Interplay between obesity and aging on myocardial geometry and function: Role of leptin-STAT3-stress signaling

BackgroundUncorrected obesity facilitates premature aging and cardiovascular anomalies. This study examined the interaction between obesity and aging on cardiac remodeling and contractile function. Methods: Cardiac echocardiographic geometry, function, morphology, intracellular Ca²⁺ handling, oxidative stress (DHE fluorescence), STAT3 and stress signaling were evaluated in young (3-mo) and old (12- and 18-mo) lean and leptin deficient ob/ob obese mice. Cardiomyocytes from young and old lean and ob/ob mice were treated with leptin (1 nM) for 4 h in vitro prior to assessment of mechanical and biochemical properties. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obese mice at young and old age were evaluated for comparison. Results: Our results displayed reduced survival in ob/ob mice. Obesity but less likely older age dampened echocardiographic, geometric, cardiomyocyte function and intracellular Ca²⁺ properties, elevated O₂^? and p^47phox NADPH oxidase levels with a more pronounced geometric change at older age. Immunoblot analysis revealed elevated p^47phox NADPH oxidase and dampened phosphorylation of STAT3, with a more pronounced response in old ob/ob mice, the effects were restored by leptin. Obesity and aging inhibited phosphorylation of Akt, eNOS, AMPK, and p38 while promoting phosphorylation of JNK and IκB. Leptin reconciled cardiomyocyte dysfunction, O₂^? yield, p^47phox upregulation, STAT3 dephosphorylation and stress signaling in ob/ob mice although its action on stress signaling cascades were lost at old age. High fat diet-induced and db/db obesity displayed aging-associated cardiomyocyte anomalies reminiscent of ob/ob model albeit lost leptin response.ConclusionsOur data suggest disparate age-associated obesity response in cardiac remodeling and contractile dysfunction due to phosphorylation of Akt, eNOS and stress signaling-related oxidative stress.     

The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice

Background

Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp^−/−) double mutant mice.

Results

Both ob/ob and double mutant ob/ob;Shp^−/− mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp^−/− mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp^−/− liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp^−/− mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp^−/− mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes.

Conclusions

Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.     

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism

Objectives

Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca²⁺ properties.

Methods

Murine cardiomyocyte contractile function and intracellular Ca²⁺ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca²⁺ decay rate. Stress signaling and Ca²⁺ regulatory proteins were assessed using Western blot analysis.

Results

In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca²⁺ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca²⁺ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca²⁺ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca²⁺ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.

Conclusions

Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca²⁺ through a NADPH oxidase-dependent mechanism.     

Gene Knock-Outs of Inositol 1,4,5-Trisphosphate Receptors Types 1 and 2 Result in Perturbation of Cardiogenesis

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R1, 2, and 3) are intracellular Ca²⁺ release channels that regulate various vital processes. Although the ryanodine receptor type 2, another type of intracellular Ca²⁺ release channel, has been shown to play a role in embryonic cardiomyocytes, the functions of the IP₃Rs in cardiogenesis remain unclear.

Methodology/Principal Findings

We found that IP₃R1^−/−-IP₃R2^−/− double-mutant mice died in utero with developmental defects of the ventricular myocardium and atrioventricular (AV) canal of the heart by embryonic day (E) 11.5, even though no cardiac defect was detectable in IP₃R1^−/− or IP₃R2^−/− single-mutant mice at this developmental stage. The double-mutant phenotype resembled that of mice deficient for calcineurin/NFATc signaling, and NFATc was inactive in embryonic hearts from the double knockout-mutant mice. The double mutation of IP₃R1/R2 and pharmacologic inhibition of IP₃Rs mimicked the phenotype of the AV valve defect that result from the inhibition of calcineurin, and it could be rescued by constitutively active calcineurin.

Conclusions/Significance

Our results suggest an essential role for IP₃Rs in cardiogenesis in part through the regulation of calcineurin-NFAT signaling.     

