Atypical cell death and insufficient matrix organization in long-bone growth plates from Tric-b-knockout mice.

TRIC-A and TRIC-B proteins form homotrimeric cation-permeable channels in the endoplasmic reticulum (ER) and nuclear membranes and are thought to contribute to counterionic flux coupled with store Ca2+ release in various cell types. Serious mutations in the TRIC-B (also referred to as TMEM38B) locus cause autosomal recessive osteogenesis imperfecta (OI), which is characterized by insufficient bone mineralization. We have reported that Tric-b-knockout mice can be used as an OI model; Tric-b deficiency deranges ER Ca2+ handling and thus reduces extracellular matrix (ECM) synthesis in osteoblasts, leading to poor mineralization. Here we report irregular cell death and insufficient ECM in long-bone growth plates from Tric-b-knockout embryos. In the knockout growth plate chondrocytes, excess pro-collagen fibers were occasionally accumulated in severely dilated ER elements. Of the major ER stress pathways, activated PERK/eIF2α (PKR-like ER kinase/ eukaryotic initiation factor 2α) signaling seemed to inordinately alter gene expression to induce apoptosis-related proteins including CHOP (CCAAT/enhancer binding protein homologous protein) and caspase 12 in the knockout chondrocytes. Ca2+ imaging detected aberrant Ca2+ handling in the knockout chondrocytes; ER Ca2+ release was impaired, while cytoplasmic Ca2+ level was elevated. Our observations suggest that Tric-b deficiency directs growth plate chondrocytes to pro-apoptotic states by compromising cellular Ca2+-handling and exacerbating ER stress response, leading to impaired ECM synthesis and accidental cell death.

Optogenetic Control of Endoplasmic Reticulum-Mitochondria Tethering.

The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER-mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER-mitochondrial contacts.

Mice lacking the intracellular cation channel TRIC-B have compromised collagen production and impaired bone mineralization.

The trimeric intracellular cation (TRIC) channels TRIC-A and TRIC-B localize predominantly to the endoplasmic reticulum (ER) and likely support Ca(2+) release from intracellular stores by mediating cationic flux to maintain electrical neutrality. Deletion and point mutations in TRIC-B occur in families with autosomal recessive osteogenesis imperfecta. Tric-b knockout mice develop neonatal respiratory failure and exhibit poor bone ossification. We investigated the cellular defect causing the bone phenotype. Bone histology indicated collagen matrix deposition was reduced in Tric-b knockout mice. Osteoblasts, the bone-depositing cells, from Tric-b knockout mice exhibited reduced Ca(2+) release from ER and increased ER Ca(2+) content, which was associated with ER swelling. These cells also had impaired collagen release without a decrease in collagen-encoding transcripts, consistent with a defect in trafficking of collagen through ER. In contrast, osteoclasts, the bone-degrading cells, from Tric-b knockout mice were similar to those from wild-type mice. Thus, TRIC-B function is essential to support the production and release of large amounts of collagen by osteoblasts, which is necessary for bone mineralization.

Generation of stomach tissue from mouse embryonic stem cells.

Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, ß-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

Mitsugumin 56 (hedgehog acyltransferase-like) is a sarcoplasmic reticulum-resident protein essential for postnatal muscle maturation.

Mitsugumin 56 (MG56), also known as the membrane-bound O-acyl-transferase family member hedgehog acyltransferase-like, was identified as a new sarcoplasmic reticulum component in striated muscle. Mg56-knockout mice grew normally for a week after birth, but shortly thereafter exhibited a suckling defect and died under starvation conditions. In the knockout skeletal muscle, regular contractile features were largely preserved, but sarcoplasmic reticulum elements swelled and further developed enormous vacuoles. In parallel, the unfolded protein response was severely activated in the knockout muscle, and presumably disrupted muscle development leading to the suckling failure. Therefore, MG56 seems essential for postnatal skeletal muscle maturation.

Lysosomal two-pore channel subtype 2 (TPC2) regulates skeletal muscle autophagic signaling.

