Altered Calcium Homeostasis Research Articles

P.321 - Ultrasonography evaluation of long-term taurine effects on cardiac and skeletal muscles of mdx mice

Taurine (Tau) is the most abundant free amino acid in heart and skeletal muscle. In these tissues Tau is involved in normal contractile function by regulating the calcium homeostasis and the calcium sensitivity of the contractile proteins. Preclinical studies in mdx mice disclosed promising effects of Tau as a countermeasure for the early alterations of skeletal muscle in Duchenne muscular dystrophy (DMD). DMD is a rare disease caused by the absence of sub-sarcolemmal protein dystrophin, progressively leading to skeletal muscle wasting and heart failure via altered calcium homeostasis abnormal inflammation and profibrotic signaling. We presently focused on the potential effectiveness of Tau supplementation on later cardiac muscle dysfunction in DMD, since this has never been investigated up to now. Tau was orally administered (1g/kg/daily) in male wild type (WT) and mdx mice at an initial age of 6 months. The treatment duration was 6 months. Tau effects were tested on WT and mdx mice by measuring body mass, in vivo force and resistance to treadmill exercise. In parallel, by using an UBM system (Vevo 2100; VisualSonics, Canada), ultrasound evaluation of the left ventricular function and hindlimb volume (V) and percentage of vascularization (%V) were also performed. Our results showed a significant reduction of stroke volume (SV; -30%), ejection fraction (EF; -18.4%) and fractional shortening (FS; -23%) in 12 months old untreated mdx mice with respect to the age-matched untreated WT. Interestingly, Tau treatment significantly and selectively counteracted the alterations of SV, EF and SF in mdx mice as the values were similar to those of untreated WT mice. No significant effects were observed on the V and %V of hindlimb skeletal muscle as well as on the other functional parameters in response to Tau. This study provide the new evidence that long term Tau supplementation could be an effective specific countermeasure for DMD cardiomyopathy (DPP-NL and PRIN-MIUR).

Early Life Exposure to Low Levels of AHR Agonist PCB126 (3,3',4,4',5-Pentachlorobiphenyl) Reprograms Gene Expression in Adult Brain.

Early life exposure to environmental chemicals can have long-term consequences that are not always apparent until later in life. We recently demonstrated that developmental exposure of zebrafish to low, nonembryotoxic levels of 3,3',4,4',5-pentachlorobiphenyl (PCB126) did not affect larval behavior, but caused changes in adult behavior. The objective of this study was to investigate the underlying molecular basis for adult behavioral phenotypes resulting from early life exposure to PCB126. We exposed zebrafish embryos to PCB126 during early development and measured transcriptional profiles in whole embryos, larvae and adult male brains using RNA-sequencing. Early life exposure to 0.3 nM PCB126 induced cyp1a transcript levels in 2-dpf embryos, but not in 5-dpf larvae, suggesting transient activation of aryl hydrocarbon receptor with this treatment. No significant induction of cyp1a was observed in the brains of adults exposed as embryos to PCB126. However, a total of 2209 and 1628 genes were differentially expressed in 0.3 and 1.2 nM PCB126-exposed groups, respectively. KEGG pathway analyses of upregulated genes in the brain suggest enrichment of calcium signaling, MAPK and notch signaling, and lysine degradation pathways. Calcium is an important signaling molecule in the brain and altered calcium homeostasis could affect neurobehavior. The downregulated genes in the brain were enriched with oxidative phosphorylation and various metabolic pathways, suggesting that the metabolic capacity of the brain is impaired. Overall, our results suggest that PCB exposure during sensitive periods of early development alters normal development of the brain by reprogramming gene expression patterns, which may result in alterations in adult behavior.

Selective serotonin reuptake inhibitor, fluoxetine, impairs E-cadherin-mediated cell adhesion and alters calcium homeostasis in pancreatic beta cells

Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed drugs for mood disorders. Long term use of SSRIs is associated with an increased risk of diabetes, but the underlying mechanism(s) remains elusive. E-cadherin-mediated cell-cell adhesion and elevated [Ca2+]i are important for insulin release and pancreatic β cell functions. This study aims to investigate whether a SSRI, fluoxetine (Prozac), induces pancreatic β cell dysfunction through affecting E-cadherin and/or [Ca2+]i. Here we show that fluoxetine significantly reduces glucose stimulated insulin secretion (GSIS). MIN6 cells, an established murine immortalized β cell line, form smaller colonies of loosely packed cells with reduced cell-cell contact after fluoxetine treatment. Immunofluorescence staining reveals that fluoxetine increases cytoplasmic accumulation of E-cadherin and reduces the membrane-localized E-cadherin probably due to increase of its endocytosis. Fluoxetine inhibits spreading of β cells on E-cad/Fc coated slides and also disrupts E-cadherin-mediated actin filaments. Additionally, fluoxetine significantly suppresses endoplasmic reticulum (ER) calcium release and store-operated calcium entry (SOCE) activation, probably through reduction of ER calcium storage and inhibition of stromal interaction molecule 1 (STIM1) trafficking. These data suggest that exposure to fluoxetine results in impaired β cell functions, occurring in concert with reduction of E-cadherin-dependent cell adhesion and alterations of calcium homeostasis.

Bile Acids Do Not Contribute to the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis.

Previously, we have found that intracellular calcium homeostasis is altered in platelets from an experimental model of liver cirrhosis, the bile-duct ligated (BDL) rat; these alterations are compatible with the existence of a hypercoagulable state and related to an enhanced intracellular calcium release evoked by thrombin and an increased amount of calcium stored in the intracellular organelles. In the present study we have investigated the role of bile acids in those alterations of the BDL cirrhotic model. Cholic acid (CA) or deoxycholic acid (DCA) did not change P-selectin expression or platelet aggregation in any group but elevated baseline platelet calcium levels. Incubation with both bile acids reduced calcium release after stimulation with thrombin in the absence of extracellular calcium. Pretreatment with CA but not with DCA reduced significantly thrombin-induced calcium entry in all three experimental groups. The capacitative calcium entry was also significantly lower in platelets pretreated with both bile acids. The simultaneous addition of thapsigargin and ionomycin to estimate the total amount of calcium in platelet internal stores was decreased by pretreatment with both CA and DCA, although these changes were significantly different in the control rats only with CA and in the BDL platelets with DCA. These results indicate that CA and DCA reduce calcium movements in platelets of control and BDL animals, thus suggesting that bile acids do not participate in the alterations observed in the BDL cirrotic model.

Calcium homeostasis modulator 1 ( CALHM1 ) polymorphisms in cattle

ABSTRACT: The calcium homeostasis modulator 1 gene (CALHM1), which is located on chromosome 10 in humans and on chromosome 26 in cattle, is a transmembrane glycoprotein that controls the cytosolic calcium concentrations. Altered calcium homeostasis has been associated with several neurodegenerative disorders, including Alzheimer’s disease (AD). In a recent study, single nucleotide polymorphisms (SNPs) of CALHM1 have been associated with sporadic Creutzfeldt-Jakob disease (CJD). The protein sequence of human CALHM1 shows 93% homology with bovine CALHM1. Although SNPs of human CALHM1 have been correlated with human prion disease, polymorphisms of the bovine CALHM1 gene have not been reported in cattle thus far. To investigate polymorphisms of the bovine CALHM1 gene in Korean native cattle, we analyzed the open reading frame (ORF) of this gene in 175 Hanwoo and 141 Holstein cattle. We observed five SNPs: c.219C>T (rs380966453), c.357C>T (rs385969338), and c.869A>G (rs516301908) within the ORF region of two exons; and c.552+92A>G (rs481706737) and c.553-3A>C (rs448524869) in the intron of bovine CALHM1. Among the three SNPs that are in the ORF region of bovine CALHM1, the genotype and allele frequencies of the c.869A>G (p.His290Arg) and c.219C>T (p.Asn73Asn) SNPs were significantly different between Hanwoo and Holstein cattle (P<0.0001). Haplotype analysis showed that haplotypes ht2, ht3 and ht5 were also significantly different in these two cattle breeds. This study provides the first genetic analysis of the bovine CALHM1 gene in cattle.

