Comparative analysis of fat composition in marrow, serum, and muscle from aging C57BL6 mice.

Osteosarcopenia is an age-related condition characterized by fragile bone and low muscle mass and function. Fat infiltration concomitantly contributes to age-related bone and muscle decline. Fat-secreted factors could be locally secreted in the muscle and bone marrow milieu affecting cell function and survival. However, the specific fat-related secretory factors that may simultaneously affect those tissues remain unknown. Using targeted-lipidomics approach, we comprehensively quantified fat composition (lipid mediators [LMs]) in bone marrow flush, gastrocnemius and serum obtained from 6-, 24- and 42-week-old C57BL6 mice. Compared to young mice (6wks), all tissues in older mice showed significantly higher levels of arachidonic acid (AA) and AA-derived eicosanoids, PGA 2, TXB 2, and 11,12-EET, which are known to affect muscle and bone function. Moreover, Lipoxin B4, another AA product and an enhancer of bone turnover and negative regulator for muscle, showed significantly lower values in older mice compared to young mice in both genders. Furthermore, eicosapentaenoic acid and docosahexaenoic acid autoxidation products (20-HDoHE, 11-HDoHE, 7-HDoHE and 4-HDoHE), and omega-3 fatty acids that negatively regulate bone and muscle health, were significantly higher in older mice. In conclusion, these results suggest that LMs could play a role in modulating musculoskeletal function during aging.

Numb is required for optimal contraction of skeletal muscle.

BACKGROUND: The role of Numb, a protein that is important for cell fate and development and that, in human muscle, is expressed at reduced levels with advanced age, was investigated; adult mice skeletal muscle and its localization and function within myofibres were determined. METHODS: Numb expression was evaluated by western blot. Numb localization was determined by confocal microscopy. The effects of conditional knock out (cKO) of Numb and the closely related gene Numb-like in skeletal muscle fibres were evaluated by in situ physiology, transmission and focused ion beam scanning electron microscopy, three-dimensional reconstruction of mitochondria, lipidomics, and bulk RNA sequencing. Additional studies using primary mouse myotubes investigated the effects of Numb knockdown on cell fusion, mitochondrial function, and calcium transients. RESULTS: Numb protein expression was reduced by ~70% (P < 0.01) at 24 as compared with 3 months of age in gastrocnemius and tibialis anterior muscle. Numb was localized within muscle fibres as bands traversing fibres at regularly spaced intervals in close proximity to dihydropyridine receptors. The cKO of Numb and Numb-like reduced specific tetanic force by 36% (P < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z-line spacing by 30% (P < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (P < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1, and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acids through lipoxygenases were up-regulated in Numb cKO skeletal muscle: 12-HEPE was increased by ~250% (P < 0.05) and 17,18-EpETE by ~240% (P < 0.05). In mouse primary myotubes, Numb knockdown reduced cell fusion (~20%, P < 0.01) and delayed the caffeine-induced rise in cytosolic calcium concentrations by more than 100% (P < 0.01). CONCLUSIONS: These findings implicate Numb as a critical factor in skeletal muscle structure and function and suggest that Numb is critical for calcium release. We therefore speculate that Numb plays critical roles in excitation-contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.

