Cytosolic Calcium Accumulation Research Articles

Blood Brain Barrier-Crossing Delivery of Felodipine Nanodrug Ameliorates Anxiety-Like Behavior and Cognitive Impairment in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder leading to cognitive decline. Excessive cytosolic calcium (Ca2+) accumulation plays a critical role in the pathogenesis of AD since it activates the NOD-like receptor family, pyrin domain containing 3 (NLRP3), switches the endoplasmic reticulum (ER) unfolded protein response (UPR) toward proapoptotic signaling and promotes Aβ seeding. Herein, a liposomal nanodrug (felodipine@LND) is developed incorporating a calcium channel antagonist felodipine for Alzheimer's disease treatment through a low-intensity pulse ultrasound (LIPUS) irradiation-assisted blood brain barrier (BBB)-crossing drug delivery. The multifunctional felodipine@LND is effectively delivered to diseased brain through applying a LIPUS irradiation to the skull, which resulted in a series of positive effects against AD. Markedly, the nanodrug treatment switched the ER UPR toward antioxidant signaling, prevented the surface translocation of ER calreticulin (CALR) in microglia, and inhibited the NLRP3 activation and Aβ seeding. In addition, it promoted the degradation of damaged mitochondria via mitophagy, thereby inhibiting the neuronal apoptosis. Therefore, the anxiety-like behavior and cognitive impairment of 5xFAD mice with AD is significantly ameliorated, which manifested the potential of LIPUS - assisted BBB-crossing delivery of felodipine@LND to serve as a paradigm for AD therapy based on the well-recognized clinically available felodipine.

SERCA2a overexpression improves muscle function in a canine Duchenne muscular dystrophy model

Excessive cytosolic calcium accumulation contributes to muscle degeneration in Duchenne muscular dystrophy (DMD). Sarco/endoplasmic reticulum calcium ATPase (SERCA) is a sarcoplasmic reticulum (SR) calcium pump that actively transports calcium from the cytosol into the SR. We previously showed that adeno-associated virus (AAV)-mediated SERCA2a therapy reduced cytosolic calcium overload and improved muscle and heart function in the murine DMD model. Here we tested whether AAV SERCA2a therapy could ameliorate muscle disease in the canine DMD model. 7.83 x 1013 viral genome particles of the AAV vector were injected into the extensor carpi ulnaris (ECU) muscles of four juvenile-affected dogs. Contralateral ECU muscles received excipient. Three months later, we observed widespread transgene expression and significantly increased SERCA2a levels in the AAV-injected muscles. Treatment improved SR calcium uptake, significantly reduced calpain activity, significantly improved contractile kinetics, and significantly enhanced resistance to eccentric contraction-induced force loss. Nonetheless, muscle histology was not improved. To evaluate the safety of AAV SERCA2a therapy, we delivered the vector to the ECU muscle of adult normal dogs. We achieved strong transgene expression without altering muscle histology and function. Our results suggest that AAV SERCA2a therapy has the potential to improve muscle performance in a dystrophic large mammal.

The GABA shunt contributes to ROS homeostasis in guard cells of Arabidopsis.

γ-Aminobutyric acid (GABA) accumulates rapidly under stress via the GABA shunt pathway, which has been implicated in reducing the accumulation of stress-induced reactive oxygen species (ROS) in plants. γ-Aminobutyric acid has been demonstrated to act as a guard-cell signal in Arabidopsis thaliana, modulating stomatal opening. Knockout of the major GABA synthesis enzyme Glutamate Decarboxylase 2 (GAD2) increases the aperture of gad2 mutants, which results in greater stomatal conductance and reduces water-use efficiency compared with wild-type plants. Here, we found that the additional loss of GAD1, GAD4, and GAD5 in gad2 leaves increased GABA deficiency but abolished the more open stomatal pore phenotype of gad2, which we link to increased cytosolic calcium (Ca2+ ) and ROS accumulation in gad1/2/4/5 guard cells. Compared with wild-type and gad2 plants, glutamate was ineffective in closing gad1/2/4/5 stomatal pores, whereas lowering apoplastic calcium, applying ROS inhibitors or complementation with GAD2 reduced gad1/2/4/5 guard-cell ROS, restored the gad2-like greater stomatal apertures of gad1/2/4/5 beyond that of wild-type. We conclude that GADs are important contributors to ROS homeostasis in guard cells likely via a Ca2+ -mediated pathway. As such, this study reveals greater complexity in GABA's role as a guard-cell signal and the interactions it has with other established signals.

