanism is achieved via changes in cytoplasmic calcium and activation of brain-specific, mitochondrial MICU3.

Effects of Osmotic Challenges on Gap Junction Communication

Gap junction communication plays a critical role in multicellular organisms, allowing for the direct transfer of ions, metabolites, and signaling molecules between cytosolic compartments to synchronize cellular activities, direct development, and maintain tissue homeostasis. The number and activity of gap junction channels are sensitive to a variety of cytoplasmic factors, including pH, calcium, 2ndmessengers, kinases, and binding proteins. The concentration of these factors is dependent on cell volume, and subject to change dramatically during volume changes associated with exposure of cells to anisotonic conditions.

Biophysical Origins of Calcium-Inhibited Membrane Binding by the C2A Domain of Synaptotagmin-Like Protein 2

Although C2 domains canonically bind membranes in response to increased cytoplasmic calcium, there are also many examples of C2 domains known to bind membranes in the absence of calcium. Most of these are calcium-independent C2 domains that lack the conserved aspartate cluster, but the C2A domain of synapotagmin-like protein 2 (Slp-2; also called exophilin-4) has been reported to be inhibited by calcium. Here, we use a combination of fluorescence-based liposome binding and kinetic assays, mass spectrometry, and NMR spectroscopy to analyze the physical-chemical origins of this unusual property. Our data confirm that the purified recombinant C2A domain indeed binds anionic lipid membranes containing phosphatidylserine and/or phosphatidylinositol-(4,5)-bisphosphate in a manner that is inhibited by sub-millimolar concentrations of calcium. Surprisingly, however, the Slp-2 C2A domain does not show detectable binding to calcium ions in solution, despite having 3 of 5 conserved aspartate residues that form the canonical calcium binding site in C2 domains. Rather, its electrostatically driven membrane binding must be inhibited through effects of calcium on membrane lipids. Because calcium is more effective at inhibiting Slp-2 C2A membrane binding than other protein domains with comparable membrane affinities, we speculate that the inhibition may arise due to a combination of electrostatic screening and other effects, e.g. on membrane structure. Overall, this unusual C2 domain competes with calcium ions for binding to its anionic lipid targets.

Dashing Through the Maze: Active Mixing in the Endoplasmic Reticulum

The endoplasmic reticulum (ER) serves as the striking example of morphological complexity in cellular organelles. It comprises a series of stacked membranous sheets in the perinuclear region and an expansive network of tubules spanning the cell periphery, interconnected by a single continuous lumen. Its many functions include protein sorting and quality control, lipid distribution, and intracellular calcium modulation. Each of these functions relies on the ability to efficiently deliver molecular components to localized regions of the cell as well as redistributing these components throughout the ER lumen and membrane. While transport within the ER is generally assumed to be diffusive in nature, recent evidenceindicates that proteins in the the peripheral network also exhibit rapid processive movements indicative of an active transport mechanism. We use physical modeling to examine both the plausible underlying causes of this processive motion and the potential effect on ER function. Specifically, we focus on calcium ion distribution through the ER as an exemplar system for transport within an active network. We show that diffusion alone places a hard physical limit on the magnitude of cytoplasmic calcium sparks that can be generated by localized release from an ER tubule, and that processive motion of calcium buffer proteins can greatly enhance these sparks. Furthermore, we demonstrate that refilling the ER lumen after global calcium release would result in a substantial latency period between sequential signaling events unless the mixing of luminal contents in the periphery is enhanced, as expected from sporadic active transport. Our results highlight the importance of calcium buffer protein mobility for the efficiency of ion distribution through the ER lumen, as well as potentially crucial roles for actively driven processive motion within ER tubules.