Pathophysiology of Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced neuropathy is a common, dose-dependent adverse effect of several antineoplastics. It can lead to detrimental dose reductions and discontinuation of treatment, and severely affects the quality of life of cancer survivors. Clinically, chemotherapy-induced peripheral neuropathy presents as deficits in sensory, motor, and autonomic function which develop in a glove and stocking distribution due to preferential effects on longer axons. The pathophysiological processes are multi-factorial and involve oxidative stress, apoptotic mechanisms, altered calcium homeostasis, axon degeneration and membrane remodeling as well as immune processes and neuroinflammation. This review focusses on the commonly used antineoplastic substances oxaliplatin, cisplatin, vincristine, docetaxel, and paclitaxel which interfere with the cancer cell cycle—leading to cell death and tumor degradation—and cause severe acute and chronic peripheral neuropathies. We discuss drug mechanism of action and pharmacokinetic disposition relevant to the development of peripheral neuropathy, the epidemiology and clinical presentation of chemotherapy-induced neuropathy, emerging insight into genetic susceptibilities as well as current understanding of the pathophysiology and treatment approaches.

Biochemical markers of bone turnover in benign paroxysmal positional vertigo.

ObjectiveSeveral studies have suggested a possible relationship between recurrent benign paroxysmal positional vertigo (BPPV) and altered calcium homeostasis in the endolymph of the inner ear. The present study aimed to evaluate the association between Ca2+ and vitamin D status and BPPV occurrence as well as the status of bone biochemical markers in osteoporotic patients who were diagnosed with idiopathic BPPV.MethodsThe study included total 132 patients who were referred to our clinic between August 2008 and October 2013. Based on the bone mineral density (BMD) results, the subjects were divided into three groups: normal BMD (n = 34), osteopenia (n = 40) and osteoporosis (n = 58). The biochemical markers of bone turnover including serum Carboxy-terminal telopeptide of type I collagen (s-CTX), osteocalcin, alkaline phosphatase (ALP) and urinary free deoxypyridinoline (u-DPD), were analyzed, along with the serum Ca2+ and vitamin D levels.ResultsThe mean serum calcium, phosphate and creatinine clearance levels were within the standard laboratory reference range. The incidence of vitamin D deficiency was 11.8% (4/34) in the normal BMD group, 15% (6/40) in the osteopenia group and 43.1% (25/58) in the osteoporosis group. There was a positive correlation between the 25(OH)D and BMD results in the patients with BPPV. Among the bone turnover markers, the osteocalcin and u-DPD levels were significantly elevated in the osteoporotic patients with BPPV. Multiple logistic regression analyses showed that osteoporosis and vitamin D deficiency were associated with BPPV.ConclusionOur findings suggest that the prevalence of BPPV in osteoporotic patients is associated with vitamin D deficiency and high bone turnover rates at systemic level, which could disturb local Ca2+ homeostasis in the inner ear.

Growth hormone secretagogues prevent dysregulation of skeletal muscle calcium homeostasis in a rat model of cisplatin-induced cachexia.

BackgroundCachexia is a wasting condition associated with cancer types and, at the same time, is a serious and dose‐limiting side effect of cancer chemotherapy. Skeletal muscle loss is one of the main characteristics of cachexia that significantly contributes to the functional muscle impairment. Calcium‐dependent signaling pathways are believed to play an important role in skeletal muscle decline observed in cachexia, but whether intracellular calcium homeostasis is affected in this situation remains uncertain. Growth hormone secretagogues (GHS), a family of synthetic agonists of ghrelin receptor (GHS‐R1a), are being developed as a therapeutic option for cancer cachexia syndrome; however, the exact mechanism by which GHS interfere with skeletal muscle is not fully understood.MethodsBy a multidisciplinary approach ranging from cytofluorometry and electrophysiology to gene expression and histology, we characterized the calcium homeostasis in fast‐twitch extensor digitorum longus (EDL) muscle of adult rats with cisplatin‐induced cachexia and established the potential beneficial effects of two GHS (hexarelin and JMV2894) at this level. Additionally, in vivo measures of grip strength and of ultrasonography recordings allowed us to evaluate the functional impact of GHS therapeutic intervention.ResultsCisplatin‐treated EDL muscle fibres were characterized by a ~18% significant reduction of the muscle weight and fibre diameter together with an up‐regulation of atrogin1/Murf‐1 genes and a down‐regulation of Pgc1‐a gene, all indexes of muscle atrophy, and by a two‐fold increase in resting intracellular calcium, [Ca2+]i, compared with control rats. Moreover, the amplitude of the calcium transient induced by caffeine or depolarizing high potassium solution as well as the store‐operated calcium entry were ~50% significantly reduced in cisplatin‐treated rats. Calcium homeostasis dysregulation parallels with changes of functional ex vivo (excitability and resting macroscopic conductance) and in vivo (forelimb force and muscle volume) outcomes in cachectic animals. Administration of hexarelin or JMV2894 markedly reduced the cisplatin‐induced alteration of calcium homeostasis by both common as well as drug‐specific mechanisms of action. This effect correlated with muscle function preservation as well as amelioration of various atrophic indexes, thus supporting the functional impact of GHS activity on calcium homeostasis.ConclusionsOur findings provide a direct evidence that a dysregulation of calcium homeostasis plays a key role in cisplatin‐induced model of cachexia gaining insight into the etiopathogenesis of this form of muscle wasting. Furthermore, our demonstration that GHS administration efficaciously prevents cisplatin‐induced calcium homeostasis alteration contributes to elucidate the mechanism of action through which GHS could potentially ameliorate chemotherapy‐associated cachexia.