Postnatal skeletal muscle mass is regulated by the balance between anabolic protein synthesis and catabolic protein degradation, and muscle atrophy occurs when protein homeostasis is disrupted. Autophagy has emerged as critical in clearing dysfunctional organelles and thus in regulating protein turnover. Here we show that endolysosomal two-pore channel subtype 2 (TPC2) contributes to autophagy signaling and protein homeostasis in skeletal muscle. Muscles derived from Tpcn2(-/-) mice exhibit an atrophic phenotype with exacerbated autophagy under starvation. Compared with wild types, animals lacking TPC2 demonstrated an enhanced autophagy flux characterized by increased accumulation of autophagosomes upon combined stress induction by starvation and colchicine treatment. In addition, deletion of TPC2 in muscle caused aberrant lysosomal pH homeostasis and reduced lysosomal protease activity. Association between mammalian target of rapamycin and TPC2 was detected in skeletal muscle, allowing for appropriate adjustments to cellular metabolic states and subsequent execution of autophagy. TPC2 therefore impacts mammalian target of rapamycin reactivation during the process of autophagy and contributes to maintenance of muscle homeostasis.

Contribution of calumin to embryogenesis through participation in the endoplasmic reticulum-associated degradation activity.

Calumin is an endoplasmic reticulum (ER)-transmembrane protein, and little is known about its physiological roles. Here we showed that calumin homozygous mutant embryos die at embryonic days (E) 10.5-11.5. At mid-gestation, calumin was expressed predominantly in the yolk sac. Apoptosis was enhanced in calumin homozygous mutant yolk sacs at E9.5, pointing to a possible link to the embryonic lethality. Calumin co-immunoprecipitated with ERAD components such as p97, BIP, derlin-1, derlin-2 and VIMP, suggesting its involvement in ERAD. Indeed, calumin knockdown in HEK 293 cells resulted in ERAD being less efficient, as demonstrated by attenuation in both degradations of a misfolded α1-antitrypsin variant and the ER-to-cytosol dislocation of cholera toxin A1 subunit. In calumin homozygous mutant yolk sac endoderm cells, ER stress-associated alterations were observed, including lipid droplet accumulation, fragmentation of the ER and dissociation of ribosomes from the ER. In this context, the ER-overload response, assumed to be cytoprotective, was also triggered in the mutant endoderm cells, but seemed to fully counteract the excessive ER stress generated due to defective ERAD. Taken together, our findings suggested that calumin serves to maintain the yolk sac integrity through participation in the ERAD activity, contributing to embryonic development.

Prohibitin 2 regulates the proliferation and lineage-specific differentiation of mouse embryonic stem cells in mitochondria.

BACKGROUND: The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.

Trimeric intracellular cation channels and sarcoplasmic/endoplasmic reticulum calcium homeostasis.

Trimeric intracellular cation channels (TRIC) represents a novel class of trimeric intracellular cation channels. Two TRIC isoforms have been identified in both the human and the mouse genomes: TRIC-A, a subtype predominantly expressed in the sarcoplasmic reticulum (SR) of muscle cells, and TRIC-B, a ubiquitous subtype expressed in the endoplasmic reticulum (ER) of all tissues. Genetic ablation of either TRIC-A or TRIC-B leads to compromised K(+) permeation and Ca(2+) release across the SR/ER membrane, supporting the hypothesis that TRIC channels provide a counter balancing K(+) flux that reduces SR/ER membrane depolarization for maintenance of the electrochemical gradient that drives SR/ER Ca(2+) release. TRIC-A and TRIC-B seem to have differential functions in Ca(2+) signaling in excitable and nonexcitable cells. Tric-a(-/-) mice display defective Ca(2+) sparks and spontaneous transient outward currents in arterial smooth muscle and develop hypertension, in addition to skeletal muscle dysfunction. Knockout of TRIC-B results in abnormal IP3 receptor-mediated Ca(2+) release in airway epithelial cells, respiratory defects, and neonatal lethality. Double knockout mice lacking both TRIC-A and TRIC-B show embryonic lethality as a result of cardiac arrest. Such an aggravated lethality indicates that TRIC-A and TRIC-B share complementary physiological functions in Ca(2+) signaling in embryonic cardiomyocytes. Tric-a(-/-) and Tric-b(+/-) mice are viable and susceptible to stress-induced heart failure. Recent evidence suggests that TRIC-A directly modulates the function of the cardiac ryanodine receptor 2 Ca(2+) release channel, which in turn controls store-overload-induced Ca(2+) release from the SR. Thus, the TRIC channels, in addition to providing a countercurrent for SR/ER Ca(2+) release, may also function as accessory proteins that directly modulate the ryanodine receptor/IP3 receptor channel functions.