Resveratrol Preconditioning Protects Against Ischemia-Induced Synaptic Dysfunction and Cofilin Hyperactivation in the Mouse Hippocampal Slice.

Perturbations in synaptic function are major determinants of several neurological diseases and have been associated with cognitive impairments after cerebral ischemia (CI). Although the mechanisms underlying CI-induced synaptic dysfunction have not been well defined, evidence suggests that early hyperactivation of the actin-binding protein, cofilin, plays a role. Given that synaptic impairments manifest shortly after CI, prophylactic strategies may offer a better approach to prevent/mitigate synaptic damage following an ischemic event. Our laboratory has previously demonstrated that resveratrol preconditioning (RPC) promotes cerebral ischemic tolerance, with many groups highlighting beneficial effects of resveratrol treatment on synaptic and cognitive function in other neurological conditions. Herein, we hypothesized that RPC would mitigate hippocampal synaptic dysfunction and pathological cofilin hyperactivation in an ex vivo model of ischemia. Various electrophysiological parameters and synaptic-related protein expression changes were measured under normal and ischemic conditions utilizing acute hippocampal slices derived from adult male mice treated with resveratrol (10mg/kg) or vehicle 48h prior. Remarkably, RPC significantly increased the latency to anoxic depolarization, decreased cytosolic calcium accumulation, prevented aberrant increases in synaptic transmission, and rescued deficits in long-term potentiation following ischemia. Additionally, RPC upregulated the expression of the activity-regulated cytoskeleton associated protein, Arc, which was partially required for RPC-mediated attenuation of cofilin hyperactivation. Taken together, these findings support a role for RPC in mitigating CI-induced excitotoxicity, synaptic dysfunction, and pathological over-activation of cofilin. Our study provides further insight into mechanisms underlying RPC-mediated neuroprotection against CI and implicates RPC as a promising strategy to preserve synaptic function after ischemia.

Biao and Ben Acupoints Combination Regulates Mitochondrial Function Through Purinergic P2X7 Receptor in Rats with Myocardial Ischemia

We aimed to investigate the possible mechanisms of electroacupuncture at the Biao and Ben acupoints in improving myocardial ischemia-induced mitochondrial dysfunction. The study examined mitochondrial structure, ATP production, cytosolic calcium accumulation, P2X7 receptor, and mitochondrial-associated oxidative stress factors in rats with a myocardial ischemia model. Eighty-four male and female SD rats were randomly divided into seven groups: Control, Sham, Model, High-dose/Low-dose ZnCl2, electroacupuncture at the Neiguan point, and the Combination of Biao and Ben acupoints. The electroacupuncture groups, including the Neiguan point and Biao and Ben acupoints group, were treated with acupuncture for 10 minutes per day for 21 days. The combination of Biao and Ben acupoints decreased the damage to the mitochondrial structure caused by myocardial ischemia, restored ATP production, and inhibited Ca2+ accumulation. Furthermore, electroacupuncture at Biao and Ben inhibited calcium accumulation and restored P2X7 receptor expression levels. The protective effect of the combination of Biao and Ben acupoints on mitochondrial function is that they restore P2X7 receptor expression, which regulates mitochondrial metabolism.