Role of homocysteine and folic acid on the altered calcium homeostasis of platelets from rats with biliary cirrhosis

Previously, we have found that intracellular calcium homeostasis is altered in platelets from an experimental model of liver cirrhosis, the bile-duct ligated (BDL) rat; these alterations are compatible with the existence of a hypercoagulable state. Different studies indicate that cholestatic diseases are associated with hyperhomocysteinemia; thus, we hypothetized that it could contribute to those platelet alterations. In the present study, we have investigated the role of homocysteine (HCY) in platelet aggregation and calcium signaling in the BDL model. The effect of chronic folic acid treatment was also analyzed. Acute treatment with HCY increased the aggregation response to ADP and calcium responses to thrombin in platelets of control and BDL rats. Capacitative calcium entry was not altered by HCY. Chronic treatment with folic acid decreased platelet aggregation in control and BDL rats, but this decrease was greater in BDL rats. In folic acid-treated rats, thrombin-induced calcium entry and release were decreased in platelet of control rats but unaltered in BDL rats; however, capacitative calcium entry was decreased in platelets of control and BDL rats treated with folic acid. Reactive oxygen species were produced at higher levels by BDL platelets after stimulation with HCY or thrombin and folic acid normalized these responses. HCY plays a role in the enhanced platelet aggregation response of BDL rats, probably through an enhanced formation of ROS. Folic acid pretreatment normalizes many of the platelet alterations shown by BDL rats.

The medical and surgical treatment in secondary and tertiary hyperparathyroidism. Review.

Hyperparathyroidism is an alteration of the pathophysiological parathyroid hormone (PTH) secretion due or an independent and abnormal release (primary or tertiary hyperparathyroidism) by the parathyroid or an alteration of calcium homeostasis that stimulates the excessive production of parathyroid hormone (secondary hyperparathyroidism). There is not a standard, clinical or surgical, treatment for hyperparathyroidism. We review current diagnostic and therapeutic methods. In secondary hyperparathyroidism (2HPT) there is a progressive hyperplasia of the parathyroid glands and an increased production of parathyroid hormone. Several causes are proposed: chronic renal insufficiency, vitamin D deficiency, malabsorption syndrome. The tertiary hyperparathyroidism (3HPT) is considered a state of excessive autonomous secretion of PTH due to long-standing 2HPT and it's usually the result of a lack of suppression in the production of PTH. The pathophysiological implications are both skeletal and extraskeletal: it damages the cardiovascular system, nervous system, immune, hematopoietic and endocrine system. The introduction of new drugs has improved the survival of these patients, allowing the inhibition of the synthesis of PTH. Indication for surgical treatment is unresponsive medical therapy. There are no large prospective studies that comparing the medical and surgical treatment. The choice is not unique and we have to consider the singolar case and the clinical condition of the patient.