N-Cadherin is a prospective cell surface marker of human mesenchymal stem cells that have high ability for cardiomyocyte differentiation.

Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. When the cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as α-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation.

Facilitated hyperpolarization signaling in vascular smooth muscle-overexpressing TRIC-A channels.

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca(2+) release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca(2+) release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca(2+) transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca(2+) sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca(2+) sparks were facilitated and cell surface Ca(2+)-dependent K(+) channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca(2+) channels were inactivated, and thus, the resting intracellular Ca(2+) levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca(2+) signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.

Larval bullfrog skin lacks amiloride-blockable epithelial transport because α-ENaC is located within intracellular vesicles in epidermal apical cells and not in the apical plasma membrane.

The epithelial Na channel (ENaC) plays an essential role in sodium transport across epithelia such as adult frog skin. Transport across the skin, measured as short-circuit current (SCC), is blocked by amiloride. Bullfrog alpha-ENaC (α-fENaC) is expressed in adult bullfrog skin, and the SCC across this skin is blocked by amiloride. In contrast, an amiloride-blockable SCC is not detected in larval bullfrog skin, even though it expresses α-fENaC. We examined the subcellular localization of α-ENaC in such larval and adult skins. Immunofluorescent and immunoelectron microscopy of apical cells in the larval epidermis revealed α-fENaC localization within intracellular vesicles, but not in the plasma membrane. In contrast, in adult skin α-fENaC was localized to the apical-side membrane and to intracellular vesicles in Stratum granulosum cells. This may support the view that amiloride-blockable SCC is absent from larval skin, but is present in adult skin.

Orai1 mediates exacerbated Ca(2+) entry in dystrophic skeletal muscle.

There is substantial evidence indicating that disruption of Ca(2+) homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca(2+) deregulation and the Ca(2+) signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca(2+) entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca(2+) storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca(2+) storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca(2+) homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca(2+) store contributes to the disrupted Ca(2+) homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.

Disrupted membrane structure and intracellular Ca²âº signaling in adult skeletal muscle with acute knockdown of Bin1.

Efficient intracellular Ca²âº ([Ca²âº]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca²âº homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short -hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca²âº release. Voltage-induced Ca²âº released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca²âº current and SR Ca²âº transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca²âº sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca²âº sparks' amplitude that was attributed to decreased total Ca²âº stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca²âº]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.

TRIC-A channels in vascular smooth muscle contribute to blood pressure maintenance.

TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.

A versatile single-plasmid system for tissue-specific and inducible control of gene expression in transgenic mice.

We describe a novel transgenic system for tissue-specific and inducible control of gene expression in mice. The system employs a tetracycline-responsive CMV promoter that controls transcription of a short-hairpin RNA (shRNA) that remains nonfunctional until an interrupting reporter cassette is excised by Cre recombinase. Insertion of Dicer and Drosha RNase processing sites within the shRNA allows generation of siRNA to knock down a target gene efficiently. Tissue-specific shRNA expression is achieved through the use of appropriate inducer mice with tissue-specific expression of Cre. We applied this system to regulate expression of junctophilins (JPs), genes essential for maintenance of membrane ultrastructure and Ca(2+) signaling in muscle. Transgenic mice with skeletal muscle-specific expression of shRNA against JP mRNAs displayed no basal change of JP expression before treatment with doxycycline (Dox), while inducible and reversible knockdown of JPs was achieved by feeding mice with Dox-containing water. Dox-induced knockdown of JPs led to abnormal junctional membrane structure and Ca(2+) signaling in adult muscle fibers, consistent with essential roles of JPs in muscle development and function. This transgenic approach can be applied for inducible and reversible gene knockdown or gene overexpression in many different tissues, thus providing a versatile system for elucidating the physiological gene function in viable animal models.