Norepinephrine Controls Both Torpor Initiation and Emergence via Distinct Mechanisms in the Mouse

Some mammals, including laboratory mice, enter torpor in response to food deprivation, and leptin can attenuate these bouts of torpor. We previously showed that dopamine β-hydroxylase knockout (Dbh −/−) mice, which lack norepinephrine (NE), do not reduce circulating leptin upon fasting nor do they enter torpor. To test whether the onset of torpor in mice during a fast requires a NE-mediated reduction in circulating leptin, double mutant mice deficient in both leptin (ob/ob) and DBH (DBL MUT) were generated. Upon fasting, control and ob/ob mice entered torpor as assessed by telemetric core T_b acquisition. While fasting failed to induce torpor in Dbh −/− mice, leptin deficiency bypassed the requirement for NE, as DBL MUT mice readily entered torpor upon fasting. These data indicate that sympathetic activation of white fat and suppression of leptin is required for the onset of torpor in the mouse. Emergence from torpor was severely retarded in DBL MUT mice, revealing a novel, leptin-independent role for NE in torpor recovery. This phenotype was mimicked by administration of a β₃ adrenergic receptor antagonist to control mice during a torpor bout. Hence, NE signaling via β₃ adrenergic receptors presumably in brown fat is the first neurotransmitter-receptor system identified that is required for normal recovery from torpor.     

Cardiomyocyte Contractile Dysfunction in the APPswe/PS1dE9 Mouse Model of Alzheimer's Disease

Objectives

Ample clinical and experimental evidence indicated that patients with Alzheimer''s disease display a high incidence of cardiovascular events. This study was designed to examine myocardial histology, cardiomyocyte shortening, intracellular Ca²⁺ homeostasis and regulatory proteins, electrocardiogram, adrenergic response, endoplasmic reticulum (ER) stress and protein carbonyl formation in C57 wild-type (WT) mice and an APPswe/PS1dE9 transgenic (APP/PS1) model for Alzheimer''s disease.

Methods

Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR), maximal velocity of shortening and relengthening (±dL/dt), intracellular Ca²⁺ transient rise and decay.

Results

Little histological changes were observed in APP/PS1 myocardium. Cardiomyocytes from APP/PS1 but not APP or PS1 single mutation mice exhibited depressed PS, reduced±dL/dt, normal TPS and TR compared with WT mice_. Rise in intracellular Ca²⁺ was lower accompanied by unchanged resting/peak intracellular Ca²⁺ levels and intracellular Ca²⁺ decay in APP/PS1 mice. Cardiomyocytes from APP/PS1 mice exhibited a steeper decline in PS at high frequencies. The responsiveness to adrenergic agonists was dampened although β₁-adrenergic receptor expression was unchanged in APP/PS1 hearts. Expression of the Ca²⁺ regulatory protein phospholamban and protein carbonyl formation were downregulated and elevated, respectively, associated with unchanged SERCA2a, Na⁺-Ca²⁺ exchanger and ER stress markers in APP/PS1 hearts. Our further study revealed that antioxidant N-acetylcysteine attenuated the contractile dysfunction in APP/PS1 mice.

Conclusions

Our results depicted overt cardiomyocyte mechanical dysfunction in the APP/PS1 Alzheimer''s disease model, possibly due to oxidative stress.     

Hypoxia Activates a Ca2+-Permeable Cation Conductance Sensitive to Carbon Monoxide and to GsMTx-4 in Human and Mouse Sickle Erythrocytes

Background

Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca²⁺] ([Ca²⁺]_i) and subsequent activation of K_Ca 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.

Methodology/Principal Findings

We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca²⁺- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 µM), dipyridamole (100 µM), DIDS (100 µM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca²⁺]_i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca²⁺]_i in mouse sickle erythrocytes did not require KCa3.1 activity.

Conclusions/Significance

The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca²⁺-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca²⁺ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.     

Guanine Nucleotides in the Meiotic Maturation of Starfish Oocytes: Regulation of the Actin Cytoskeleton and of Ca2+ Signaling

Background

Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca²⁺ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca²⁺ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation.