The Ycx1 protein encoded by the yeast YDL206W gene plays a role in calcium and calcineurin signaling

Calcium is ubiquitously present in all living cells and plays important regulatory roles in a wide variety of biological processes. In yeast, many effects of calcium are mediated via the action of calcineurin, a calcium/calmodulin-dependent protein phosphatase. Proper signaling of calcium and calcineurin is important in yeast, and the calcineurin pathway has emerged as a valuable target for developing novel antifungal drugs. Here, we report a role of YDL206W in calcium and calcineurin signaling in yeast. YDL206W is an uncharacterized gene in yeast, encoding a protein with two sodium/calcium exchange domains. Disrupting the YDL206W gene leads to a diminished level of calcium-induced activation of calcineurin and a reduced accumulation of cytosolic calcium. Consistent with a role of calcineurin in regulating pheromone and cell wall integrity signaling, the ydl206wΔ mutants display an enhanced growth arrest induced by pheromone treatment and poor growth at elevated temperature. Subcellular localization studies indicate that YDL206W is localized in endoplasmic reticulum and Golgi. Together, our results reveal YDL206W as a new regulator for calcineurin signaling in yeast and suggest a role of the endoplasmic reticulum and Golgi in regulating cytosolic calcium in yeast.

Uptake‐independent killing of macrophages by extracellular Mycobacterium tuberculosis aggregates

Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular Mtb replication induces the death of the infected macrophages and the release of bacterial aggregates. Here, we show that these aggregates can evade phagocytosis by killing macrophages in a contact‐dependent but uptake‐independent manner. We use time‐lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytosolic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb ESX‐1 secretion system, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface‐exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that blocking ESX‐1‐mediated secretion of the EsxA/EsxB virulence factors does not eliminate the uptake‐independent killing of macrophages and that the 50‐kDa isoform of the ESX‐1‐secreted protein EspB can mediate killing in the absence of EsxA/EsxB secretion. Treatment with an ESX‐1 inhibitor reduces uptake‐independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti‐phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung.

Lipid ROS- and Iron-Dependent Ferroptotic Cell Death in Unicellular Algae Chlamydomonas reinhardtii.

The phenomenon of heat stress leading to ferroptosis-like cell death has recently been observed in bacteria as well as plant cells. Despite recent findings, the evidence of ferroptosis, an iron-dependent cell death remains unknown in microalgae. The present study aimed to investigate if heat shock could induce reactive oxygen species (ROS) and iron-dependent ferroptotic cell death in Chlamydomonas reinhardtii in comparison with RSL3-induced ferroptosis. After RSL3 and heat shock (50 °C) treatments with or without inhibitors, Chlamydomonas cells were evaluated for cell viability and the induction of ferroptotic biomarkers. Both the heat shock and RSL3 treatment were found to trigger ferroptotic cell death, with hallmarks of glutathione-ascorbic acid depletion, GPX5 downregulation, mitochondrial dysfunction, an increase in cytosolic calcium, ROS production, lipid peroxidation, and intracellular iron accumulation via heme oxygenase-1 activation (HO-1). Interestingly, the cells preincubated with ferroptosis inhibitors (ferrostatin-1 and ciclopirox) significantly reduced RSL3- and heat-induced cell death by preventing the accumulation of Fe2+ and lipid ROS. These findings reveal that ferroptotic cell death affects the iron homeostasis and lipid peroxidation metabolism of Chlamydomonas, indicating that cell death pathways are evolutionarily conserved among eukaryotes.

Calcineurin-mediated intracellular organelle calcium homeostasis is required for the survival of fungal pathogens upon extracellular calcium stimuli

ABSTRACT In eukaryotes, calcium not only is an essential mineral nutrient but also serves as an intracellular second messenger that is necessary for many physiological processes. Previous studies showed that the protein phosphatase-calcineurin protects fungi from toxicity caused by the extracellular calcium; however, little is known about how calcineurin mediates the cellular physiology process for this function. In this study, by monitoring intracellular calcium, particularly by tracking vacuolar calcium dynamics in living cells through a novel procedure using modified aequorin, we found that calcineurin dysfunction systematically caused abnormal intracellular calcium homeostasis in cytosol, mitochondria, and vacuole, leading to drastic autophagy, global organelle fragmentation accompanied with the increased expression of cell death-related enzymes, and cell death upon extracellular calcium stimuli. Notably, all detectable defective phenotypes seen with calcineurin mutants can be significantly suppressed by alleviating a cytosolic calcium overload or increasing vacuolar calcium storage capacity, suggesting toxicity of exogenous calcium to calcineurin mutants is tightly associated with abnormal cytosolic calcium accumulation and vacuolar calcium storage capacity deficiency. Our findings provide insights into how the original recognized antifungal drug target-calcineurin regulates intracellular calcium homeostasis for cell survival and may have important implications for antifungal therapy and clinical drug administration.

Assessing the Efficacy of Dietary Selenomethionine Supplementation in the Setting of Cardiac Ischemia/Reperfusion Injury.

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury. This partly arises from the presence of activated phagocytes releasing myeloperoxidase (MPO) and its production of hypochlorous acid (HOCl). The dietary supplement selenomethionine (SeMet) has been shown to bolster endogenous antioxidant processes as well as readily react with MPO-derived oxidants. The aim of this study was to assess whether supplementation with SeMet could modulate the extent of cellular damage observed in an in vitro cardiac myocyte model exposed to (patho)-physiological levels of HOCl and an in vivo rat model of cardiac I/R injury. Exposure of the H9c2 cardiac myoblast cell line to HOCl resulted in a dose-dependent increase in necrotic cell death, which could be prevented by SeMet supplementation and was attributed to SeMet preventing the HOCl-induced loss of mitochondrial inner trans-membrane potential, and the associated cytosolic calcium accumulation. This protection was credited primarily to the direct oxidant scavenging ability of SeMet, with a minor contribution arising from the ability of SeMet to bolster cardiac myoblast glutathione peroxidase (GPx) activity. In vivo, a significant increase in selenium levels in the plasma and heart tissue were seen in male Wistar rats fed a diet supplemented with 2 mg kg−1 SeMet compared to controls. However, SeMet-supplementation demonstrated only limited improvement in heart function and did not result in better heart remodelling following I/R injury. These data indicate that SeMet supplementation is of potential benefit within pathological settings where excessive HOCl is known to be generated but has limited efficacy as a therapeutic agent for the treatment of heart attack.

Induction of an MLKL mediated non-canonical necroptosis through reactive oxygen species by tanshinol A in lung cancer cells

Recent discoveries revealed several types of programmed necrosis, such as necroptosis, ferroptosis, pyroptosis, etc. Necroptosis is mediated by signaling complexes with receptor-interacting protein kinases (RIPs) and mixed lineage kinase domain-like protein (MLKL). Here, we described an MLKL mediated non-canonical necroptosis through reactive oxygen species (ROS) in lung cancer cells triggered by a natural compound, tanshinol A (TSA). Morphologically, TSA-induced necrotic cell death is characterized by increased cell volume, transparent of cytoplasm, and rupture of the cell membrane. Biochemically, it induces intracellular ATP depletion and PI penetration. Molecularly, TSA-induced cell death is mediated by MLKL but independent of RIP1 and RIP3. Furthermore, TSA induces MLKL phosphorylation and membrane translocation, and cytosolic calcium accumulation. However, calcium shows no effect on TSA-induced cell death. Especially, TSA induces intracellular ROS generation, which was found to be the upstream of MLKL. Collectively, our data indicated that TSA triggers a novel type of programmed necrosis mediated by MLKL in lung cancer cells, which might have therapeutic potentials for lung cancer treatment.

Correction to Butylated Hydroxyanisole Exerts Neurotoxic Effects by Promoting Cytosolic Calcium Accumulation and Endoplasmic Reticulum Stress in Astrocytes

Correction to Butylated Hydroxyanisole Exerts Neurotoxic Effects by Promoting Cytosolic Calcium Accumulation and Endoplasmic Reticulum Stress in Astrocytes

Butylated Hydroxyanisole Exerts Neurotoxic Effects by Promoting Cytosolic Calcium Accumulation and Endoplasmic Reticulum Stress in Astrocytes.

Astrocytes provide nutritional support, regulate inflammation, and perform synaptic functions in the human brain. Although butylated hydroxyanisole (BHA) is a well-known antioxidant, several studies in animals have indicated BHA-mediated liver toxicity, retardation in reproductive organ development and learning, and sleep deficit. However, the specific effects of BHA on human astrocytes and the underlying mechanisms are yet unclear. Here, we investigated the antigrowth effects of BHA through cell cycle arrest and downregulation of regulatory protein expression. The typical cell proliferative signaling pathways, phosphoinositide 3-kinase/protein kinase B and extracellular signal-regulated kinase 1/2, were downregulated in astrocytes after BHA treatment. BHA increased the levels of pro-apoptotic proteins, such as BAX, cytochrome c, cleaved caspase 3, and cleaved caspase 9, and decreased the level of anti-apoptotic protein BCL-XL. It also increased the cytosolic calcium level and the expression of endoplasmic reticulum stress proteins. Treatment with BAPTA-AM, a calcium chelator, attenuated the increased levels of ER stress proteins and cleaved members of the caspase family. We further performed an in vivo evaluation of the neurotoxic effect of BHA on zebrafish embryos and glial fibrillary acidic protein, a representative astrocyte biomarker, in a gfap:eGFP zebrafish transgenic model. Our results provide clear evidence of the potent cytotoxic effects of BHA on human astrocytes, which lead to disruption of the brain and nerve development.

Characterization of the cellular effects of myeloperoxidase-derived oxidants on H9c2 cardiac myoblasts

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury, which is in part due to the release of the enzyme myeloperoxidase (MPO) from activated infiltrating leukocytes, and the subsequent production of the oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Although exposure of various cell types to either oxidant is known to cause cellular dysfunction within a variety of pathological settings, the precise role of HOCl and HOSCN in the initiation of tissue damage evident following cardiac I/R injury remains unclear. In this study, we have employed the use of the cardiac myoblast cell line H9c2 as a model for cardiac myocytes and demonstrate that exposure to either oxidant elicits a dose-dependent increase in cytosolic calcium accumulation, depletion of the cellular thiol pool, reduction of glutathione (GSH) levels and loss of mitochondrial inner trans-membrane potential, concomitant with increased necrotic cell death. H9c2 cell recovery from the initial oxidant exposure involves the initiation of cell survival signalling pathways centred around Nrf2-antioxidant response element (ARE) and activator protein 1 (AP-1) activation, with cell survival accompanied by restoration of mitochondrial function following exposure to HOSCN, but not HOCl. These data highlight the cellular responses elicited by HOCl and HOSCN in cardiac myocytes furthering our understanding of the pathogenesis of oxidant injury following cardiac I/R injury.

Increased Cytosolic Calcium Contributes to Hydrogen-Rich Water-Promoted Anthocyanin Biosynthesis Under UV-A Irradiation in Radish Sprouts Hypocotyls.

Our previous studies showed that hydrogen-rich water (HRW) promoted the biosynthesis of anthocyanin under UV-A in radish. However, molecular mechanism involved in the regulation of the anthocyanin biosynthesis is still unclear. In this study, the role of calcium (Ca2+) in HRW-promoted anthocyanin biosynthesis in radish sprouts hypocotyls under UV-A was investigated. The results showed that a positive effect of HRW on the content of cytosolic calcium and anthocyanin accumulation, mimicking the effects of induced CaCl2. Exogenous addition of Ca2+ chelator bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and inositol 1,4,5-trisphosphate (IP3) synthesis inhibitor neomycin partially reversed the facilitated effect of HRW. The positive effects of HRW on activity of anthocyanin biosynthetic-enzymes (L-phenylalanine ammonia-lyase, PAL; chalcone isomerase, CHI; dihydroflavonol 4-reductase, DFR and UDP glc-flavonoid 3-O-glucosyl transferase, UFGT) were reversed by EGTA and neomycin. Further tests confirmed that the upregulation of anthocyanin biosynthetic related genes induced by HRW was substantially inhibited by calcium antagonists. The possible involvement of CaM in HRW-regulated anthocyanin biosynthesis was also preliminarily investigated in this study. Taken together, our results indicate that IP3-dependent calcium signaling pathway might be involved in HRW-regulated anthocyanin biosynthesis under UV-A irradiation.