Development of Ca²(+) signaling mechanisms and cell motility in presumptive ectodermal cells during amphibian gastrulation.

This study investigated the development of Ca²(+) signaling mechanisms and their role in initiating morphogenetic cell movement in the presumptive ectoderm of Japanese newt (Cynops pyrrhogaster) during gastrulation. Histochemical staining using fluorescently labeled ryanodine and dihydropyridine probes revealed that dihydropyridine receptor (L-type Ca²(+) channels) appeared in stage 12b embryos, while ryanodine receptors were expressed in both stage 11 and 12b embryos. Transmission electron microscopy of stage 12b embryos showed abundant peripheral couplings, which are couplings of the endoplasmic reticulum and cell membrane with an approximate 12 nm gap. Caffeine increased the intracellular free Ca²(+) concentration ([Ca²(+)](i)) in presumptive ectodermal cells isolated from both stage 11 and 12b embryos, while (±)-Bay K 8644 ((±)-BayK) increased [Ca²(+)](i) in cells isolated from stage 12b embryos, but not in cells isolated from stage 11 embryos. Dantrolene and nifedipine completely inhibited increases in [Ca²(+)](i) after treatment with caffeine and (±)-BayK, respectively. Caffeine activated the motility of cells isolated from both stage 11 and 12b embryos, but (±)-BayK only activated the motility of cells isolated from stage 12b embryos. These findings suggested that formation of the Ca²(+) -induced Ca²(+) release system in presumptive ectodermal cells during gastrulation plays an important role in the initiation and execution of epibolic extension.

Claudin5 genes encoding tight junction proteins are required for Xenopus heart formation.

Claudin proteins are the major components of tight junctions connecting adjacent cells, where they regulate a variety of cellular activities. In the present paper we identified two Xenopus claudin5 genes (cldn5a and 5b), which are expressed early in the developing cardiac region. Precocious cldn5 expression was observed in explants of non-heart-forming mesoderm under inhibition of the canonical Wnt pathway. Cardiogenesis was severely perturbed by antisense oligonucleotides against cldn5 or by Cldn5 proteins lacking the cytoplasmic domain. Results of light- and electron-microscopic observations suggested that cldn5a and 5b are required for Xenopus heart tube formation through epithelialization of the precardiac mesoderm.

Ca2+ overload and sarcoplasmic reticulum instability in tric-a null skeletal muscle.

The sarcoplasmic reticulum (SR) of skeletal muscle contains K(+), Cl(-), and H(+) channels may facilitate charge neutralization during Ca(2+) release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca(2+) release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a(-/-) skeletal muscle showed Ca(2+) overload inside the SR with frequent formation of Ca(2+) deposits compared with the wild type muscle. This elevated SR Ca(2+) pool in the tric-a(-/-) muscle could be released by caffeine, whereas the elemental Ca(2+) release events, e.g. osmotic stress-induced Ca(2+) spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of "alternan" behavior with isolated tric-a(-/-) skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca(2+ ATPase) function could lead to aggravation of the stress-induced alternans in the tric-a(-/-) muscle. Our data suggests that absence of TRIC-A may lead to Ca(2+) overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca(2+) movement across the SR membrane. The observed alternan behavior with the tric-a(-/-) muscle may reflect a skeletal muscle version of store overload-induced Ca(2+) release that has been reported in the cardiac muscle under stress conditions.

Inhibitory Smad proteins promote the differentiation of mouse embryonic stem cells into ependymal-like ciliated cells.

Motile cilia play crucial roles in the maintenance of homeostasis in vivo. Defects in the biosynthesis of cilia cause immotile cilia syndrome, also known as primary ciliary dyskinesia (PCD), which is associated with a variety of complex diseases. In this study, we found that inhibitory Smad proteins, Smad7 and Smad6, significantly promoted the differentiation of mouse embryonic stem (ES) cells into ciliated cells. Moreover, these Smad proteins specifically induced morphologically distinct Musashi1-positive ciliated cells. These results suggest that inhibitory Smad proteins could be important regulators not only for the regulation of ciliated cell differentiation, but also for the subtype specification of ciliated cells during differentiation from mouse ES cells.