Methodology/Principal Findings

By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca²⁺ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca²⁺; iii) GDPβS has little effect on the amplitude of the Ca²⁺ peak, but significantly expedites the initial Ca²⁺ waves induced by InsP₃ photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca²⁺ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca²⁺ signaling.

Conclusions/Significance

Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca²⁺ release.     

Involvement of AMPK in Alcohol Dehydrogenase Accentuated Myocardial Dysfunction Following Acute Ethanol Challenge in Mice

Objectives

Binge alcohol drinking often triggers myocardial contractile dysfunction although the underlying mechanism is not fully clear. This study was designed to examine the impact of cardiac-specific overexpression of alcohol dehydrogenase (ADH) on ethanol-induced change in cardiac contractile function, intracellular Ca²⁺ homeostasis, insulin and AMP-dependent kinase (AMPK) signaling.

Methods

ADH transgenic and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Oral glucose tolerance test, cardiac AMP/ATP levels, cardiac contractile function, intracellular Ca²⁺ handling and AMPK signaling (including ACC and LKB1) were examined.

Results

Ethanol exposure led to glucose intolerance, elevated plasma insulin, compromised cardiac contractile and intracellular Ca²⁺ properties, downregulated protein phosphatase PP2A subunit and PPAR-γ, as well as phosphorylation of AMPK, ACC and LKB1, all of which except plasma insulin were overtly accentuated by ADH transgene. Interestingly, myocardium from ethanol-treated FVB mice displayed enhanced expression of PP2Cα and PGC-1α, decreased insulin receptor expression as well as unchanged expression of Glut4, the response of which was unaffected by ADH. Cardiac AMP-to-ATP ratio was significantly enhanced by ethanol exposure with a more pronounced increase in ADH mice. In addition, the AMPK inhibitor compound C (10 µM) abrogated acute ethanol exposure-elicited cardiomyocyte mechanical dysfunction.

Conclusions

In summary, these data suggest that the ADH transgene exacerbated acute ethanol toxicity-induced myocardial contractile dysfunction, intracellular Ca²⁺ mishandling and glucose intolerance, indicating a role of ADH in acute ethanol toxicity-induced cardiac dysfunction possibly related to altered cellular fuel AMPK signaling cascade.     

Fusion-Activated Ca2+ Entry: An “Active Zone” of Elevated Ca2+ during the Postfusion Stage of Lamellar Body Exocytosis in Rat Type II Pneumocytes

Background

Ca²⁺ is essential for vesicle fusion with the plasma membrane in virtually all types of regulated exocytoses. However, in contrast to the well-known effects of a high cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) in the prefusion phase, the occurrence and significance of Ca²⁺ signals in the postfusion phase have not been described before.

Methodology/Principal Findings

We studied isolated rat alveolar type II cells using previously developed imaging techniques. These cells release pulmonary surfactant, a complex of lipids and proteins, from secretory vesicles (lamellar bodies) in an exceptionally slow, Ca²⁺- and actin-dependent process. Measurements of fusion pore formation by darkfield scattered light intensity decrease or FM 1-43 fluorescence intensity increase were combined with analysis of [Ca²⁺]_c by ratiometric Fura-2 or Fluo-4 fluorescence measurements. We found that the majority of single lamellar body fusion events were followed by a transient (t_1/2 of decay = 3.2 s) rise of localized [Ca²⁺]_c originating at the site of lamellar body fusion. [Ca²⁺]_c increase followed with a delay of ∼0.2–0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at ∼10 µm/s). Removal of Ca²⁺ from, or addition of Ni²⁺ to the extracellular solution, strongly inhibited these [Ca²⁺]_c transients, whereas Ca²⁺ store depletion with thapsigargin had no effect. Actin-GFP fluorescence around fused LBs increased several seconds after the rise of [Ca²⁺]_c. Both effects were reduced by the non-specific Ca²⁺ channel blocker {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365.