Nampt activator P7C3 ameliorates diabetes and improves skeletal muscle function modulating cell metabolism and lipid mediators.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt), a key enzyme in NAD salvage pathway is decreased in metabolic diseases, and its precise role in skeletal muscle function is not known. We tested the hypothesis, Nampt activation by P7C3 (3,6-dibromo-α-[(phenylamino)methyl]-9H-carbazol-9-ethanol) ameliorates diabetes and muscle function. METHODS: We assessed the functional, morphometric, biochemical, and molecular effects of P7C3 treatment in skeletal muscle of type 2 diabetic (db/db) mice. Nampt+/- mice were utilized to test the specificity of P7C3. RESULTS: Insulin resistance increased 1.6-fold in diabetic mice compared with wild-type mice and after 4 weeks treatment with P7C3 rescued diabetes (P < 0.05). In the db-P7C3 mice fasting blood glucose levels decreased to 0.96-fold compared with C57Bl/6J wild-type naïve control mice. The insulin and glucose tolerance tests blood glucose levels were decreased to 0.6-fold and 0.54-folds, respectively, at 120 min along with an increase in insulin secretion (1.76-fold) and pancreatic ß-cells (3.92-fold) in db-P7C3 mice. The fore-limb and hind-limb grip strengths were increased to 1.13-fold and 1.17-fold, respectively, together with a 14.2-fold increase in voluntary running wheel distance in db-P7C3 mice. P7C3 treatment resulted in a 1.4-fold and 7.1-fold increase in medium-sized and larger-sized myofibres cross-sectional area, with a concomitant 0.5-fold decrease in smaller-sized myofibres of tibialis anterior (TA) muscle. The transmission electron microscopy images also displayed a 1.67-fold increase in myofibre diameter of extensor digitorum longus muscle along with 2.9-fold decrease in mitochondrial area in db-P7C3 mice compared with db-Veh mice. The number of SDH positive myofibres were increased to 1.74-fold in db-P7C3 TA muscles. The gastrocnemius and TA muscles displayed a decrease in slow oxidative myosin heavy chain type1 (MyHC1) myofibres expression (0.46-fold) and immunostaining (6.4-fold), respectively. qPCR analysis displayed a 2.9-fold and 1.3-fold increase in Pdk4 and Cpt1, and 0.55-fold and 0.59-fold decrease in Fgf21 and 16S in db-P7C3 mice. There was also a 3.3-fold and 1.9-fold increase in Fabp1 and CD36 in db-Veh mice. RNA-seq differential gene expression volcano plot displayed 1415 genes to be up-regulated and 1726 genes down-regulated (P < 0.05) in db-P7C3 mice. There was 1.02-fold increase in serum HDL, and 0.9-fold decrease in low-density lipoprotein/very low-density lipoprotein ratio in db-P7C3 mice. Lipid profiling of gastrocnemius muscle displayed a decrease in inflammatory lipid mediators n-6; AA (0.83-fold), and n-3; DHA (0.69-fold) and EPA (0.81-fold), and a 0.66-fold decrease in endocannabinoid 2-AG and 2.0-fold increase in AEA in db-P7C3 mice. CONCLUSIONS: Overall, we demonstrate that P7C3 activates Nampt, improves type 2 diabetes and skeletal muscle function in db/db mice.

The toxic effects of chloroquine and hydroxychloroquine on skeletal muscle: a systematic review and meta-analysis.

The aim of this systematic review was to perform qualitative and quantitative analysis on the toxic effects of chloroquine (CQ) and hydroxychloroquine (HCQ) on skeletal muscles. We designed the study according to PRISMA guidelines. Studies for qualitative and quantitative analyses were selected according to the following inclusion criteria: English language; size of sample (> 5 patients), adult (> age of 18) patients, treated with CQ/HCQ for inflammatory diseases, and presenting and not presenting with toxic effects on skeletal muscles. We collected data published from 1990 to April 2020 using PubMed, Cochrane Library, EMBASE, and SciELO. Risk of bias for observational studies was assessed regarding the ROBIN-I scale. Studies with less than five patients (case reports) were selected for an additional qualitative analysis. We used the software Comprehensive Meta-Analysis at the confidence level of 0.05. We identified 23 studies for qualitative analysis (17 case-reports), and five studies were eligible for quantitative analysis. From case reports, 21 patients presented muscle weakness and confirmatory biopsy for CQ/HCQ induced myopathy. From observational studies, 37 patients out of 1,367 patients from five studies presented muscle weakness related to the use of CQ/HCQ, and 252 patients presented elevated levels of muscle enzymes (aldolase, creatine phosphokinase, and lactate dehydrogenase). Four studies presented data on 34 patients with confirmatory biopsy for drug-induced myopathy. No study presented randomized samples. The chronic use of CQ/HCQ may be a risk for drug-induced myopathy. There is substantiated need for proper randomized trials and controlled prospective studies needed to assess the clinical and subclinical stages of CQ/HCQ -induced muscle myopathy.

A simple model of immune and muscle cell crosstalk during muscle regeneration.

Muscle injury during aging predisposes skeletal muscles to increased damage due to reduced regenerative capacity. Some of the common causes of muscle injury are strains, while other causes are more complex muscle myopathies and other illnesses, and even excessive exercise can lead to muscle damage. We develop a new mathematical model based on ordinary differential equations of muscle regeneration. It includes the interactions between the immune system, healthy and damaged myonuclei as well as satellite cells. Our new mathematical model expands beyond previous ones by accounting for 21 specific parameters, including those parameters that deal with the interactions between the damaged and dead myonuclei, the immune system, and the satellite cells. An important assumption of our model is the replacement of only damaged parts of the muscle fibers and the dead myonuclei. We conduce systematic sensitivity analysis to determine which parameters have larger effects on the model and therefore are more influential for the muscle regeneration process. We propose additional validation for these parameters. We further demonstrate that these simulations are species-, muscle-, and age-dependent. In addition, the knowledge of these parameters and their interactions, may suggest targeting or selecting these interactions for treatments that accelerate the muscle regeneration process.