17β-Estradiol and/or estrogen receptor alpha signaling blocks protein phosphatase 1 mediated ISO induced cardiac hypertrophy.

Earlier studies have shown that estrogen possess protective function against the development of pathological cardiac hypertrophy. However, the molecular mechanisms of estrogens (E2) protective effect are poorly understood. Additionally, abnormal activation of β-adrenergic signaling have been implicated in the development of pathological cardiac remodeling. However, the role of serine/threonine protein phosphatase 1 (PP1) in pathological cardiac remodeling under the influence of β-adrenergic signaling have been sparsely investigated. In this study, we assessed the downstream effects of abnormal activation of PP1 upon isoproterenol (ISO) induced pathological cardiac changes. We found that pre-treatment of 17β-estradiol (E2), tet-on estrogen receptor-α, or both significantly inhibited ISO-induced increase in cell size, hypertrophy marker gene expression and cytosolic calcium accumulation in H9c2 cells. Additionally, treatment with estrogen receptor inhibitor (ICI) reversed those effects, implicating role of E2 in inhibiting pathological cardiac remodeling. However, specific inhibition of ERα using melatonin, reduced ISO-induced PP1c expression and enhanced the level of ser-16 phosphorylated phospholamban (PLB), responsible for regulation of sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Furthermore, hypertrophic effect caused by overexpression of PP1cα was reduced by treatment with specific inhibitor of ERα. Collectively, we found that estrogen and estrogen receptor-α have protective effect against pathological cardiac changes by suppressing PP1 expression and its downstream signaling pathway, which further needs to be elucidated.

P-159 - The effect of chlorinated nucleosides on vascular cell function

During atherosclerosis, leukocytes infiltrate the vessel wall and release myeloperoxidase, which forms hypochlorous acid (HOCl). HOCl is highly reactive and forms chlorinated DNA and RNA products that are present within atherosclerotic plaques. In this study, the effect of various chlorinated nucleoside products on endothelial and smooth muscle cell function was examined, as these cells are dysregulated in atherosclerosis. Human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) were exposed to different chlorinated nucleosides. 8-chloroadenosine (8ClA) was readily incorporated into the RNA and nucleotide pool, resulting in the accumulation of 8ClATP. In HCAEC, 8ClA activated the unfolded protein response leading to cytosolic calcium accumulation, altered cellular respiration and apoptotic cell death. 8ClA affected the proliferation rate of HCASMC but did not activate the unfolded protein response. HCASMC are more susceptible to 5-chorodeoxycytidine (5CldC), with exposure to this nucleoside activating inflammatory pathways, which culminates in elevated matrix metalloproteinase expression. These data support a role of chlorinated nucleosides in promoting vascular cell dysfunction in atherosclerosis.

Cardiac myosin activators for heart failure therapy: focus on omecamtiv mecarbil

Heart failure continues to be a major global health problem with a pronounced impact on morbidity and mortality and very limited drug treatment options especially with regard to inotropic therapy. Omecamtiv mecarbil is a first-in-class cardiac myosin activator, which increases the proportion of myosin heads that are tightly bound to actin and creates a force-producing state that is not associated with cytosolic calcium accumulation. Phase I and phase II studies have shown that it is safe and well tolerated. It produces dose-dependent increases in systolic ejection time (SET), stroke volume (SV), left ventricular ejection fraction (LVEF), and fractional shortening. In the ATOMIC-AHF trial, intravenous (IV) omecamtiv mecarbil did not improve dyspnoea overall but may have improved it in a high-dose group of acute heart failure patients. It did, however, increase SET, decrease left ventricular end-systolic diameter, and was well tolerated. The COSMIC-HF trial showed that a pharmacokinetic-based dose-titration strategy of oral omecamtiv mecarbil improved cardiac function and reduced ventricular diameters compared to placebo and had a similar safety profile. It also significantly reduced plasma N-terminal-pro B-type natriuretic peptide compared with placebo. The GALACTIC-HF trial is now underway and will compare omecamtiv mecarbil with placebo when added to current heart failure standard treatment in patients with chronic heart failure and reduced LVEF. It is expected to be completed in January 2021. The ongoing range of preclinical and clinical research on omecamtiv mecarbil will further elucidate its full range of pharmacological effects and its clinical usefulness in heart failure.

