Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale

In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).

Objective

To characterize RyR functional properties in relation to TT proximity, at baseline and after MI.

Methods

Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca²⁺ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca²⁺ release, F>F₅₀ within 20 ms) or their absence (delayed areas). Spontaneous Ca²⁺ release events during diastole, Ca²⁺ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.

Results

In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na⁺/Ca²⁺ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca²⁺ influx via Ca²⁺ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca²⁺ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca²⁺ removal by NCX at the membrane was significantly lower in MI.

Conclusion

TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca²⁺ loss and raise SR Ca²⁺ content, but may promote Ca²⁺ waves.     

Biphasic effect of extracellular ATP on human and rat airways is due to multiple P2 purinoceptor activation

Background

Extracellular ATP may modulate airway responsiveness. Studies on ATP-induced contraction and [Ca²⁺]_i signalling in airway smooth muscle are rather controversial and discrepancies exist regarding both ATP effects and signalling pathways. We compared the effect of extracellular ATP on rat trachea and extrapulmonary bronchi (EPB) and both human and rat intrapulmonary bronchi (IPB), and investigated the implicated signalling pathways.

Methods

Isometric contraction was measured on rat trachea, EPB and IPB isolated rings and human IPB isolated rings. [Ca²⁺]_i was monitored fluorimetrically using indo 1 in freshly isolated and cultured tracheal myocytes. Statistical comparisons were done with ANOVA or Student''s t tests for quantitative variables and χ² tests for qualitative variables. Results were considered significant at P < 0.05.

Results

In rat airways, extracellular ATP (10^-6–10^-3 M) induced an epithelium-independent and concentration-dependent contraction, which amplitude increased from trachea to IPB. The response was transient and returned to baseline within minutes. Similar responses were obtained with the non-hydrolysable ATP analogous ATP-γ-S. Successive stimulations at 15 min-intervals decreased the contractile response. In human IPB, the contraction was similar to that of rat IPB but the time needed for the return to baseline was longer. In isolated myocytes, ATP induced a concentration-dependent [Ca²⁺]_i response. The contractile response was not reduced by thapsigargin and RB2, a P2Y receptor inhibitor, except in rat and human IPB. By contrast, removal of external Ca²⁺, external Na⁺ and treatment with D600 decreased the ATP-induced response. The contraction induced by α-β-methylene ATP, a P2X agonist, was similar to that induced by ATP, except in IPB where it was lower. Indomethacin and H-89, a PKA inhibitor, delayed the return to baseline in extrapulmonary airways.

Conclusion

Extracellular ATP induces a transient contractile response in human and rat airways, mainly due to P2X receptors and extracellular Ca²⁺ influx in addition with, in IPB, P2Y receptors stimulation and Ca²⁺ release from intracellular Ca²⁺ stores. Extracellular Ca²⁺ influx occurs through L-type voltage-dependent channels activated by external Na⁺ entrance through P2X receptors. The transience of the response cannot be attributed to ATP degradation but to purinoceptor desensitization and, in extrapulmonary airways, prostaglandin-dependent PKA activation.     

Ageing‐associated increase in SGLT2 disrupts mitochondrial/sarcoplasmic reticulum Ca2+ homeostasis and promotes cardiac dysfunction

The prevalence of death from cardiovascular disease is significantly higher in elderly populations; the underlying factors that contribute to the age‐associated decline in cardiac performance are poorly understood. Herein, we identify the involvement of sodium/glucose co‐transporter gene (SGLT2) in disrupted cellular Ca²⁺‐homeostasis, and mitochondrial dysfunction in age‐associated cardiac dysfunction. In contrast to younger rats (6‐month of age), older rats (24‐month of age) exhibited severe cardiac ultrastructural defects, including deformed, fragmented mitochondria with high electron densities. Cardiomyocytes isolated from aged rats demonstrated increased reactive oxygen species (ROS), loss of mitochondrial membrane potential and altered mitochondrial dynamics, compared with younger controls. Moreover, mitochondrial defects were accompanied by mitochondrial and cytosolic Ca²⁺ ([Ca²⁺]_i) overload, indicative of disrupted cellular Ca²⁺‐homeostasis. Interestingly, increased [Ca²⁺]_i coincided with decreased phosphorylation of phospholamban (PLB) and contractility. Aged‐cardiomyocytes also displayed high Na⁺/Ca²⁺‐exchanger (NCX) activity and blood glucose levels compared with young‐controls. Interestingly, the protein level of SGLT2 was dramatically increased in the aged cardiomyocytes. Moreover, SGLT2 inhibition was sufficient to restore age‐associated defects in [Ca²⁺]_i‐homeostasis, PLB phosphorylation, NCX activity and mitochondrial Ca²⁺‐loading. Hence, the present data suggest that deregulated SGLT2 during ageing disrupts mitochondrial function and cardiac contractility through a mechanism that impinges upon [Ca²⁺]_i‐homeostasis. Our studies support the notion that interventions that modulate SGLT2‐activity can provide benefits in maintaining [Ca²⁺]_i and cardiac function with advanced age.     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