A comparative study on silicon nitride, titanium and polyether ether ketone on mouse pre-osteoblast cells.

The current study provides more insights about the surface bioactivity of the silicon nitride (Si3N4) as a potential candidate for bone regeneration in craniofacial and orthopaedic applications compared with conventional implantation materials. Current skeletal reconstructive materials such as titanium and polyether ether ketone (PEEK) are limited by poor long-term stability, biocompatibility and prolonged healing. Si3N4 is an FDA-approved material for an intervertebral spacer in spinal fusion applications. It is biocompatible and has antimicrobial properties. Here, we hypothesize that Si3N4 was found to be an osteoconductive material and conducts the growth, differentiation of MC3T3-E1 cells for extracellular matrix deposition, mineralization and eventual bone regeneration for craniofacial and orthopaedic applications. MC3T3-E1 cells were used to study the osteoblastic differentiation and mineralization on sterile samples of Si3N4, titanium alloy and PEEK. The samples were then analysed for extracellular matrix deposition and mineralization by FTIR, Raman spectroscopy, SEM, EDX, Alizarin Red, qRT-PCR and ELISA. The in vitro study indicates the formation of collagen fibres and mineral deposition on all three sample surfaces. There was more profound and faster ECM deposition and mineralization on Si3N4 surface as compared to titanium and PEEK. The FTIR and Raman spectroscopy show formation of collagen and mineral deposition at 30 days for Si3N4 and titanium and not PEEK. The peaks shown by Raman for Si3N4 resemble closely to natural bone. Results also indicate the upregulation of osteogenic transcription factors such as RUNX2, SP7, collagen type I and osteocalcin. The authors concluded that Si3N4 rapidly conducts mineralized tissue formation via extracellular matrix deposition and biomarker expression in mouse calvarial pre-osteoblast cells. Thus, this study confirms that the bioactive Si3N4 could be a potential material for craniofacial and orthopaedic applications leading to rapid bone regeneration that resemble the natural bone structure.

New Surgical Model for Bone-Muscle Injury Reveals Age and Gender-Related Healing Patterns in the 5 Lipoxygenase (5LO) Knockout Mouse.

Signaling lipid mediators released from 5 lipoxygenase (5LO) pathways influence both bone and muscle cells, interfering in their proliferation and differentiation capacities. A major limitation to studying inflammatory signaling pathways in bone and muscle healing is the inadequacy of available animal models. We developed a surgical injury model in the vastus lateralis (VL) muscle and femur in 129/SvEv littermates mice to study simultaneous musculoskeletal (MSK) healing in male and female, young (3 months) and aged (18 months) WT mice compared to mice lacking 5LO (5LOKO). MSK defects were surgically created using a 1-mm punch device in the VA muscle followed by a 0.5-mm round defect in the femur. After days 7 and 14 post-surgery, the specimens were removed for microtomography (microCT), histopathology, and immunohistochemistry analyses. In addition, non-injured control skeletal muscles along with femur and L5 vertebrae were analyzed. Bones were microCT phenotyped, revealing that aged female WT mice presented reduced BV/TV and trabecular parameters compared to aged males and aged female 5LOKO mice. Skeletal muscles underwent a customized targeted lipidomics investigation for profiling and quantification of lipid signaling mediators (LMs), evidencing age, and gender related-differences in aged female 5LOKO mice compared to matched WT. Histological analysis revealed a suitable bone-healing process with osteoid deposition at day 7 post-surgery, followed by woven bone at day 14 post-surgery, observed in all young mice. Aged WT females displayed increased inflammatory response at day 7 post-surgery, delayed bone matrix maturation, and increased TRAP immunolabeling at day 14 post-surgery compared to 5LOKO females. Skeletal muscles of aged animals showed higher levels of inflammation in comparison to young controls at day 14 post-surgery; however, inflammatory process was attenuated in aged 5LOKO mice compared to aged WT. In conclusion, this new model shows that MSK healing is influenced by age, gender, and the 5LO pathway, which might serve as a potential target to investigate therapeutic interventions and age-related MSK diseases. Our new model is suitable for bone-muscle crosstalk studies.