25 - Role of selenomethionine supplementation in the modulation of damage during inflammation and myocardial ischemia/reperfusion injury

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury. This partly arises from the presence of activated phagocytes releasing the heme enzyme myeloperoxidase (MPO) and the associated production of hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). The dietary supplement selenomethionine (SeMet) has been shown to both bolster endogenous antioxidant processes and readily react with MPO-derived oxidants, yet its ability to protect against cardiac I/R injury is unknown. The aim of this study was to assess whether supplementation with SeMet could modulate the extent of cellular damage observed in an in vitro cardiac myocyte model exposed to (patho)-physiological levels of HOCl and HOSCN and an in vivo rat model of cardiac I/R injury. Exposure of cardiac myocytes to HOCl resulted in a dose-dependent increase in necrotic cell death, which could be prevented by SeMet supplementation (25 µM). This was attributed to SeMet preventing the HOCl-induced loss of mitochondrial inner trans-membrane potential, and the associated cytosolic calcium accumulation, together with bolstering the activity of the selenium-dependent antioxidant enzyme glutathione peroxidase. In contrast, no apparent protection of SeMet supplementation was evident upon exposure of cells to HOSCN, where higher oxidant concentrations (100 – 125 µM) were required to induce cell death. In vivo, a significant increase in selenium levels in the plasma, brain, kidney, and heart tissue were seen in rats fed a diet supplemented with 2 mg/kg SeMet compared to controls. Echocardiograph assessment of LV function at 4 weeks post-surgical intervention in the I/R injury cohort demonstrated limited improvement in fractional shortening and ejection fraction in SeMet-supplemented fed rats compared to controls. However, a more marked improvement was observed in the sham injury cohort, indicating SeMet supplementation’s potential cardiac health benefit.

Localized accumulation of cytosolic calcium near the fused sperm is associated with the calcium- and voltage-dependent block of sperm entry in the sea urchin egg.

Interaction of the sperm and egg depolarizes the egg membrane, allowing the sperm to enter; however, if the egg membrane is not allowed to depolarize from its resting potential (e.g., by voltage-clamp), the sperm will not enter. Previous studies demonstrated that sperm entry into sea urchin eggs that are voltage-clamped at negative membrane potentials is regulated both by the egg's membrane potential and a voltage-dependent influx of calcium into the egg. In these cases, electrical or cytoplasmic continuity (sperm-egg membrane fusion) occurs at negative membrane potentials, but subsequent loss of cytoplasmic continuity results in failure of sperm entry (unfusion). The work presented herein examined where, in relation to the sperm, and when, in relation to the sperm-induced electrophysiological events, the egg's calcium influx occurs, and how these events relate to successful or failed sperm entry. When sperm entered the egg, elevation of intracellular calcium concentration ([Ca2+ ]i ) began near the fused sperm on average 5.9 s after sperm-egg membrane fusion. Conversely, when sperm failed to enter the egg, [Ca2+ ]i elevated near the site of sperm-egg fusion on average 0.7 s after sperm-egg membrane fusion, which is significantly earlier than in eggs for which sperm entered. Therefore, the accumulation of calcium near the site of sperm-egg fusion is spatially and temporally consistent with the mechanism that may be responsible for loss of cytoplasmic continuity and failure of sperm entry.