Fingolimod Limits Acute A\u03b2 Neurotoxicity and Promotes Synaptic Versus Extrasynaptic NMDA Receptor Functionality in Hippocampal Neurons

Fingolimod, also known as FTY720, is an analogue of the sphingolipid sphingosine, which has been proved to be neuroprotective in rodent models of Alzheimer’s disease (AD). Several cellular and molecular targets underlying the neuroprotective effects of FTY720 have been recently identified. However, whether the drug directly protects neurons from toxicity of amyloid-beta (Aβ) still remains poorly defined. Using a combination of biochemical assays, live imaging and electrophysiology we demonstrate that FTY720 induces a rapid increase in GLUN2A-containing neuroprotective NMDARs on the surface of dendritic spines in cultured hippocampal neurons. In addition, the drug mobilizes extrasynaptic GLUN2B-containing NMDARs, which are coupled to cell death, to the synapses. Altered ratio of synaptic/extrasynaptic NMDARs decreases calcium responsiveness of neurons to neurotoxic soluble Aβ 1–42 and renders neurons resistant to early alteration of calcium homeostasis. The fast defensive response of FTY720 occurs through a Sphingosine-1-phosphate receptor (S1P-R) -dependent mechanism, as it is lost in the presence of S1P-R1 and S1P-R3 antagonists. We propose that rapid synaptic relocation of NMDARs might have direct impact on amelioration of cognitive performance in transgenic APPswe/PS1dE9 AD mice upon sub-chronic treatment with FTY720.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

Tissue transglutaminase (TG2) and mitochondrial function and dysfunction.

Mitochondria are the cell's power plant to satisfy the energy demands. However, dysfunctional mitochondria can cause overproduction of reactive oxygen species (ROS), oxidative stress, and alteration of calcium homeostasis, which are the hallmarks of mitochondrial diseases. Under prolong oxidative stress, repeated cytosolic calcium elevations even only transiently, can lead to activation of some enzymes. One calcium-activated enzyme with demonstrated pathophysiological important in mitochondrial disease is tissue transglutaminase (TG2). TG2 is known as a post-translational modification (PTM) enzyme that is induced by oxidative stress. Compared to other types of PTMs, the physiological significance of TG2 mediated PTM is just beginning to be understood. Once activated, TG2 can modulate transcription, inactivate metabolic enzymes, and cause aggregation of critical proteins. Recent data indicate that TG2's activity not only can modulate the assembly of respiratory chain complexes but can also modulate the transcription of critical genes including PGC-1alpha and cytochrome C that are important for function and biogenesis of mitochondria. Here, we summarize dysfunctional mitochondria in diseases such as in neurodegenerative disorders can modulate TG2's activity and function. TG2 is also important for normal function of mitochondria.

INFLUENCE OF ZINC CHLORIDE INTRAGASTRIC AND PARENTERAL ADMINISTRATION ON SYSTEMIC HEMODYNAMICS IN A CHANGING CALCIUM HOMEOSTASIS

The objective of the research work is to study the influence of altered calcium homeostasis on the hemodynamic effects of zinc chloride. The experimental hypercalcaemiawas created in Wistar rats by intragastric administration of calcium chloride. Zinc chloride was administered intragastrically via a probe and subcutaneously, at a dose of 20 mg /kg daily for one month. At the end of the experiment the functional state of the cardiovascular system was investigated, the basic parameters of systemic hemodynamics were determined. The following parameters were determined: blood pressure – invasively (bloody way) by introducing a plastic catheter into the femoral artery. For measuring a minute volume of blood thermistor was inserted through the left common carotid artery to the aortic arch. Indications were recorded on the MX-04 monitor. Mean arterial pressure was calculated by a special formula, heart rate –by means of a surgical monitor; cardiac index, stroke index and the specific peripheral vascular resistance were calculated by special formulas. Studies have shown that in subcutaneous and intragastric administration changes in systemic hemodynamics in the form of hypertension was observed. In the experimental hypercalcaemia, induced by administration of calcium chloride, metal intragastric administration produced significantly less hypertensive reaction. In the group with subcutaneous administration of metal on the background of hypercalcaemia the changes were not pronounced.

Direct Myosin Activation by Omecamtiv Mecarbil for Heart Failure with Reduced Ejection Fraction.