Amorphous Silicon Oxynitrophosphide-Coated Implants Boost Angiogenic Activity of Endothelial Cells.

Lack of osteointegration is a major cause of aseptic loosening and failure of implants used in bone replacement. Implants coated with angiogenic biomaterials can improve osteointegration and potentially reduce these complications. Silicon- and phosphorus-based materials have been shown to upregulate expression of angiogenic factors and improve endothelial cell functions. In the present study, we hypothesize that implants coated with amorphous silica-based coatings in the form of silicon oxynitrophosphide (SiONP) by using plasma-enhanced chemical vapor deposition (PECVD) technique could enhance human umbilical vein endothelial cell angiogenic properties in vitro. The tested groups were: glass coverslip (GCS), tissue culture plate, SiON, SiONP1 (O: 7.3 at %), and SiONP2 (O: 14.2 at %) implants. The SiONP2 composition demonstrated 3.5-fold more fibronectin deposition than the GCS (p < 0.001). The SiONP2 group also presented a significant improvement in the capillary tubule length and thickness compared with the other groups (p < 0.01). At 24 h, we observed at least a twofold upregulation of vascular endothelial growth factor A, hypoxia-inducible factor-1α, angiopoietin-1, and nesprin-2, more evident in the SiONP1 and SiONP2 groups. In conclusion, the studied amorphous silica-coated implants, especially the SiONP2 composition, could enhance the endothelial cell angiogenic properties in vitro and may induce faster osteointegration and healing. Impact Statement In this study, we report for the first time the significant enhancement of human umbilical vein endothelial cell angiogenic properties (in vitro) by the amorphous silica-based coatings in the form of silicon oxynitrophosphide (SiONP). The SiONP2 demonstrated 3.5-fold more fibronectin deposition than the glass coverslip and presented a significant improvement in the capillary tubule length and thickness. At 24 h, SiONP reported twofold upregulation of vascular endothelial growth factor A, hypoxia-inducible factor-1α, angiopoietin-1, and nesprin-2. The studied amorphous silica-coated implants enhance the endothelial cell angiogenic properties in vitro and may induce faster osteointegration and healing.

Primum non nocere - Are chloroquine and hydroxychloroquine safe prophylactic/treatment options for SARS-CoV-2 (covid-19)?

ABSTRACT Chloroquine (CQ) and its analog hydroxychloroquine (HCQ) were recently included in several clinical trials as potential prophylactic and therapeutic options for SARS-COV-2 infection/covid-19. However, drug effectiveness in preventing, treating, or slowing the progression of the disease is still unknown. Despite some initial promising in vitro results, rigorous pre-clinical animal studies and randomized clinical trials have not been performed yet. On the other hand, while the potential effectiveness of CQ/HCQ is, at best, hypothetical, their side effects are factual and most worrisome, particularly when considering vulnerable groups of patients being treated with these drugs. in this comment, we briefly explain the possible mechanisms of action of CQ/HCQ for treating other diseases, possible actions against covid-19, and their potent side effects, in order to reinforce the necessity of evaluating the benefit-risk balance when widely prescribing these drugs for SARS-COV-2 infection/covid-19. We conclude by strongly recommending against their indiscriminate use.

ß-aminoisobutyric Acid, l-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor.

Exercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor ß-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of a bone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS). l-BAIBA was as or more protective than estrogen or N-acetyl cysteine, signaling through the Mas-Related G Protein-Coupled Receptor Type D (MRGPRD) to prevent the breakdown of mitochondria due to ROS. BAIBA supplied in drinking water prevented bone loss and loss of muscle function in the murine hindlimb unloading model, a model of osteocyte apoptosis. The protective effect of BAIBA was lost with age, not due to loss of the muscle capacity to produce BAIBA but likely to reduced Mrgprd expression with aging. This has implications for understanding the attenuated effect of exercise on bone with aging.

Targeted quantification of lipid mediators in skeletal muscles using restricted access media-based trap-and-elute liquid chromatography-mass spectrometry.