Conclusions/Significance

Fusion-activated Ca²⁺ entry (FACE) is a new mechanism that leads to [Ca²⁺]_c transients at the site of vesicle fusion. Substantial evidence from this and previous studies indicates that fusion-activated Ca²⁺ entry enhances localized surfactant release from type II cells, but it may also play a role for compensatory endocytosis and other cellular functions.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.     

FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice

The metabolic syndrome is a worldwide problem mainly caused by obesity. FTO was found to be a obesity-risk gene in humans and FTO deficiency in mice led to reduction in adipose tissue. Thus, FTO is an important factor for the development of obesity. Leptin-deficient mice are a well characterized model for analysing the metabolic syndrome. To determine the relevance of FTO for the development of the metabolic syndrome we analysed different parameters in combined homozygous deficient mice (Lep^ob/ob;Fto^−/−). Lep^ob/ob;Fto^−/− mice showed an improvement in analysed hallmarks of the metabolic syndrome in comparison to leptin-deficient mice wild type or heterozygous for Fto. Lep^ob/ob;Fto^−/− mice did not develop hyperglycaemia and showed an improved glucose tolerance. Furthermore, extension of beta-cell mass was prevented in Lep^ob/ob;Fto^−/−mice and accumulation of ectopic fat in the liver was reduced. In conclusion this study demonstrates that FTO deficiency has a protective effect not only on the development of obesity but also on the metabolic syndrome. Thus, FTO plays an important role in the development of metabolic disorders and is an interesting target for therapeutic agents.     

Absence of adipose differentiation related protein upregulates hepatic VLDL secretion,relieves hepatosteatosis,and improves whole body insulin resistance in leptin-deficient mice

We previously showed that adipose differentiation related protein (Adfp)-deficient mice display a 60% reduction in hepatic triglyceride (TG) content. In this study, we investigated the role of ADFP in lipid and glucose homeostasis in a genetic obesity model, Lep^ob/ob mice. We bred Adfp^−/− mice with Lep^ob/ob mice to create Lep^ob/ob/Adfp^−/− and Lep^ob/ob/Adfp^+/+ mice and analyzed the hepatic lipids, lipid droplet (LD) morphology, LD protein composition and distribution, lipogenic gene expression, and VLDL secretion, as well as insulin sensitivity of the two groups of mice. Compared with Lep^ob/ob/Adfp^+/+ mice, Lep^ob/ob/Adfp^−/− mice displayed an increased VLDL secretion rate, a 25% reduction in hepatic TG associated with improvement in fatty liver grossly and microscopically with a change of the size of LDs in a proportion of the hepatocytes and a redistribution of major LD-associated proteins from the cytoplasmic compartment to the LD surface. There was no detectable change in lipogenic gene expression. Lep^ob/ob/Adfp^−/− mice also had improved glucose tolerance and insulin sensitivity in both liver and muscle. The alteration of LD size in the liver of Lep^ob/ob/Adfp^−/− mice despite the relocation of other LDPs to the LD indicates a nonredundant role for ADFP in determining the size and distribution of hepatic LDs.     

Cyclic Nucleotide-Gated Channels Contribute to Thromboxane A2-Induced Contraction of Rat Small Mesenteric Arteries

Background

Thromboxane A₂ (TxA₂)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA₂-induced Ca²⁺ influx, which resulted in vascular contraction via Ca²⁺-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA₂-induced Ca²⁺ influx in vascular smooth muscle cells.

Methodology/Principal Findings

Application of U-46619, a thromboxane A₂ mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca²⁺, because removal of extracellular Ca²⁺ abolished the constriction. This constriction was partially inhibited by an L-type Ca²⁺ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.

Conclusions/Significance

These data suggest a functional role of CNG channels in U-46619-induced Ca²⁺ influx and contraction of smooth muscle cells.     

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

Background

The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell''s diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.

Methodology/Principal Findings

The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K⁺ and Cl⁻ currents were strictly dependent on the presence of Ca²⁺. The Ca²⁺-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca²⁺ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca²⁺ permeability pathway leading to increased [Ca²⁺]_i, secondary activation of Ca²⁺-sensitive K⁺ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.

