Calcineurin Activity Is Required for the Initiation of Skeletal Muscle Differentiation

Differentiation of skeletal muscle myoblasts follows an ordered sequence of events: commitment, cell cycle withdrawal, phenotypic differentiation, and finally cell fusion to form multinucleated myotubes. The molecular signaling pathways that regulate the progression are not well understood. Here we investigate the potential role of calcium and the calcium-dependent phosphatase calcineurin in myogenesis. Commitment, phenotypic differentiation, and cell fusion are identified as distinct calcium-regulated steps, based on the extracellular calcium concentration required for the expression of morphological and biochemical markers specific to each of these stages. Furthermore, differentiation is inhibited at the commitment stage by either treatment with the calcineurin inhibitor cyclosporine A (CSA) or expression of CAIN, a physiological inhibitor of calcineurin. Retroviral-mediated gene transfer of a constitutively active form of calcineurin is able to induce myogenesis only in the presence of extracellular calcium, suggesting that multiple calcium-dependent pathways are required for differentiation. The mechanism by which calcineurin initiates differentiation includes transcriptional activation of myogenin, but does not require the participation of NFAT. We conclude that commitment of skeletal muscle cells to differentiation is calcium and calcineurin-dependent, but NFAT-independent.

Download Full-text

MO092THE ROLE OF CALCIUM IN UROMODULIN EXPRESSION AND SECRETION FROM RENAL MEDULLARY EPITHELIAL CELLS OF HYPERTENSIVE AND NORMOTENSIVE RATS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab106.001 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Philipp Boder ◽

Sheon Mary ◽

Lesley Graham ◽

Christian Delles

Keyword(s):

Blood Pressure ◽

Epithelial Cells ◽

Mrna Level ◽

Extracellular Calcium ◽

Female Rats ◽

Thick Ascending Limb ◽

Molecular Signaling ◽

Extracellular Secretion ◽

Wistar Kyoto

Abstract Background and Aims Uromodulin (UMOD) is the most abundantly secreted protein found within the urine, primarily produced by medullary thick ascending limb (mTAL) epithelial cells of the kidneys. There is accruing genetic evidence implicating UMOD in blood pressure regulation and consequently hypertension. The molecular signaling induced by calcium in the kidney and its influence on blood pressure are not well understood. The aim of this study was to investigate the potential role of extracellular calcium and the calcium-sensing receptor (CaSR) in mTAL on UMOD production and secretion in TAL cells with the hope of defining novel clinical targets for the treatment of hypertension. Method Kidneys were harvested from normotensive Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) female rats. To determine the effect of extracellular calcium on UMOD secretion, mTAL tubules were incubated in media with and without 1mM calcium, nifedipine (10µM), NPS2143 (1 or 5 µM) and spermine (2mM). Extracellular and intracellular UMOD protein levels were detected by Western blot. Gene expression of Umod was determined by qRT-PCR. Results Calcium increased mTAL tubule UMOD secretion in WKY and SHRSP. Nifedipine slightly decreased UMOD secretion in WKY without calcium. In both strains, NPS2143 increased calcium-induced UMOD secretion, with an enhanced effect in SHRSP. Stimulation of CaSR with spermine decreased UMOD secretion in WKY. Analysis of intracellular UMOD levels in these conditions demonstrated increased accumulation when extracellular secretion was low, and vice versa. Incubation of primary mTAL cells with calcium confirmed increased localisation of UMOD at the membrane compared to the cytosol, without any major differences in cell morphology. The Umod mRNA level changes were not statistically significant among conditions. Conclusion Trafficking of UMOD in the mTAL is influenced by the type of CaSR ligand and the biased nature of G-protein coupled CaSR signalling. Unravelling the signalling events post-calcium will be necessary for identification of key regulators of UMOD secretion and provide new sites for therapeutic intervention in hypertension.

Download Full-text

FGF-2–dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021013118 ◽

2021 ◽

Vol 118 (37) ◽

pp. e2021013118 ◽

Cited By ~ 1

Author(s):

Sebastian Mathes ◽

Alexandra Fahrner ◽

Umesh Ghoshdastider ◽

Hannes A. Rüdiger ◽

Michael Leunig ◽

...

Keyword(s):

Skeletal Muscle ◽

Transcriptional Activation ◽

Human Skeletal Muscle ◽

Muscle Growth ◽

Muscle Cells ◽

Adeno Associated Virus ◽

Adult Skeletal Muscle

Aged skeletal muscle is markedly affected by fatty muscle infiltration, and strategies to reduce the occurrence of intramuscular adipocytes are urgently needed. Here, we show that fibroblast growth factor-2 (FGF-2) not only stimulates muscle growth but also promotes intramuscular adipogenesis. Using multiple screening assays upstream and downstream of microRNA (miR)-29a signaling, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and adeno-associated virus–mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular adipose tissue (IMAT) formation in vivo. Skeletal muscle from human donors aged >75 y versus <55 y showed activation of FGF-2–dependent signaling and increased IMAT. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a pathway to combat fat accumulation in aged human skeletal muscle.