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.     

Computational Model of Ca2+ Wave Propagation in Human Retinal Pigment Epithelial ARPE-19 Cells

Objective

Computational models of calcium (Ca²⁺) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca²⁺ signaling model for RPE based on our experimental data of mechanically induced Ca²⁺ wave in the in vitro model of RPE, the ARPE-19 monolayer.

Methods

We combined six essential Ca²⁺ signaling components into a model: stretch-sensitive Ca²⁺ channels (SSCCs), P₂Y₂ receptors, IP₃ receptors, ryanodine receptors, Ca²⁺ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca²⁺ waves reproduced our control experimental data and data where gap junctions were blocked.

Results

Our model was able to explain Ca²⁺ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca²⁺ through plasma membrane SSCCs and gap junctions conduct the Ca²⁺ and IP³ between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca²⁺ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP₃ receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca²⁺ signal in the distance.

Conclusions

The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P₂Y₂ receptors or accelerate P₂Y₂ receptor phosphorylation, which may partially be the reason for Ca²⁺ wave attenuation by suramin. Being the first RPE Ca²⁺ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.     

Structure and Functional Analysis of a Ca2+ Sensor Mutant of the Na+/Ca2+ Exchanger

The mammalian Na⁺/Ca²⁺ exchanger, NCX1.1, serves as the main mechanism for Ca²⁺ efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca²⁺, NCX1.1 activity is also strongly regulated by Ca²⁺ binding to two intracellular regulatory domains, CBD1 and CBD2. The structures of both of these domains have been solved by NMR spectroscopy and x-ray crystallography, greatly enhancing our understanding of Ca²⁺ regulation. Nevertheless, the mechanisms by which Ca²⁺ regulates the exchanger remain incompletely understood. The initial NMR study showed that the first regulatory domain, CBD1, unfolds in the absence of regulatory Ca²⁺. It was further demonstrated that a mutation of an acidic residue involved in Ca²⁺ binding, E454K, prevents this structural unfolding. A contradictory result was recently obtained in a second NMR study in which Ca²⁺ removal merely triggered local rearrangements of CBD1. To address this issue, we solved the crystal structure of the E454K-CBD1 mutant and performed electrophysiological analyses of the full-length exchanger with mutations at position 454. We show that the lysine substitution replaces the Ca²⁺ ion at position 1 of the CBD1 Ca²⁺ binding site and participates in a charge compensation mechanism. Electrophysiological analyses show that mutations of residue Glu-454 have no impact on Ca²⁺ regulation of NCX1.1. Together, structural and mutational analyses indicate that only two of the four Ca²⁺ ions that bind to CBD1 are important for regulating exchanger activity.Cardiac contraction/relaxation relies upon Ca²⁺ fluxes across the plasma membrane (sarcolemma) of cardiomyocytes. Rapid Ca²⁺ influx (primarily through L-type Ca²⁺ channels) triggers the release of additional Ca²⁺ from the sarcoplasmic reticulum (SR),⁴ resulting in cardiomyocyte contraction. Removal of cytosolic Ca²⁺ by reuptake into the SR (through the SR Ca²⁺-ATPase) and expulsion from the cell (primarily through the Na⁺/Ca²⁺ exchanger, NCX1.1) results in relaxation (1). Altered Ca²⁺ cycling is observed in a number of pathophysiological situations including ischemia, hypertrophy, and heart failure (2). Understanding the function and regulation of NCX1.1 is thus of fundamental importance to understand cardiac physiology.NCX1.1 utilizes the electrochemical potential of the Na⁺ gradient to extrude Ca²⁺ in a ratio of three Na⁺ ions to one Ca²⁺ ion (3). In addition to transporting both Na⁺ and Ca²⁺, NCX1.1 is also strongly regulated by these two