Conclusions/Significance

The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca²⁺-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca²⁺ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.     

CXCL14 Deficiency in Mice Attenuates Obesity and Inhibits Feeding Behavior in a Novel Environment

Background

CXCL14 is a chemoattractant for macrophages and immature dendritic cells. We recently reported that CXCL14-deficient (CXCL14 ^−/−) female mice in the mixed background are protected from obesity-induced hyperglycemia and insulin resistance. The decreased macrophage infiltration into visceral adipose tissues and the increased insulin sensitivity of skeletal muscle contributed to these phenotypes.

Methodology/Principal Findings

In this study, we performed a comprehensive study for the body weight control of CXCL14 ^−/− mice in the C57BL/6 background. We show that both male and female CXCL14 ^−/− mice have a 7–11% lower body weight compared to CXCL14 ^+/− and CXCL14 ^+/+ mice in adulthood. This is mainly caused by decreased food intake, and not by increased energy expenditure or locomotor activity. Reduced body weight resulting from the CXCL14 deficiency was more pronounced in double mutant CXCL14^−/− ob/ob and CXCL14 ^−/−A^y mice. In the case of CXCL14 ^−/−A^y mice, oxygen consumption was increased compared to CXCL14 ^+/−A^y mice, in addition to the reduced food intake. In CXCL14 ^−/− mice, fasting-induced up-regulation of Npy and Agrp mRNAs in the hypothalamus was blunted. As intracerebroventricular injection of recombinant CXCL14 did not change the food intake of CXCL14 ^−/− mice, CXCL14 could indirectly regulate appetite. Intriguingly, the food intake of CXCL14 ^−/− mice was significantly repressed when mice were transferred to a novel environment.

Conclusions/Significance

We demonstrated that CXCL14 is involved in the body weight control leading to the fully obese phenotype in leptin-deficient or A^y mutant mice. In addition, we obtained evidence indicating that CXCL14 may play an important role in central nervous system regulation of feeding behavior.     

Frog α- and β-Ryanodine Receptors Provide Distinct Intracellular Ca2+ Signals in a Myogenic Cell Line

Background

In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, α-RyR and β-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/Principal Findings

In this study, we expressed either or both α-RyR and β-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that α-RyR and β-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca²⁺ imaging studies, each isoform exhibited caffeine-induced Ca²⁺ transients, an indicative of Ca²⁺-induced Ca²⁺ release (CICR). However, the fashion of Ca²⁺ release events was fundamentally different: α-RyR mediated graded and sustained Ca²⁺ release observed uniformly throughout the cytoplasm, whereas β-RyR supported all-or-none type regenerative Ca²⁺ oscillations and waves. α-RyR but not β-RyR exhibited Ca²⁺ transients triggered by membrane depolarization with high [K⁺]_o that were nifedipine-sensitive, indicating that only α-RyR mediates depolarization-induced Ca²⁺ release. Myotubes co-expressing α-RyR and β-RyR demonstrated high [K⁺]_o-induced Ca²⁺ transients which were indistinguishable from those with myotubes expressing α-RyR alone. Furthermore, procaine did not affect the peak height of high [K⁺]_o-induced Ca²⁺ transients, suggesting minor amplification of Ca²⁺ release by β-RyR via CICR in 1B5 myotubes.

Conclusions/Significance

These findings suggest that α-RyR and β-RyR provide distinct intracellular Ca²⁺ signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.     

Cannabinoid CB2 Receptor Potentiates Obesity-Associated Inflammation,Insulin Resistance and Hepatic Steatosis

Background

Obesity-associated inflammation is of critical importance in the development of insulin resistance and non-alcoholic fatty liver disease. Since the cannabinoid receptor CB2 regulates innate immunity, the aim of the present study was to investigate its role in obesity-induced inflammation, insulin resistance and fatty liver.

Methodology

Murine obesity models included genetically leptin-deficient ob/ob mice and wild type (WT) mice fed a high fat diet (HFD), that were compared to their lean counterparts. Animals were treated with pharmacological modulators of CB2 receptors. Experiments were also performed in mice knock-out for CB2 receptors (Cnr2 −/−).