Myosin is the indispensable molecular motor that utilizes chemical energy to produce force for contraction within the cardiac myocyte. Myosin activity is gated by intracellular calcium levels which are regulated by multiple upstream signaling cascades that can be altered for clinical utility using inotropic medications. In contrast to clinically available cardiac inotropes, omecamtiv mecarbil is a novel direct myosin activator developed to augment left ventricular systolic function without the undesirable secondary effects of altered calcium homeostasis. Its identification and synthesis followed high-throughput screening of a reconstituted sarcomere, deliberate optimization, exquisite biochemical evaluation, and subsequently promising effects in animal models were demonstrated. Physiologically, it prolonged the duration of left ventricular systole in animal models, healthy adults, and patients with heart failure with reduced ejection fraction (HFrEF) without changing the velocity of pressure development, as assessed in animal models. It has been formulated for both intravenous and oral administration, and in both acute and chronic settings produced similar alterations in the duration of systole associated with beneficial increases in cardiac output, improvements in left ventricular volumes, and reductions in heart rate and often of natriuretic peptides. Small, asymptomatic increases in troponin were also observed in the absence of clinically evident ischemia. Clinically, the question remains as to whether the possible harm of this minimal troponin release is outweighed by the potential benefits of reduced neurohormonal activation, increased stroke volume and cardiac output, and improved ventricular remodeling in patients treated with omecamtiv mecarbil. The resolution of this question is being addressed by a phase III outcomes trial of this potential novel therapy for heart failure.

Neuroprotective effect of Spilanthes acmella Murr. on pesticide-induced neuronal cells death.

To investigate protective effects of Spilanthes acmella (S.acmella) Murr. extracts against pesticide-induced neuronal cells death and to elucidate the underlying molecular mechanism in dopaminergic (SH-SY5Y) cells lines. Cell viability of SH-SY5Y cells was studied by treating the cells with various concentration of pirimicarb for 24h. Neuroprotective effect of S.acmella Murr. extracts was investigated by adding the plant extracts to the medium for 24h prior to the incubation with 100μM H2O2 or with pirimicarb for 24h. Control-untreated cells were incubated with the culture medium. Cell viability was measured by MTT assay, calpainand calpastatin expressions were analyzed by Western blotting and immunocytochemistry. Pretreatment of SH-SY5Y cells with S.acmella Murr. extracts (1μg/mL) for 24h significantly increased the dopaminergic neurons in pirimicarb-induced neurotoxicity. In addition, pretreatment with the S.acmella Murr. extracts led to decreased calpain but increased calpastatin protein levels. S.acmella Murr. extracts exerted neuroprotective effect, via an alteration of calcium homeostasis, against pirimicarb induced neurotoxicity. The S.acmella Murr. might be a potential natural candidate with neuroprotective activity.

Molecular Understanding of Aβ-hIAPP Cross-Seeding Assemblies on Lipid Membranes.

Amyloid-β (Aβ) and human islet polypeptide (hIAPP) are the causative agents responsible for Alzheimer's disease (AD) and type II diabetes (T2D), respectively. While numerous studies have reported the cross-seeding behavior of Aβ and hIAPP in solution, little effort has been made to examine the cross-seeding of Aβ and hIAPP in the presence of cell membranes, which is more biologically relevant to the pathological link between AD and T2D. In this work, we computationally study the cross-seeding and adsorption behaviors of Aβ and hIAPP on zwitterionic POPC and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) mixed bilayers using all-atom molecular dynamics (MD) simulations, particularly aiming to the effects of the initial orientation of the Aβ-hIAPP assembly and the lipid composition of cell membranes on mutual structural and interaction changes in both Aβ-hIAPP assembly and lipid bilayers at the atomic level. Aβ-hIAPP cross-seeding assembly always preferred to adopt a specific orientation and interface to associate with both lipid bilayers strongly via the N-terminal strands of Aβ. Such membrane-bound orientation explains experimental observation that hybrid Aβ-hIAPP fibrils on cell membranes showed similar morphologies to pure hIAPP fibrils. Moreover, Aβ-hIAPP assembly, regardless of its initial orientations, interacted more strongly with POPC/POPG bilayer than POPC bilayer, indicating that electrostatic interactions are the major forces governing peptide-lipid interactions. Strong electrostatic interactions were also attributed to the formation of Ca2+ bridges connecting both negatively charged Glu of Aβ and PO4 head groups of lipids, which facilitate the association of Aβ-hIAPP with the POPC/POPG bilayer. It was also found that the strong peptide-lipid binding reduced lipid fluidity. Both facts imply that Aβ-hIAPP assembly may induce cell damage by altering calcium homeostasis and cell membrane phase. This work provides a better fundamental understanding of cross-seeding of Aβ and hIAPP on cell membranes and a potential pathological link between AD and T2D.

Targeting integrin α5β1 ameliorates severe airway hyperresponsiveness in experimental asthma.