ions. Intracellular Na⁺ can induce NCX1.1 to enter an inactivated state, whereas Ca²⁺ bound to regulatory sites removes Na⁺-dependent inactivation and also activates Na⁺/Ca²⁺ exchange (3). These regulatory sites are located on a large cytoplasmic loop (∼500 residues located between transmembrane helices V and VI) containing two calcium binding domains (CBD1 and CBD2), which sense cytosolic Ca²⁺ levels. We have previously shown that Ca²⁺ binding to the primary site in CBD2 is required for full exchange regulation (4); CBD1, however, is a site of higher affinity and appears to dominate the activation of exchange activity by Ca²⁺.Both CBDs have an immunoglobulin fold formed from two antiparallel β sheets generating a β sandwich with a differing number of Ca²⁺ ions coordinated at the tip of the domain (4, 5). CBD1 binds four Ca²⁺ ions, whereas CBD2 binds only two Ca²⁺ ions. An initial NMR study revealed a local unfolding of the upper portion of CBD1 upon release of Ca²⁺ (6). In contrast, CBD2 did not display an unfolding response upon Ca²⁺ removal. A comparative analysis between CBDs revealed a difference in charge at residues in equivalent positions near the Ca²⁺ coordination site; Glu-454 in CBD1 is replaced by Lys-585 in CBD2. The unstructuring of CBD1 upon Ca²⁺ removal was alleviated by reversing the charge of the acidic residue (E454K) involved in Ca²⁺ coordination (6). Previously, we solved the structures of the Ca²⁺-bound and -free conformations of CBD2 and revealed a charge compensation mechanism involving Lys-585 (4). The positively charged lysine residue assumes the position of one of the Ca²⁺ ions upon Ca²⁺ depletion, permitting CBD2 to retain its overall fold (4). A similar phenomenon is predicted to take place in E454K-CBD1 mutant. In addition, Hilge et al. (6) showed that the E454K mutation of CBD1 decreases Ca²⁺ affinity to a level similar to that of CBD2 and suggested that the E454K mutation would cause the loss of primary regulation of NCX1.1 by CBD1.The significance of some of these observations is unclear as a recent NMR study (7) of CBD1 under more physiologically relevant conditions revealed no significant alteration in tertiary structure in the absence of Ca²⁺. It was hypothesized that Ca²⁺ binding induces localized conformational and dynamic changes involving several of the binding site residues. To clarify this issue, we solved the crystal structure of the E454K-CBD1 mutant and examined the functional effects of different CBD1 mutations in the full-length NCX1.1. The results indicate that charge compensation is indeed provided by the residue Lys-454 to replace one Ca²⁺, whereas the overall E454K-CBD1 structure is only slightly perturbed. The charge compensation, however, has no impact on Ca²⁺ regulation of NCX1.1.     

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na⁺, Ca²⁺) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca²⁺ at the expense of the Na⁺ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca²⁺ extrusion and thus to the recovery from intracellular Ca²⁺ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na⁺ to 1 Ca²⁺, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na⁺ concentration or minor depolarisations switch it into the “reverse mode” (Ca²⁺ import/Na⁺ export). Notably, transient Na⁺ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na⁺-permeable ion channels in response to neuronal activity. Activity-related Na⁺ transients result in NCX reversal, which mediates Ca²⁺ influx from the extracellular space, thereby generating astrocyte Ca²⁺ signalling independent from InsP₃-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca²⁺ overload, while dampening Na⁺ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na⁺-driven, reverse NCX-mediated astrocyte Ca²⁺ signals are involved neurone-glia interaction. Na⁺ transients, translated by the NCX into Ca²⁺ elevations, thereby emerge as a new signalling pathway in astrocytes.     

A SGLT2 inhibitor dapagliflozin suppresses prolonged ventricular-repolarization through augmentation of mitochondrial function in insulin-resistant metabolic syndrome rats

Background

Metabolic syndrome (MetS) is a prevalent risk factor for cardiac dysfunction. Although SGLT2-inhibitors have important cardioprotective effects in hyperglycemia, their underlying mechanisms are complex and not completely understood. Therefore, we examined mechanisms of a SGLT2-inhibitor dapagliflozin (DAPA)-related cardioprotection in overweight insulin-resistant MetS-rats comparison with insulin (INSU), behind its glucose-lowering effect.

Methods

A 28-week high-carbohydrate diet-induced MetS-rats received DAPA (5 mg/kg), INSU (0.15 mg/kg) or vehicle for 2 weeks. To validate MetS-induction, we monitored all animals weekly by measuring body weight, blood glucose and HOMO-IR index, electrocardiograms, heart rate, systolic and diastolic pressures.

Results

DAPA-treatment of MetS-rats significantly augmented the increased blood pressure, prolonged Q–R interval, and low heart rate with depressed left ventricular function and relaxation of the aorta. Prolonged-action potentials were preserved with DAPA-treatment, more prominently than INSU-treatment, at most, through the augmentation in depressed voltage-gated K⁺-channel currents. DAPA, more prominently than INSU-treatment, preserved the depolarized mitochondrial membrane potential, and altered mitochondrial protein levels such as Mfn-1, Mfn-2, and Fis-1 as well as provided significant augmentation in cytosolic Ca²⁺-homeostasis. Furthermore, DAPA also induced significant augmentation in voltage-gated Na⁺-currents and intracellular pH, and the cellular levels of increased oxidative stress, protein-thiol oxidation and ADP/ATP ratio in cardiomyocytes from MetS rats. Moreover, DAPA-treatment normalized the increases in the mRNA level of SGLT2 in MetS-rat heart.

Conclusions

Overall, our data provided a new insight into DAPA-associated cardioprotection in MetS rats, including suppression of prolonged ventricular-repolarization through augmentation of mitochondrial function and oxidative stress followed by improvement of fusion–fission proteins, out of its glucose-lowering effect.

     

The Plasma Membrane Ca2+ ATPase and the Plasma Membrane Sodium Calcium Exchanger Cooperate in the Regulation of Cell Calcium

Calcium is an ambivalent signal: it is essential for the correct functioning of cell life, but may also become dangerous to it. The plasma membrane Ca²⁺ ATPase (PMCA) and the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) are the two mechanisms responsible for Ca²⁺ extrusion. The NCX has low Ca²⁺ affinity but high capacity for Ca²⁺ transport, whereas the PMCA has a high Ca²⁺ affinity but low transport capacity for it. Thus, traditionally, the PMCA pump has been attributed a housekeeping role in maintaining cytosolic Ca²⁺, and the NCX the dynamic role of counteracting large cytosolic Ca²⁺ variations (especially in excitable cells). This view of the roles of the two Ca²⁺ extrusion systems has been recently revised, as the specific functional properties of the numerous PMCA isoforms and splicing variants suggests that they may have evolved to cover both the basal Ca²⁺ regulation (in the 100 nM range) and the Ca²⁺ transients generated by cell stimulation (in the μM range).Ca²⁺ controls critical cellular responses in all eukaryotic organisms. It controls both short-term biological processes that occur in milliseconds, such as muscle contraction, as well as long-term processes that require longer times, such as cell proliferation and organ development. The specificity of cellular Ca²⁺ signals is controlled by a sophisticated “toolkit” comprising numerous ion channels, pumps, and exchangers that drive the fluxes of Ca²⁺ ions across the plasma membrane and across the membranes of intracellular organelles (Berridge et al. 2003).The plasma membrane contains several types of channels that mediate Ca²⁺ entry from the extracellular ambient, and two systems for Ca²⁺ extrusion: a low affinity, high capacity Na⁺/Ca²⁺ exchanger (NCX), and a high-affinity, low-capacity Ca²⁺-ATPase (the plasma membrane Ca²⁺ pump (PMCA)) (Fig. 1). The type of channels and the relative proportions of NCX and PMCA vary with the cell type, the NCX being particularly abundant in excitable tissues, e.g., heart and brain. The regulated opening of the Ca²⁺ channels by either voltage gating, interaction with ligands or the emptying of intracellular stores, allows a limited amount of Ca²⁺ to enter the cell to transmit signals to its designated targets. Thereafter, the Ca²⁺ transients must be dissipated: its extrusion from the cell is mediated by the NCX and the PMCA pump, but Ca²⁺ is also restored to basal levels by sequestration in the endo/sarcoplasmic reticulum via the SERCA pump and in the mitochondria by the electrophoretic uniporter. The NCX has also been found at the inner membrane of the nuclear envelope (NE) and has been proposed to mediate Ca²⁺ flux between the nucleoplasm and the NE (Xie et al. 2002), and then to the ER (Wu et al. 2009) in neuronal and certain other cell types. Ca²⁺ binding proteins also contributed to Ca²⁺ buffering: In this review, we will not cover them, as we will only discuss the systems that extrude Ca²⁺ out of the cell.Open in a separate windowFigure 1.A schematic representation of the structures involved in cellular Ca²⁺ homeostasis. The model shows a cell with its Ca²⁺-transporting systems: Ca²⁺-ATPases (plasma membrane and sarco/endoplasmic reticulum, PMCA and SERCA), plasma membrane (PM) Ca²⁺ channels, Na⁺/Ca²⁺ exchangers (NCX and NCLX), 1,4,5-triphosphate receptor (IP₃R) and ryanodine receptor (RyR), the electrophoretic mitochondrial uptake uniporter (U). Mitochondria are drawn as yellow ellipses, nucleus as orange circle and endoplasmic reticulum is colored in red. The different Ca²⁺-transporting systems cooperate to maintain the Ca²⁺ concentration gradient between the extracellular and the intracellular ambient.The PMCA pump is a minor component of the total protein of the plasma membrane (less than 0.1% of it). Quantitatively, it is overshadowed by the more powerful NCX in excitable tissue like heart; however, even cells in which the NCX predominates, the PMCA pump is likely to be the fine tuner of cytosolic Ca²⁺, as it can operate in a concentration range in which the low affinity NCX is relatively very inefficient.The PMCA was discovered in erythrocytes (Schatzmann 1966), and was then described and characterized in numerous other cell types. It was purified in 1979 using a calmodulin affinity column (Niggli et al. 1979), and cloned about 10 years later (Shull and Greeb 1988; Verma et al. 1988). It shows the same essential membrane topology properties of the SERCA pump. Molecular modeling work using the structure of the SERCA pump as a template (Toyoshima et al. 2000) predicts the same general features of the latter, with 10 transmembrane domains and the large cytosolic headpiece divided into the three main cytosolic A, N, and P domains. The Na⁺/Ca²⁺ cotransport process was discovered at about the same time as PMCA by two independent groups working on heart (Reuter and Seitz 1968) and on the squid giant axon (Baker et al. 1969). The exchanger was cloned in 1990 (Nicoll et al. 1990). The sequence was initially predicted to correspond to a protein with 11 transmembrane domains and one large cytosolic loop linking transmembrane domain five and six but a revised model predicting only nine transmembrane domains is now generally accepted.     

23Na Magnetic Resonance Imaging of the Lower Leg of Acute Heart Failure Patients during Diuretic Treatment

Objective

Na⁺ can be stored in muscle and skin without commensurate water accumulation. The aim of this study was to assess Na⁺ and H₂O in muscle and skin with MRI in acute heart failure patients before and after diuretic treatment and in a healthy cohort.

Methods

Nine patients (mean age 78 years; range 58–87) and nine age and gender-matched controls were studied. They underwent ²³Na/¹H-MRI at the calf with a custom-made knee coil. Patients were studied before and after diuretic therapy. ²³Na-MRI gray-scale measurements of Na⁺-phantoms served to quantify Na⁺-concentrations. A fat-suppressed inversion recovery sequence was used to quantify H₂O content.

Results

Plasma Na⁺-levels did not change during therapy. Mean Na⁺-concentrations in muscle and skin decreased after furosemide therapy (before therapy: 30.7±6.4 and 43.5±14.5 mmol/L; after therapy: 24.2±6.1 and 32.2±12.0 mmol/L; p˂0.05 and p˂0.01). Water content measurements did not differ significantly before and after furosemide therapy in muscle (p = 0.17) and only tended to be reduced in skin (p = 0.06). Na⁺-concentrations in calf muscle and skin of patients before and after diuretic therapy were significantly higher than in healthy subjects (18.3±2.5 and 21.1±2.3 mmol/L).

Conclusions

²³Na-MRI shows accumulation of Na⁺ in muscle and skin in patients with acute heart failure. Diuretic treatment can mobilize this Na⁺-deposition; however, contrary to expectations, water and Na⁺-mobilization are poorly correlated.     

Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia

Background

The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) into insulin-producing cells (IPCs) for autologous transplantation may alleviate those limitations.

Methods

hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10⁶ differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ)-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice.

Results

The differentiated IPCs were characterized by Dithizone (DTZ) positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca²⁺ channel activity and similar changes in intracellular Ca²⁺ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo.

Conclusions

IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.     

Store-Operated Ca2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

Aims

Endothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.

Methods and Results

We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca²⁺ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

Conclusions

These data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca²⁺ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.     

The Selective SGLT2 Inhibitor Ipragliflozin Has a Therapeutic Effect on Nonalcoholic Steatohepatitis in Mice

Background & Aims

In recent years, nonalcoholic steatohepatitis (NASH) has become a considerable healthcare burden worldwide. Pathogenesis of NASH is associated with type 2 diabetes mellitus (T2DM) and insulin resistance. However, a specific drug to treat NASH is lacking. We investigated the effect of the selective sodium glucose cotransporter 2 inhibitor (SGLT2I) ipragliflozin on NASH in mice.

Methods

We used the Amylin liver NASH model (AMLN), which is a diet-induced model of NASH that results in obesity and T2DM. AMLN mice were fed an AMLN diet for 20 weeks. SGLT2I mice were fed an AMLN diet for 12 weeks and an AMLN diet with 40 mg ipragliflozin/kg for 8 weeks.

Results

AMLN mice showed steatosis, inflammation, and fibrosis in the liver as well as obesity and insulin resistance, features that are recognized in human NASH. Ipragliflozin improved insulin resistance and liver injury. Ipragliflozin decreased serum levels of free fatty acids, hepatic lipid content, the number of apoptotic cells, and areas of fibrosis; it also increased lipid outflow from the liver.

Conclusions

Ipragliflozin improved the pathogenesis of NASH by reducing insulin resistance and lipotoxicity in NASH-model mice. Our results suggest that ipragliflozin has a therapeutic effect on NASH with T2DM.     

Regulation of TRPM7 Function by IL-6 through the JAK2-STAT3 Signaling Pathway

Aims

Previous studies have demonstrated that expression of the TRPM7 channel, which may induce delayed cell death by mediating calcium influx, is precisely regulated. However, functional regulation of TRPM7 channels by endogenous molecules has not been elucidated. The proinflammatory cytokine IL-6 contributes to regulation of Ca²⁺ influx in cerebral ischemia, but the role of IL-6 in regulating TRPM7 functioning is unknown. Thus, we here investigated the interaction between IL-6 and TRPM7 channels and the relevant mechanisms.

Materials and Methods

Using whole-cell patch-clamping, we first investigated the effect of IL-6 on TRPM7-like currents in primary cultured cortical neurons. Next, TRPM7-overexpressing HEK293 cells were used to confirm the effect of IL-6/sIL-6R on TRPM7. Finally, we used specific signaling pathway inhibitors to investigate the signaling pathways involved.

Results

IL-6 or IL-6/sIL-6R dose-dependently inhibited inward TRPM7 currents, in both primary cultured neurons and HEK293 cells overexpressing TRPM7. In intracellular Mg²⁺-free conditions, extracellular Ca²⁺ or the α-kinase domain of TRPM7 did not participate in this regulation. The inhibitory effect of IL-6 on TRPM7 could be blocked by specific inhibitors of the JAK2−STAT3 pathway, but not of the PI3K, ERK1/2, or PLC pathways.

Conclusions

IL-6 inhibits the inward TRPM7 current via the JAK2−STAT3 signaling pathway.     

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

Background

This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As⁺³) and cadmium (Cd⁺²)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd⁺²-and As⁺³-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.

Methods

Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As⁺³-and Cd⁺²-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.

Results

It was shown that the As⁺³-and Cd⁺²-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.

Conclusions

Tumor initiating cells isolated from SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.