Lipid mediators (LMs) are a class of bioactive metabolites of the essential polyunsaturated fatty acids (PUFA), which are involved in many physiological processes. Their quantification in biological samples is critical for understanding their functions in lifestyle and chronic diseases, such as diabetes, as well allergies, cancers, and in aging processes. We developed a rapid, and sensitive LC-MS/MS method to quantify the concentrations of 14 lipid mediators of interest in mouse skeletal muscle tissue without time-consuming liquid-liquid or solid-phase extractions. A restricted-access media (RAM) based trap was used prior to LC-MS as cleanup process to prevent the analytical column from clogging and deterioration. The system enabled automatic removal of residual proteins and other biological interferences presented in the tissue extracts; the target analytes were retained in the trap and then eluted to an analytical column for separation. Matrix evaluation tests demonstrated that the use of the combined RAM trap and chromatographic separation efficiently eliminated the biological or chemical matrix interferences typically encountered in bioanalytical analysis. Using 14 LM standards and 12 corresponding deuterated compounds as internal standards, the five-point calibration curves, established over the concentration range of 0.031-320 ng mL-1, demonstrated good linearity of r2 > 0.9903 (0.9903-0.9983). The lower detection limits obtained were 0.016, 0.031, 0.062, and 0.31 ng mL-1 (0.5, 1, 2, and 10 pg on column), respectively, depending on the specific compounds. Good accuracy (87.1-114.5%) and precision (<13.4%) of the method were observed for low, medium, and high concentration quality control samples. The method was applied to measure the amount of 14 target LMs in mouse skeletal muscle tissues. All 14 analytes in this study were successfully detected and quantified in the gastrocnemius muscle samples, which provided crucial information for both age and gender-related aspects of LMs signaling in skeletal muscles previously unknown. This method could be applied to advance the understanding of skeletal muscle pathophysiology to study the role of LMs in health and disease. Furthermore, we will expand the application of this methodology to humans and other tissues/matrices in the near future.

Histone methylase MLL1 coordinates with HIF and regulate lncRNA HOTAIR expression under hypoxia.

Hypoxia signaling plays a critical role in tumor growth, angiogenesis, metastasis cancer, and aging. Under hypoxia, hypoxia-inducible factors (HIFs) are stabilized and they coordinate the process of hypoxia-induced gene expression and cell signaling leading to increased tumor growth. Recent studies indicate that non-coding RNAs which are closely associated with cancer are abnormally expressed under hypoxia. Here, we have investigated the transcriptional regulation of a cancer associated long non-coding RNA (lncRNA), HOTAIR, under hypoxic conditions. Our studies demonstrate that HOTAIR expression is upregulated under hypoxia in colon cancer and several other types of cancer cells. HOTAIR transcription is regulated by HIF1α which binds to the hypoxia response elements (HRE) present in the HOTAIR promoter under hypoxia. HIF1α knockdown results in decreased HOTAIR expression under hypoxia. Along with HIF1α, histone methylases MLL1 and histone acetylase p300 are enriched at the HOTAIR promoter under hypoxia. The levels of H3K4-trimethylation and histone acetylation are also enriched at the HOTAIR promoter. Furthermore, knockdown of MLL1 downregulated the hypoxia-induced HOTAIR expression, indicating key roles of MLL1 in hypoxia-induced HOTAIR expression. Overall, our studies demonstrate that histone methyl-transferase MLL1 coordinates with HIF1α and histone acetyltransferase p300 and regulate hypoxia-induced HOTAIR expression. The hypoxia-induced upregulation of HOTAIR expression may contribute to its roles in tumorigenesis.

Skeletal Muscle, but not Cardiovascular Function, Is Altered in a Mouse Model of Autosomal Recessive Hypophosphatemic Rickets.