Cytokinesis Triggers Two Separate Spikes of Intracellular Calcium

Cytokinesis is the final stage of cell division during which two daughter cells separate. In some animal embryos including those of sea urchin and zebrafish, cytoplasmic calcium concentration increases sharply (spike) at the cell division plane during cytokinesis. However, it remained unclear how evolutionally conserved this process is and whether the intracellular calcium is important to cytokinesis. Here we employed GCaMP6s, a genetically encoded calcium reporter, to measure intracellular calcium concentration during cytokinesis of the model organism Schizosacchromyces pombe. First, we validated this novel fission yeast calcium reporter by quantifying the calcium concentration of single cells with live fluorescence microscopy. As expected, although the calcium concentration remained constant in most of the unstimulated cells, external stimuli triggered frequent spikes of intracellular calcium concentration. In the presence of excessive extracellular calcium, the intracellular calcium concentration spiked frequently. Hypo-osmotic shocks, applied through microfluidics, triggered a transient but sharp increase in the frequency of calcium spikes. We then measured the intracellular calcium concentration throughout cell division and found cytokinesis triggered two calcium spikes. The first followed the initiation of the cleavage furrow ingression. The second spike accompanied the initiation of cell separation, exhibiting asymmetrically in two daughter cells. Both spikes are global, not restricted to the cell division plane. Lastly, we examined whether these calcium spikes are essential to cytokinesis. Inhibition of these spikes reduced the rate of ring closure and triggered frequent post-cytokinesis cell lysis. Our study demonstrated that cytokinesis triggered calcium spikes are evolutionally conserved. We propose that this transient increase of intracellular calcium concentration is essential for turgor regulation to support the furrow ingression and the cell separation.

Inhibition of inositol kinase B controls acute and chronic graft-versus-host disease

AbstractT-cell activation releases inositol 1,4,5-trisphosphate (IP3), inducing cytoplasmic calcium (Ca2+) influx. In turn, inositol 1,4,5-trisphosphate 3-kinase B (Itpkb) phosphorylates IP3 to negatively regulate and thereby tightly control Ca2+ fluxes that are essential for mature T-cell activation and differentiation and protection from cell death. Itpkb pathway inhibition increases intracellular Ca2+, induces apoptosis of activated T cells, and can control T-cell–mediated autoimmunity. In this study, we employed genetic and pharmacological approaches to inhibit Itpkb signaling as a means of controlling graft-versus-host disease (GVHD). Murine-induced, Itpkb-deleted (Itpkb−/−) T cells attenuated acute GVHD in 2 models without eliminating A20-luciferase B-cell lymphoma graft-versus-leukemia (GVL). A highly potent, selective inhibitor, GNF362, ameliorated acute GVHD without impairing GVL against 2 acute myeloid leukemia lines (MLL-AF9-eGFP and C1498-luciferase). Compared with FK506, GNF362 more selectively deleted donor alloreactive vs nominal antigen-responsive T cells. Consistent with these data and as compared with FK506, GNF362 had favorable acute GVHD and GVL properties against MLL-AF9-eGFP cells. In chronic GVHD preclinical models that have a pathophysiology distinct from acute GVHD, Itpkb−/− donor T cells reduced active chronic GVHD in a multiorgan system model of bronchiolitis obliterans (BO), driven by germinal center reactions and resulting in target organ fibrosis. GNF362 treatment reduced active chronic GVHD in both BO and scleroderma models. Thus, intact Itpkb signaling is essential to drive acute GVHD pathogenesis and sustain active chronic GVHD, pointing toward a novel clinical application to prevent acute or treat chronic GVHD.

The Mechanosensitive Ion Channel MSL10 Potentiates Responses to Cell Swelling in Arabidopsis Seedlings

The ability to respond to unanticipated increases in volume is a fundamental property of cells, essential for cellular integrity in the face of osmotic challenges. Plants must manage cell swelling during flooding, rehydration, and pathogenesis–but little is known about the mechanisms by which this occurs. It has been proposed that plant cells could sense and respond to cell swelling through the action of mechanosensitive ion channels. Here we develop and characterize a new assay to study the effects of cell swelling on Arabidopsis thaliana seedlings and to test the contributions of the mechanosensitive ion channel MscS-Like10 (MSL10). The assay incorporates both cell wall softening and hypo-osmotic treatment to induce cell swelling. We show that MSL10 is required for previously demonstrated responses to hypo-osmotic shock, including a cytoplasmic calcium transient within the first few seconds, accumulation of ROS within the first 30 minutes, and increased transcript levels of mechano-inducible genes within 60 minutes. We also show that cell swelling induces programmed cell death within 3 hours, also in a MSL10-dependent manner. Finally, we show that MSL10 is unable to potentiate cell swelling-induced death when phosphomimetic residues are introduced into its soluble N-terminus. Thus, MSL10 functions as a phospho-regulated membrane-based sensor that connects the perception of cell swelling to a downstream signaling cascade and programmed cell death.