Treatment options are limited for severe asthma, and the need for additional therapies remains great. Previously, we demonstrated that integrin αvβ6-deficient mice are protected from airway hyperresponsiveness, due in part to increased expression of the murine ortholog of human chymase. Here, we determined that chymase protects against cytokine-enhanced bronchoconstriction by cleaving fibronectin to impair tension transmission in airway smooth muscle (ASM). Additionally, we identified a pathway that can be therapeutically targeted to mitigate the effects of airway hyperresponsiveness. Administration of chymase to human bronchial rings abrogated IL-13-enhanced contraction, and this effect was not due to alterations in calcium homeostasis or myosin light chain phosphorylation. Rather, chymase cleaved fibronectin, inhibited ASM adhesion, and attenuated focal adhesion phosphorylation. Disruption of integrin ligation with an RGD-containing peptide abrogated IL-13-enhanced contraction, with no further effect from chymase. We identified α5β1 as the primary fibronectin-binding integrin in ASM, and α5β1-specific blockade inhibited focal adhesion phosphorylation and IL-13-enhanced contraction, with no additional effect from chymase. Delivery of an α5β1 inhibitor into murine airways abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. Finally, α5β1 blockade enhanced the effect of the bronchodilator isoproterenol on airway relaxation. Our data identify the α5β1 integrin as a potential therapeutic target to mitigate the severity of airway contraction in asthma.

No association between frequent apheresis donation and risk of fractures: a retrospective cohort analysis from Sweden.

Citrate anticoagulation during apheresis induces transient alterations in calcium homeostasis. It is unknown whether the repeated, transient alterations in calcium homeostasis experienced by repeated apheresis donors affects bone turnover to increase fracture risk. Our aim was to investigate the risk of osteoporotic and nonosteoporotic fracture among voluntary, frequent apheresis donors. All apheresis donors were identified from the Scandinavian Donations and Transfusions database (SCANDAT2), which includes information on over 1.6 million blood donors from Sweden and Denmark from the years 1968 and 1981, respectively. Only data from Sweden were used for these analyses. Information on fractures was obtained by linking SCANDAT2 to hospital registers. Poisson regression was used to compute incidence rate ratios of fractures in relation to the cumulative number of apheresis donations, both overall and in fixed time windows. In total, 140,289 apheresis donors (67,970 women and 72,319 men) were identified from the SCANDAT2 database and were followed for up to 23 years. We observed no association between the frequency of apheresis donation and the risk of fracture either in the overall study period or during fixed-length time windows. The incidence rate ratio of fractures in donors who had made 100 or more cumulative apheresis donations was 0.99 (95% confidence interval, 0.92-1.06) compared with donors who had made from 9 to 24 donations. The results were similar in analyses stratified by sex and restricted to postmenopausal women. The absence of an association between repeated apheresis donation and fracture risk indicates that apheresis collection is safe with regard to bone health.

Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy.

ABSTRACTAutosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy characterized by centrally located nuclei in muscle fibers. CNM results from mutations in the gene encoding dynamin 2 (DNM2), a large GTPase involved in endocytosis, intracellular membrane trafficking, and cytoskeleton regulation. We developed a knock-in mouse model expressing the most frequent DNM2-CNM mutation; i.e. the KI-Dnm2R465W model. Heterozygous (HTZ) KI-Dnm2 mice progressively develop muscle atrophy, impairment of contractile properties, histopathological abnormalities, and elevated cytosolic calcium concentration. Here, we aim at better characterizing the calcium homeostasis impairment in extensor digitorum longus (EDL) and soleus muscles from adult HTZ KI-Dnm2 mice. We demonstrate abnormal contractile properties and cytosolic Ca2+ concentration in EDL but not soleus muscles showing that calcium impairment is correlated with muscle weakness and might be a determinant factor of the spatial muscle involvement. In addition, the elevated cytosolic Ca2+ concentration in EDL muscles is associated with an increased sarcolemmal permeability to Ca2+ and releasable Ca2+ content from the sarcoplasmic reticulum. However, amplitude and kinetics characteristics of the calcium transient appear unchanged. This suggests that calcium defect is probably not a primary cause of decreased force generation by compromised sarcomere shortening but may be involved in long-term deleterious consequences on muscle physiology. Our results highlight the first pathomechanism which may explain the spatial muscle involvement occurring in DNM2-related CNM and open the way toward development of a therapeutic approach to normalize calcium content.