Autosomal recessive hypophosphatemic rickets (ARHR) is a heritable disorder characterized by hypophosphatemia, osteomalacia, and poor bone development. ARHR results from inactivating mutations in the DMP1 gene with the human phenotype being recapitulated in the Dmp1 null mouse model which displays elevated plasma fibroblast growth factor 23. While the bone phenotype has been well-characterized, it is not known what effects ARHR may also have on skeletal, cardiac, or vascular smooth muscle function, which is critical to understand in order to treat patients suffering from this condition. In this study, the extensor digitorum longus (EDL-fast-twitch muscle), soleus (SOL-slow-twitch muscle), heart, and aorta were removed from Dmp1 null mice and ex-vivo functional tests were simultaneously performed in collaboration by three different laboratories. Dmp1 null EDL and SOL muscles produced less force than wildtype muscles after normalization for physiological cross sectional area of the muscles. Both EDL and SOL muscles from Dmp1 null mice also produced less force after the addition of caffeine (which releases calcium from the sarcoplasmic reticulum) which may indicate problems in excitation contraction coupling in these mice. While the body weights of the Dmp1 null were smaller than wildtype, the heart weight to body weight ratio was higher. However, there were no differences in pathological hypertrophic gene expression compared to wildtype and maximal force of contraction was not different indicating that there may not be cardiac pathology under the tested conditions. We did observe a decrease in the rate of force development generated by cardiac muscle in the Dmp1 null which may be related to some of the deficits observed in skeletal muscle. There were no differences observed in aortic contractions induced by PGF2α or 5-HT or in endothelium-mediated acetylcholine-induced relaxations or endothelium-independent sodium nitroprusside-induced relaxations. In summary, these results indicate that there are deficiencies in both fast twitch and slow twitch muscle fiber type contractions in this model of ARHR, while there was less of a phenotype observed in cardiac muscle, and no differences observed in aortic function. These results may help explain skeletal muscle weakness reported by some patients with osteomalacia and need to be further investigated.

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation.

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E2 (PGE2), a bioactive lipid mediator in various physiological or pathological conditions. PGE2 is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE2 signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE2 or specific agonists. All four PGE2 receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE2 and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE2 (17-PT PGE2) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE2 signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.

Extracellular Membrane Vesicles Derived from 143B Osteosarcoma Cells Contain Pro-Osteoclastogenic Cargo: A Novel Communication Mechanism in Osteosarcoma Bone Microenvironment.

The bone microenvironment (BME) is the main hub of all skeletal related pathological events in osteosarcoma leading to tumor induced bone destruction, and decreasing overall bone quality and bone strength. The role of extra-cellular membrane vesicles (EMVs) as mediators of intercellular communication in modulating osteosarcoma-BME is unknown, and needs to be investigated. It is our hypothesis that osteosarcoma-EMVs contain pro-osteoclastogenic cargo which increases osteoclastic activity, and dysregulated bone remodeling in the osteosarcoma-BME. In this study, EMVs were isolated from the conditioned media of 143B and HOS human osteosarcoma cell cultures using differential ultracentrifugation. Nano-particle tracking analysis determined EMVs in the size range of 50-200 nm in diameter. The EMV yield from 143B cells was relatively higher compared to HOS cells. Transmission electron microscopy confirmed the ultrastructure of 143B-EMVs and detected multivesicular bodies. Biochemical characterization of 143B-EMVs detected the expression of bioactive pro-osteoclastic cargo including matrix metalloproteinases-1 and -13 (MMP-1, -13), transforming growth factor-ß (TGF-ß), CD-9, and receptor activator of nuclear factor kappa-ß ligand (RANKL). Detection of a protein signature that is uniquely pro-osteoclastic in 143B-EMVs is a novel finding, and is significant as EMVs represent an interesting mechanism for potentially mediating bone destruction in the osteosarcoma-BME. This study further demonstrates that 143B cells actively mobilize calcium in the presence of ionomycin, and forskolin, and induce cytoskeleton rearrangements leading to vesicular biogenesis. In conclusion, this study demonstrates that 143B osteosarcoma cells generate EMVs mainly by mechanisms involving increased intracellular calcium or cAMP levels, and contain pro-osteoclastic cargo.

SH3BP2 cherubism mutation potentiates TNF-α-induced osteoclastogenesis via NFATc1 and TNF-α-mediated inflammatory bone loss.