Download Full-text

Extracellular Calcium-Dependent Modulation of Endothelium Relaxation in Rat Mesenteric Small Artery: The Role of Potassium Signaling

BioMed Research International ◽

10.1155/2015/758346 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Lise Hangaard ◽

Peter B. Jessen ◽

Dmitrii Kamaev ◽

Christian Aalkjaer ◽

Vladimir V. Matchkov

Keyword(s):

Extracellular Calcium ◽

Small Artery ◽

Small Arteries ◽

Calcium Dependent ◽

Dependent Pathway

The nature of NO- and COX-independent endothelial hyperpolarization (EDH) is not fully understood but activation of small- and intermittent-conductance Ca2+-activated K+channels (SKCaandIKCa) is important. Previous studies have suggested that the significance ofIKCadepends onCa2+out. Also it has been suggested that K+is important through localizedK+outsignaling causing activation of the Na+,K+-ATPase and inward-rectifying K+channels (Kir). Here we tested the hypothesis that the modulating effect ofCa2+outon the EDH-like response depends onK+out. We addressed this possibility using isometric myography of rat mesenteric small arteries. WhenK+outwas 4.2 mM, relaxation to acetylcholine (ACh) was stronger at 2.5 mMCa2+outthan at 1 mMCa2+out. Inhibition ofIKCawith TRAM34 suppressed the relaxations but did not change the relation between the relaxations at the low and highCa2+out. ThisCa2+out-dependence disappeared at 5.9 mMK+outand in the presence of ouabain or BaCl2. Our results suggest thatIKCaare involved in the localizedK+outsignaling which acts through the Na+,K+-ATPase andKirchannels and that the significance of this endothelium-dependent pathway is modulated byCa2+out.

Download Full-text

Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00233-14 ◽

2015 ◽

Vol 14 (3) ◽

pp. 265-277 ◽

Cited By ~ 6

Author(s):

Javier Palma-Guerrero ◽

Jiuhai Zhao ◽

A. Pedro Gonçalves ◽

Trevor L. Starr ◽

N. Louise Glass

Keyword(s):

Neurospora Crassa ◽

Somatic Cell ◽

Cell Fusion ◽

Cell Lysis ◽

Extracellular Calcium ◽

Data Set ◽

Content Type ◽

Calcium Dependent ◽

Somatic Cell Fusion ◽

Dependent Cell

ABSTRACTThe molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungusNeurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. InN. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1or Δlfd-1germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, forlatefusiondefect-2) that showed a calcium-dependent cell lysis phenotype.lfd-2encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter,fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion.

Download Full-text

Glucose utilization in heart, diaphragm and skeletal muscle during the fed-to-starved transition

Biochemical Journal ◽

10.1042/bj2700245 ◽

1990 ◽

Vol 270 (1) ◽

pp. 245-249 ◽

Cited By ~ 34

Author(s):

M J Holness ◽

M C Sugden

Keyword(s):

Skeletal Muscle ◽

Fatty Acid ◽

Glucose Uptake ◽

Glucose Utilization ◽

Active Form ◽

Rapid Decline ◽

Acute Elevation ◽

Glucose Fatty Acid Cycle ◽

Skeletal Muscle Glucose Uptake

The progressive effects of starvation on muscle glucose utilization were studied in the conscious resting rat. High rates of glucose uptake and phosphorylation in constantly working cardiothoracic (heart, diaphragm) and postural skeletal muscles (soleus, adductor longus) were maintained for at least 9 h of starvation. A rapid decline in cardiac glucose utilization was observed during the period 9-24 h of starvation, but for the other muscles the decline was more gradual. Consequently, even after 24 h, rates of glucose utilization in these muscles remained quantitatively significant. In both cardiothoracic and working (postural) skeletal muscle, glucose uptake and phosphorylation and activity of the active form of pyruvate dehydrogenase exhibited differential sensitivities to starvation and also to acute elevation of fatty acid concentrations during acute (4-9 h) starvation, such that pyruvate oxidation was more rapidly suppressed than glucose uptake and phosphorylation. The results are discussed in relation to the role of the glucose/fatty acid cycle in glucose conservation during the fed-to-starved transition.

Download Full-text