Principal Findings

In both HFD-fed WT mice and ob/ob mice, Cnr2 expression underwent a marked induction in the stromal vascular fraction of epididymal adipose tissue that correlated with increased fat inflammation. Treatment with the CB2 agonist JWH-133 potentiated adipose tissue inflammation in HFD-fed WT mice. Moreover, cultured fat pads isolated from ob/ob mice displayed increased Tnf and Ccl2 expression upon exposure to JWH-133. In keeping, genetic or pharmacological inactivation of CB2 receptors decreased adipose tissue macrophage infiltration associated with obesity, and reduced inductions of Tnf and Ccl2 expressions. In the liver of obese mice, Cnr2 mRNA was only weakly induced, and CB2 receptors moderately contributed to liver inflammation. HFD-induced insulin resistance increased in response to JWH-133 and reduced in Cnr2 −/− mice. Finally, HFD-induced hepatic steatosis was enhanced in WT mice treated with JWH-133 and blunted in Cnr2 −/− mice.

Conclusion/Significance

These data unravel a previously unrecognized contribution of CB2 receptors to obesity-associated inflammation, insulin resistance and non-alcoholic fatty liver disease, and suggest that CB2 receptor antagonists may open a new therapeutic approach for the management of obesity-associated metabolic disorders.     

Age-Related Reference Intervals of the Main Biochemical and Hematological Parameters in C57BL/6J, 129SV/EV and C3H/HeJ Mouse Strains

Background

Although the mouse is the animal model most widely used to study the pathogenesis and treatment of human diseases, reference values for biochemical parameters are scanty or lacking for the most frequently used strains. We therefore evaluated these parameters in the C57BL/6J, 129SV/EV and C3H/HeJ mice.

Methodology/Principal Findings

We measured by dry chemistry 26 analytes relative to electrolyte balance, lipoprotein metabolism, and muscle/heart, liver, kidney and pancreas functions, and by automated blood counter 5 hematological parameters in 30 animals (15 male and 15 female) of each mouse strain at three age ranges: 1–2 months, 3–8 months and 9–12 months. Whole blood was collected from the retro-orbital sinus. We used quality control procedures to investigate analytical imprecision and inaccuracy. Reference values were calculated by non parametric methods (median and 2.5^th and 97.5^th percentiles). The Mann-Whitney and Kruskal-Wallis tests were used for between-group comparisons. Median levels of GLU, LDH, Chol and BUN were higher, and LPS, AST, ALP and CHE were lower in males than in females (p range: 0.05–0.001). Inter-strain differences were observed for: (1) GLU, t-Bil, K⁺, Ca⁺⁺, PO₄ ⁻ (p<0.05) and for TAG, Chol, AST, Fe⁺⁺ (p<0.001) in 4–8 month-old animals; (2) for CK, Crea, Mg⁺⁺, Na⁺⁺, K⁺, Cl⁻ (p<0.05) and BUN (p<0.001) in 2- and in 10–12 month-old mice; and (3) for WBC, RBC, HGB, HCT and PLT (p<0.05) during the 1 year life span.

Conclusion/Significance

Our results indicate that metabolic variations in C57BL/6J, 129SV/EV and C3H/HeJ mice after therapeutic intervention should be evaluated against gender- and age-dependent reference intervals.     

Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP?/? Mice

Background

The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.

Principal Findings

We used intact olfactory epithelium obtained from WT and OMP^−/− mice to monitor the Ca²⁺ dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca²⁺ channels, or Ca²⁺ stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca²⁺-homeostasis in these neurons by influencing the activity of the plasma membrane Na⁺/Ca²⁺-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca²⁺ elevation by stimulating the reverse mode of NCX in both WT and OMP^−/− mice. The resulting Ca²⁺ responses indicate that OMP facilitates NCX activity and allows rapid Ca²⁺ extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).

Conclusions

Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions.