Proper Ca2 Research Articles

SERCA2a gene therapy can improve symptomatic heart failure in δ-sarcoglycan-deficient animals.

The loss of dystrophin or its associated proteins results in the development of muscle wasting frequently associated with cardiomyopathy. Contractile cardiac tissue is injured and replaced by fibrous tissue or fatty infiltrates, leading to a progressive decrease of the contractile force and finally to end-stage heart failure. At the time symptoms appear, restoration of a functional allele of the causative gene might not be sufficient to prevent disease progression. Alterations in Ca(2+) transport and intracellular calcium levels have been implicated in many types of pathological processes, especially in heart disease. On the basis of a gene transfer strategy, we analyzed the therapeutic efficacy of primary gene correction in a δ-sarcoglycan (δ-SG)-deficient animal model versus gene transfer of the Ca(2+) pump hSERCA2a (human sarco-endoplasmic reticulum calcium ATPase 2a), at a symptomatic stage of heart disease. Our results strongly suggest that restoration of δ-SG at this stage of disease will not lead to improved clinical outcome. However, restoration of proper Ca(2+) handling by means of amplifying SERCA2a expression in the myocardium can lead to functional improvement. Abnormalities in Ca(2+) handling play an important role in disease progression toward heart failure, and increased SERCA2a levels appear to significantly improve cardiac contraction and relaxation. Beneficial effects persist at least over a period of 6 months, and the evolution of cardiac functional parameters paralleled those of normal controls. Furthermore, we demonstrate that a plasmid formulation based on amphiphilic block copolymers can provide a safe and efficient platform for myocardial gene therapies. The use of synthetic formulations for myocardial gene transfer might thus overcome one of the major hurdles linked to viral vectors, that is, repeat administrations.

Elevation of glucose to levels associated with type I diabetes impairs bone cell mechanosignaling (1180.10)

It has been shown that patients with insulin dependent diabetes mellitus (IDDM; Type 1 diabetes) manifest a range of bone problems, including increased risk for bone fractures and defective bone healing. However, the underlying mechanisms are still not well understood. Given that the maintenance of skeletal integrity in response to daily physical activity relies homeostasis between bone forming cells, osteoblasts, and the primary load sensing cells, osteocytes, we hypothesized that bone loss in Type 1 diabetes is primarily triggered by exposure to high glucose levels which in turn compromises ability of bone cells to perceive and respond to mechanical loading as a result of altered expression of mechanosignaling mediators: specific purinergic receptor (P2Rs) and pannexin 1 (Panx1) channels. Our in situ (Akita, Type 1 diabetic and age‐matched wild type mouse model) morphometric analysis and in vitro (MLO‐Y4 osteocytes and MOB‐C osteoblasts) findings indicate that exposure to elevated glucose (10 days in α‐MEM containing 450 mg/dL (25 mM) glucose) alters P2R (specifically P2Y2R, P2Y4R and P2X7R) and Panx1 expression in both osteoblastic and osteocytic cells and blunts proper Ca2+ signaling in response to oscillatory fluid shear stress mechanical loading. Our findings indicate that Type 1 diabetes impairs the proper response of bone cells to mechanical loads, which is required to maintain bone health.Grant Funding Source: Supported by DK091466; DK081435; AR057139

Ion channels and anti-cancer immunity

The outcome of a malignant disease depends on the efficacy of the immune system to destroy cancer cells. Key steps in this process, for example the generation of a proper Ca(2+) signal induced by recognition of a specific antigen, are regulated by various ion channel including voltage-gated Kv1.3 and Ca(2+)-activated KCa3.1 K(+) channels, and the interplay between Orai and STIM to produce the Ca(2+)-release-activated Ca(2+) (CRAC) current required for T-cell proliferation and function. Understanding the immune cell subset-specific expression of ion channels along with their particular function in a given cell type, and the role of cancer tissue-dependent factors in the regulation of operation of these ion channels are emerging questions to be addressed in the fight against cancer disease. Answering these questions might lead to a better understanding of the immunosuppression phenomenon in cancer tissue and the development of drugs aimed at skewing the distribution of immune cell types towards killing of the tumour cells.

Tunable Regulation of the Serca/Phospholamban Complex by Single-Stranded DNA Sequences

Heart disease is a leading cause of death in the Western world.1 Failing hearts have been shown to exhibit abnormal calcium cycling within cardiomyocytes.2-5 While there are several proteins that are involved with the calcium pathway in cardiomyocytes, the sarcoplasmic reticulum Ca2+ -ATPase (SERCA) and phospholamban (PLN) function in the important role of calcium reuptake into the SR for storage. SERCA is inhibited by PLN, a 52 amino acid, single-pass membrane protein. Upon PLN phosphorylation at Ser16, this inhibition is relieved and SERCA's calcium affinity is restored to normal levels. It has been proposed that by reversing PLN's inhibition on SERCA in patients with heart failure proper Ca2+ homeostasis and muscle contractility may be restored.6 Using biophysical, biochemical, and analytical techniques including; enzyme coupled activity assays, solid and solution state nuclear magnetic resonance (NMR), fluorescence polarization, and affinity capillary electrophoresis we show that single stranded DNA sequences (ssDNA) target the SERCA/PLN complex and fully restore the ATPase function. Inportantly, ssDNA sequences bind PLN at a nanomolar level of affinity. The restoration of SERCA activity by ssDNA is elicited in a length-dependent manner. Studying the unique interactions between the PLN and ssDNA will give us insight into this chemical and molecular mechanism that allows for restoration of SERCA activity and will aid in the development of small molecules able to interfere with the SERCA/PLN complex for possible therapeutic interventions.

Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.

The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

The L-type Ca2+ channels blocker nifedipine represses mesodermal fate determination in murine embryonic stem cells.

Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca2+ channels of different cell types. Moreover, influx of Ca2+ through L-type Ca2+ channels (LTCCs) activates Ca2+ signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca2+ transient and reduction of LTCCs current density (I CaL) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca2+ channel α1C (CaV1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca2+ concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

Protein Components of the Ryanodine Receptor Complex Traffic Directly from Rough ER to Concentrate in Junctional SR

Interactions of the junctional SR (jSR) proteins calsequestrin (CSQ2), triadin (TRD), and junctin (JCT) with the ryanodine receptor have been demonstrated in vitro as essential for proper SR Ca2+ release. However, the intracellular pathway(s) by which these critical proteins traffic to jSR sites remains unknown. To investigate pathways of trafficking for jSR proteins in adult cardiomyocytes, wild-type canine CSQ2, TRD, and JCT were overexpressed in cultured adult rat cardiomyocytes using recombinant adenoviruses. The overexpressed proteins were detected using either species-specific antibodies, or by fusion with fluorescent proteins. After 24h and 48h, cardiomyocytes were examined using confocal fluorescence microscopy. By the time overexpressed CSQ2 was first detected by immunofluorescence (24h), it was already distributed across jSR sites. In contrast, TRD and JCT exhibited patterns of perinuclear accumulation after 24h. By 48h, they became increasingly concentrated along a transverse pathway that extended from perinuclear puncta towards the cell surface. This novel transverse jSR trafficking pathway co-localized with desmin, a prominent protein component of cardiac intermediate filaments. It was previously found that CSQ2-DsRed forms a polymeric complex that prematurely polymerizes at perinuclear sites of rough ER. We reasoned that this proximal CSQ2 polymer should then bind to newly synthesized TRD and JCT in situ, leading to their enhanced concentration around the nucleus. Indeed, when canine TRD and JCT were co-overexpressed with CSQ2-DsRed, both of these small transmembrane proteins bound to perinuclear CSQ2-DsRed, and failed to exhibit anterograde trafficking to peripheral jSR puncta. In contrast, TRD in which CSQ2 binding sites were deleted readily trafficked towards the cell periphery. Rates of anterograde trafficking of CSQ2 and other jSR proteins may depend upon the assembly of CSQ2 into polymers, which may be regulated by luminal Ca2+ concentrations.

Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation.

The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca2+ uptake capacity. This defect is neither a consequence of global alteration in cellular Ca2+ homeostasis nor of the reduced driving force for Ca2+ internalization within mitochondria, since cytosolic Ca2+ transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca2+ uptake speed in Afg3l2−/− cells, indicating the presence of functional Ca2+ uptake machinery. Our results show that the defective Ca2+ handling in Afg3l2−/− cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca2+ elevations, hampering the proper Ca2+ diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2−/− MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca2+ buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca2+ homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Usingheterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.

Computational Modeling and Numerical Methods for Spatiotemporal Calcium Cycling in Ventricular Myocytes

Intracellular calcium (Ca) cycling dynamics in cardiac myocytes is regulated by a complex network of spatially distributed organelles, such as sarcoplasmic reticulum (SR), mitochondria, and myofibrils. In this study, we present a mathematical model of intracellular Ca cycling and numerical and computational methods for computer simulations. The model consists of a coupled Ca release unit (CRU) network, which includes a SR domain and a myoplasm domain. Each CRU contains 10 L-type Ca channels and 100 ryanodine receptor channels, with individual channels simulated stochastically using a variant of Gillespie’s method, modified here to handle time-dependent transition rates. Both the SR domain and the myoplasm domain in each CRU are modeled by 5 × 5 × 5 voxels to maintain proper Ca diffusion. Advanced numerical algorithms implemented on graphical processing units were used for fast computational simulations. For a myocyte containing 100 × 20 × 10 CRUs, a 1-s heart time simulation takes about 10 min of machine time on a single NVIDIA Tesla C2050. Examples of simulated Ca cycling dynamics, such as Ca sparks, Ca waves, and Ca alternans, are shown.

Lecture

The physiology of paracellular permeation of ions and solutes in the kidney is pivotally important but poorly understood. Claudins are the key components of the paracellular pathway. Defects in claudin function result in a broad range of renal diseases, including hypomagnesemia, hypercalciuria and nephrolithiasis. This review describes recent findings on the physiological function of claudins underlying paracellular transport mechanisms with a focus on renal Ca2+ handling. We have uncovered a molecular mechanism underlying paracellular Ca2+ transport in the thick ascending limb of Henle (TAL) that involves the functional interplay of three important claudin genes: claudin-14, -16 and -19, all of which are associated with human kidney diseases with hypercalciuria, nephrolithiasis and bone mineral loss. The Ca2+ sensing receptor (CaSR) signaling in the kidney has long been a mystery. By analyzing small non-coding RNA molecules in the kidney, we have uncovered a novel microRNA based signaling pathway downstream of CaSR that directly regulates claudin-14 gene expression and establishes the claudin-14 molecule as a key regulator for renal Ca2+ homeostasis. The molecular cascade of CaSR-microRNAs-claudins forms a regulatory loop to maintain proper Ca2+ homeostasis in the kidney.

Abstract P231: Dispensability of Dmitofusin in Calcium Signaling in Intact Drosophila Heart Tubes

Sarcoplasmic Reticulum (SR) drives cardiomyocyte excitation-contraction coupling through the release and storage of Ca2+. Phasic fluctuations of Ca2+ require ATP. Mitochondria occupy ∼30% of the volume of cardiomyocytes and provide cardiomyocytes with ATP. Perturbations of mitochondria therefore have a detrimental effect on the heart. Mitochondrial homeostasis in mammals is regulated in part through fusion and fission by outer mitochondrial membrane proteins mitofusin 1 and 2 (Mfn1 and Mfn2) and in Drosophila by the homolog dMfn. Mutations in Mfn2 show impaired contractile function in humans (hMfn), mice (mMfn) and RNAi suppression in Drosophila (dMfn). Additionally Mfn2, independently of Mfn1, tethers the SR to mitochondria allowing for efficient transfer of Ca2+ between the organelles. It is currently not known whether mitochondria-SR tethering is required for proper Ca2+ cycling in cardiomyocytes. We monitored calcium transients in heart tubes of live flies, using the genetically encoded calcium indicator (GECI) GCaMP3.0 to study dynamic Ca2+ oscillations. Intensity signals from GCaMP3.0 in fly heart tubes are comparable to isolated cardiomyocytes in their rate cycling amplitude, and their response to L-type calcium channel antagonist, nifedipine. Additionally, non-calcium responsive GFP does not produce a signal that mimics the Ca2+ oscillations observed with GCaMP3.0. Comparisons between Wt Drosophila hearts and cardiomyopathic RNAi dMfn hearts expressing GCaMP3.0 have normal amplitude and time to decay constant Ca2+ signals. This study shows that suppression of dMfn with RNAi in the fly heart tube leads to cardiomyopathy. Monitoring intercellular Ca2+ levels with the (GECI) GCaMP3.0 demonstrates that Ca2+ signaling is not affected when mitochondria-SR tethering is disrupted.

Regucalcin is broadly expressed in male reproductive tissues and is a new androgen-target gene in mammalian testis

Regucalcin (RGN) is a calcium (Ca(2)(+))-binding protein which regulates intracellular Ca(2)(+) homeostasis by modulating the activity of enzymes regulating Ca(2)(+) concentration and enhancing Ca(2)(+)-pumping activity. Several studies have described the pivotal role of proper Ca(2)(+) homeostasis regulation to spermatogenesis and male fertility. Recently, RGN was identified as a sex steroid-regulated gene in prostate and breast; however, a possible role of RGN in spermatogenesis has not been examined. In this study, the expression and localization of RGN in rat and human testis, and other rat reproductive tissues was analyzed. Moreover, we studied whether RGN protein was present in seminiferous tubule fluid (STF). Finally, we examined the effect of 5α-dihydrotestosterone (DHT) on the expression of Rgn mRNA in rat seminiferous tubules (SeT) cultured ex vivo. The results presented in this study show that RGN is expressed in Leydig and Sertoli cells, as well as in all types of germ cells of both rat and human testis. RGN is also expressed in rat prostate, epididymis, and seminal vesicles. Moreover, RGN protein is present in rat STF. The results also demonstrate that Rgn expression is age dependent in rat testis, and is upregulated by the non-aromatizable androgen DHT in rat SeT cultured ex vivo. Taken together, these findings indicate that Rgn is a novel androgen-target gene in rat testis and that it may have a role in male reproductive function, particularly in the control of spermatogenesis.

The otoferlin interactome in neurosensory hair cells: Significance for synaptic vesicle release and trans-Golgi network (Review)

Sound perception in terrestrial vertebrates relies on a structure in the inner ear consisting of the utriculus, sacculus and lagena. In mammals, the lagena has developed into the cochlea where mechanotransduction at ciliated cells leads to ion influx via regulated ion channels. To maintain proper Ca2+ concentration many cellular systems use a variety of functional proteins; the neurosensory systems use calcium-sensors like hippocalcin, visinin or recoverin. In cochlear hair cells the 230 kDa protein otoferlin has been suggested to play this role. While several observations support this hypothesis additional data argue for a more expanded functional profile of otoferlin. Evidence for otoferlin's multiple roles and newer results on otoferlin's interacting partners are presented and the existence of a protein complex as a functional unit ('interactome') in the cochlea and further tissues is suggested.

Heterogeneous N-Terminal Acylation of Retinal Proteins Results from the Retina’s Unusual Lipid Metabolism

Protein N-myristoylation occurs by a covalent attachment of a C14:0 fatty acid to the N-terminal Gly residue. This reaction is catalyzed by a N-myristoyltransferase that uses myristoyl-coenzyme A as substrate. But proteins in the retina also undergo heterogeneous N-acylation with C14:2, C14:1, and C12:0 fatty acids. The basis and the role of this retina-specific phenomenon are poorly understood. We studied guanylate cyclase-activating protein 1 (GCAP1) as an example of retina-specific heterogeneously N-acylated protein. The types and the abundance of fatty acids bound to bovine retinal GCAP1 were C14:2, 37.0%; C14:0, 32.4%; C14:1, 22.3%; and C12:0, 8.3% as quantified by liquid chromatography coupled mass spectrometry. We also devised a method for N-acylating proteins in vitro and used it to modify GCAP1 with acyl moieties of different lengths. Analysis of these GCAPs both confirmed that N-terminal acylation of GCAP1 is critical for its high activity and proper Ca(2+)-dependent response and revealed comparable functionality for GCAP1 with acyl moieties of various lengths. We also tested the hypothesis that retinal heterogeneous N-acylation results from retinal enrichment of unusual N-myristoyltransferase substrates. Thus, acyl-coenzyme A esters were purified from both bovine retina and brain and analyzed by liquid chromatography coupled mass spectrometry. Substantial differences in acyl-coenzyme A profiles between the retina and brain were detected. Importantly, the ratios of uncommon N-acylation substrates--C14:2- and C14:1-coenyzme A to C14:0-coenzyme A--were higher in the retina than in the brain. Thus, our results suggest that heterogeneous N-acylation, responsible for expansion of retinal proteome, reflects the unique character of retinal lipid metabolism. Additionally, we propose a new hypothesis explaining the physiological relevance of elevated retinal ratios of C14:2- and C14:1-coenzyme A to C14:0-coenzyme A.

STIM1 as a key regulator for Ca2+ homeostasis in skeletal-muscle development and function

Stromal interaction molecules (STIM) were identified as the endoplasmic-reticulum (ER) Ca2+ sensor controlling store-operated Ca2+ entry (SOCE) and Ca2+-release-activated Ca2+ (CRAC) channels in non-excitable cells. STIM proteins target Orai1-3, tetrameric Ca2+-permeable channels in the plasma membrane. Structure-function analysis revealed the molecular determinants and the key steps in the activation process of Orai by STIM. Recently, STIM1 was found to be expressed at high levels in skeletal muscle controlling muscle function and properties. Novel STIM targets besides Orai channels are emerging.Here, we will focus on the role of STIM1 in skeletal-muscle structure, development and function. The molecular mechanism underpinning skeletal-muscle physiology points toward an essential role for STIM1-controlled SOCE to drive Ca2+/calcineurin/nuclear factor of activated T cells (NFAT)-dependent morphogenetic remodeling programs and to support adequate sarcoplasmic-reticulum (SR) Ca2+-store filling. Also in our hands, STIM1 is transiently up-regulated during the initial phase of in vitro myogenesis of C2C12 cells. The molecular targets of STIM1 in these cells likely involve Orai channels and canonical transient receptor potential (TRPC) channels TRPC1 and TRPC3. The fast kinetics of SOCE activation in skeletal muscle seem to depend on the triad-junction formation, favoring a pre-localization and/or pre-formation of STIM1-protein complexes with the plasma-membrane Ca2+-influx channels. Moreover, Orai1-mediated Ca2+ influx seems to be essential for controlling the resting Ca2+ concentration and for proper SR Ca2+ filling. Hence, Ca2+ influx through STIM1-dependent activation of SOCE from the T-tubule system may recycle extracellular Ca2+ losses during muscle stimulation, thereby maintaining proper filling of the SR Ca2+ stores and muscle function. Importantly, mouse models for dystrophic pathologies, like Duchenne muscular dystrophy, point towards an enhanced Ca2+ influx through Orai1 and/or TRPC channels, leading to Ca2+-dependent apoptosis and muscle degeneration. In addition, human myopathies have been associated with dysfunctional SOCE. Immunodeficient patients harboring loss-of-function Orai1 mutations develop myopathies, while patients suffering from Duchenne muscular dystrophy display alterations in their Ca2+-handling proteins, including STIM proteins. In any case, the molecular determinants responsible for SOCE in human skeletal muscle and for dysregulated SOCE in patients of muscular dystrophy require further examination.

Coupling and competition between ferroelectric and antiferroelectric states in Ca-doped Sr0.9−xBa0.1CaxTiO3: Multipolar states

We prepare a series of polycrystalline Sr(0.9-x)Ba(0.1)Ca(x)TiO(3) (SBCT) with relatively low Ba- and Ca-doping levels, and their microstructural, ferroelectric, dielectric, and lattice dynamic properties are investigated. It is surprisingly found that a proper Ca(2+) doping, which usually favors the antiferroelectric state in SrTiO(3), can remarkably enhance the ferroelectricity of SBCT. The coupling and competition between the ferroelectric state induced by Ba(2+) doping and antiferroelectric state induced by Ca(2+) doping lead to a sequence of multipolarized states, such as the enhanced polarization state, the cooperative polarization state, and the antiferroelectric state.

Non-Proteolytic Functions of Calpain-3 in Sarcoplasmic Reticulum in Skeletal Muscles

Mutations in CAPN3/Capn3, which codes for skeletal muscle-specific calpain-3/p94 protease, are responsible for limb-girdle muscular dystrophy type 2A. Using “knock-in” (referred to as Capn3 CS/CS ) mice, in which the endogenous calpain-3 is replaced with a mutant calpain-3:C129S, which is a proteolytically inactive but structurally intact calpain-3, we demonstrated in our previous studies that loss of calpain-3 protease activity causes muscular dystrophy [Ojima, K. et al. (2010) J. Clin. Invest. 120, 2672–2683]. However, compared to Capn3-null ( Capn3 −/− ) mice, Capn3 CS/CS mice showed less severe dystrophic symptoms. This suggests that calpain-3 also has a non-proteolytic function. This study aimed to elucidate the non-proteolytic functions of calpain-3 through comparison of Capn3 CS/CS mice with Capn3 −/− mice. We found that calpain-3 is a component of the sarcoplasmic reticulum (SR), and that calpain-3 interacts with, but does not proteolyze, typical SR components such as ryanodine receptor and calsequestrin. Furthermore, Capn3 CS/CS mice showed that the nonenzymatic role of calpain-3 is required for proper Ca 2+ efflux from the SR to cytosol during muscle contraction. These results indicate that calpain-3 functions as a nonenzymatic element for the Ca 2+ efflux machinery in the SR, rather than as a protease. Thus, defects in the nonenzymatic function of calpain-3 must also be involved in the pathogenesis of limb-girdle muscular dystrophy type 2A.

Adequate calcium nutrition and quality of egg shell and bones in layers: Innovative approach

Calcium metabolism (Ca) in layers depends on many factors which can be divided into factors influencing the intake, absorption, retention and/or excretion of Ca. Varying of any of above mentioned factors presented and reviewed in this study, exhibit their impact on quality of bones in layers and egg shell quality. For the purpose of maintaining of the egg quality and quality of layer hens? bones, the influence of mentioned factors on production should be taken into consideration in the practice, and on that basis not only the proper diet Ca level determined, but also adequate source and size of Ca particles, as well as mutual balance with other nutrients in the diet, primarily phosphorus, vitamin D and some micro elements. Recent studies have the direction of finding the more complex approach to solving of the issues of egg shell quality, also bone quality and ensuring adequate nutrition for modern layer hybrids.

Emanuel Strehler’s work on calcium pumps and calcium signaling

Cells are equipped with mechanisms to control tightly the influx, efflux and resting level of free calcium (Ca(2+)). Inappropriate Ca(2+) signaling and abnormal Ca(2+) levels are involved in many clinical disorders including heart disease, Alzheimer's disease and stroke. Ca(2+) also plays a major role in cell growth, differentiation and motility; disturbances in these processes underlie cell transformation and the progression of cancer. Accordingly, research in the Strehler laboratory is focused on a better understanding of the molecular "toolkit" needed to ensure proper Ca(2+) homeostasis in the cell, as well as on the mechanisms of localized Ca(2+) signaling. A long-term focus has been on the plasma membrane calcium pumps (PMCAs), which are linked to multiple disorders including hearing loss, neurodegeneration, and heart disease. Our work over the past 20 years or more has revealed a surprising complexity of PMCA isoforms with different functional characteristics, regulation, and cellular localization. Emerging evidence shows how specific PMCAs contribute not only to setting basal intracellular Ca(2+) levels, but also to local Ca(2+) signaling and vectorial Ca(2+) transport. A second major research area revolves around the calcium sensor protein calmodulin and an enigmatic calmodulin-like protein (CALML3) that is linked to epithelial differentiation. One of the cellular targets of CALML3 is the unconventional motor protein myosin-10, which raises new questions about the role of CALML3 and myosin-10 in cell adhesion and migration in normal cell differentiation and cancer.