Cherubism (OMIM# 118400) is a genetic disorder with excessive jawbone resorption caused by mutations in SH3 domain binding protein 2 (SH3BP2), a signaling adaptor protein. Studies on the mouse model for cherubism carrying a P416R knock-in (KI) mutation have revealed that mutant SH3BP2 enhances tumor necrosis factor (TNF)-α production and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in myeloid cells. TNF-α is expressed in human cherubism lesions, which contain a large number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, and TNF-α plays a critical role in inflammatory bone destruction in homozygous cherubism mice (Sh3bp2(KI/KI) ). The data suggest a pathophysiological relationship between mutant SH3BP2 and TNF-α-mediated bone loss by osteoclasts. Therefore, we investigated whether P416R mutant SH3BP2 is involved in TNF-α-mediated osteoclast formation and bone loss. Here, we show that bone marrow-derived M-CSF-dependent macrophages (BMMs) from the heterozygous cherubism mutant (Sh3bp2(KI/+) ) mice are highly responsive to TNF-α and can differentiate into osteoclasts independently of RANKL in vitro by a mechanism that involves spleen tyrosine kinase (SYK) and phospholipase Cγ2 (PLCγ2) phosphorylation, leading to increased nuclear translocation of NFATc1. The heterozygous cherubism mutation exacerbates bone loss with increased osteoclast formation in a mouse calvarial TNF-α injection model as well as in a human TNF-α transgenic mouse model (hTNFtg). SH3BP2 knockdown in RAW264.7 cells results in decreased TRAP-positive multinucleated cell formation. These findings suggest that the SH3BP2 cherubism mutation can cause jawbone destruction by promoting osteoclast formation in response to TNF-α expressed in cherubism lesions and that SH3BP2 is a key regulator for TNF-α-induced osteoclastogenesis. Inhibition of SH3BP2 expression in osteoclast progenitors could be a potential strategy for the treatment of bone loss in cherubism as well as in other inflammatory bone disorders.

Visual gene-network analysis reveals the cancer gene co-expression in human endometrial cancer.

BACKGROUND: Endometrial cancers (ECs) are the most common form of gynecologic malignancy. Recent studies have reported that ECs reveal distinct markers for molecular pathogenesis, which in turn is linked to the various histological types of ECs. To understand further the molecular events contributing to ECs and endometrial tumorigenesis in general, a more precise identification of cancer-associated molecules and signaling networks would be useful for the detection and monitoring of malignancy, improving clinical cancer therapy, and personalization of treatments. RESULTS: ECs-specific gene co-expression networks were constructed by differential expression analysis and weighted gene co-expression network analysis (WGCNA). Important pathways and putative cancer hub genes contribution to tumorigenesis of ECs were identified. An elastic-net regularized classification model was built using the cancer hub gene signatures to predict the phenotypic characteristics of ECs. The 19 cancer hub gene signatures had high predictive power to distinguish among three key principal features of ECs: grade, type, and stage. Intriguingly, these hub gene networks seem to contribute to ECs progression and malignancy via cell-cycle regulation, antigen processing and the citric acid (TCA) cycle. CONCLUSIONS: The results of this study provide a powerful biomarker discovery platform to better understand the progression of ECs and to uncover potential therapeutic targets in the treatment of ECs. This information might lead to improved monitoring of ECs and resulting improvement of treatment of ECs, the 4th most common of cancer in women.

Hyperthermia: from diagnostic and treatments to new discoveries.

Hyperthermia is an important approach for the treatment of several diseases. Hyperthermia is also thought to induce hypertrophy of skeletal muscles in vitro and in vivo, and has been used as a therapeutic tool for millennia. In the first part of our work, we revise several relevant patents related to the utilization of hyperthermia for the treatment and diagnostic of human diseases. In the second part, we present exciting new data on the effects of forced and natural overexpression of HSP72, using murine in vitro (muscle cells) and ex vivo (primary skeletal muscles) models. These studies help to demonstrate that hyperthermia effects are orchestrated by tight coupling between gene expression, protein function, and intracellular Ca2+ signaling pathways with a key role for calcium-induced calcium release. We hope that the review of current patents along with previous unknown information on molecular signaling pathways that underlie the hypertrophy response to hyperthermia in skeletal muscles may trigger the curiosity of scientists worldwide to explore new inventions that fully utilize hyperthermia for the treatment of muscle diseases.

Fluorescence-based measurement of store-operated calcium entry in live cells: from cultured cancer cell to skeletal muscle fiber.

Store operated Ca(2+) entry (SOCE), earlier termed capacitative Ca(2+) entry, is a tightly regulated mechanism for influx of extracellular Ca(2+) into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca(2+) stores. Since Ca(2+) is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear. The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca(2+) measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca(2+) using the ratio of F(340nm;) and F(380nm;) (510 nm for emission wavelength) of the ratiometric Ca(2+) indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca(2+) release and Ca(2+) extrusion, a Mn(2+) quenching assay is frequently used. Mn(2+) is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca(2+) pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn(2+) into the cells represents a measurement of activity of SOCE. Ratiometric measurement and the Mn(+2) quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca(2+) indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules.

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.