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.
We have recently reported that left atrial injections of the thromboxane A(2) (TXA(2)) mimetic, (5Z)-7-(1R,4S,5S,6R)-6-(1E,3S)-3-hydroxy-1-octenyl-2 -oxabicyclo2.2.1hept-5-yl-5-heptenoic acid ...(U46619), induced ventricular arrhythmias in the anesthetized rabbit. Data from this study led us to hypothesize that TXA(2) may be inducing direct actions on the myocardium to induce these arrhythmias. The aim of this study was to further elucidate the mechanism responsible for these arrhythmias. We report that TXA(2)R is expressed at both the gene and protein levels in atrial and ventricular samples of adult rabbits. In addition, TXA(2)R mRNA was identified in single, isolated ventricular cardiac myocytes. Furthermore, treatment of isolated cardiac myocytes with U46619 increased intracellular calcium in a dose-dependent manner and these increases were blocked by the specific TXA(2)R antagonist, 7-(3-((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ29548). Pretreatment of myocytes with an inhibitor of inositol trisphosphate (IP(3)) formation, gentamicin, or with an inhibitor of IP(3) receptors, 2-aminoethoxydiphenylborate (2-APB), blocked the increase in intracellular calcium. In vivo pretreatment of anesthetized rabbits with either gentamicin or 2-APB subsequently inhibited the formation of ventricular arrhythmias elicited by U46619. These data support the hypothesis that TXA(2) can induce arrhythmias via a direct action on cardiac myocytes. Furthermore, these arrhythmogenic actions were blocked by inhibitors of the IP(3) pathway. In summary, this study provides novel evidence for direct TXA(2)-induced cardiac arrhythmias and provides a rationale for IP(3) as a potential target for the treatment of TXA(2)-mediated arrhythmias.
Recent studies in anaesthesia and intensive care indicate that a team's ability to adapt its coordination activities to changing situational demands is crucial for effective teamwork and thus, safe ...patient care. This study addresses the relationship between adaptation of team coordination and markers of clinical performance in response to a critical event, particularly regarding which types of coordination activities are used and which team member engages in those coordination activities.
Video recordings of 15 two-person anaesthesia teams (anaesthesia trainee plus anaesthesia nurse) performing a simulated induction of general anaesthesia were coded, using a structured observation system for coordination activities. The simulation involved a critical event'asystole during laryngoscopy. Clinical performance was assessed using two separate reaction times related to the critical event.
Analyses of variance revealed a significant effect of the critical event on team coordination: after the occurrence of the asystole, team members adapted their coordination activities by spending more time on information management'a specific type of coordination activity (F1,28=15.17, P=0.001). No significant effect was found for task management. The increase in information management was related to faster decisions regarding how to respond to the critical event, but only for trainees and not for nurses.
Our findings support the claim that adaptation of coordination activities is related to improved team performance in healthcare. Moreover, adaptation and its relationship to team performance were found to vary with regard to type of coordination activities and team member.
Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, ...however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.
Abstract
Background:
The involvement of aquaporin (AQP) water and small solute channels in the etiology of several diseases, including cancer, neuromyelitis optica and body fluid imbalance disorders, ...has been suggested previously. Furthermore, results obtained in a mouse model suggested that AQP9 function contributes to hyperglycemia in type-2 diabetes. In addition, the physiological role of several AQP family members remains poorly understood. Small molecule inhibitors of AQPs are therefore desirable to further study AQP physiological and pathophysiological functions.
Methods:
The binding of recently established AQP9 inhibitors to a homology model of AQP9 was investigated by molecular dynamics simulations and molecular docking. Putative inhibitor binding sites identified with this procedure were modified by site-directed mutagenesis. Active compounds were measured in a mammalian cell water permeability assay of mutated AQP9 isoforms and tested for changes in inhibitory effects.
Controls:
Three independent cell lines were established for each mutated AQP9 isoform and functionality of mutant isoforms was established.
Principal findings:
We have identified putative binding sites of recently established AQP9 inhibitors. This information facilitated successful identification of novel AQP9 inhibitors with low micromolar IC50 values in a cell based assay by in silico screening of a compound library targeting specifically this binding site.
Significance:
We have established a successful strategy for AQP small molecule inhibitor identification. AQP inhibitors may be relevant as experimental tools, to enhance our understanding of AQP function, and in the treatment of various diseases.
FGF23 production by osteocytes Bonewald, Lynda F.; Wacker, Michael J.
Pediatric nephrology (Berlin, West),
04/2013, Volume:
28, Issue:
4
Journal Article
Peer reviewed
Open access
Fibroblast Growth Factor 23 (FGF23), a known regulator of phosphate homeostasis, is produced by cells residing in bone, namely, osteocytes, to target a distant organ, the kidney. Elevated FGF23 ...levels have recently been found systemically and in osteocytes in patients and animal models of chronic kidney disease. Associations between serum FGF23 level and vascular dysfunction, vascular calcification, and increased risk of cardiovascular disease have also been observed. In this review we discuss FGF23 expression in osteocytes and the potential means to regulate expression and function of this protein at the osteocyte level.
Transgenic mice are widely used to delete or overexpress genes in a cell specific manner to advance knowledge of bone biology, function and disease. While numerous Cre models exist to target gene ...recombination in osteoblasts and osteoclasts, few target osteocytes specifically, particularly mature osteocytes. Our goal was to create a spatial and temporal conditional Cre model using tamoxifen to induce Cre activity in mature osteocytes using a Bac construct containing the 5’ and 3’ regions of the Sost gene (Sost ERT2 Cre). Four founder lines were crossed with the Ai9 Cre reporter mice. One founder line showed high and specific activity in mature osteocytes. Bones and organs were imaged and fluorescent signal quantitated. While no activity was observed in 2 day old pups, by 2 months of age some osteocytes were positive as osteocyte Cre activity became spontaneous or ‘leaky’ with age. The percentage of positive osteocytes increased following tamoxifen injection, especially in males, with 43% to 95% positive cells compared to 19% to 32% in females. No signal was observed in any bone surface cell, bone marrow, nor in muscle with or without tamoxifen injection. No spontaneous signal was observed in any other organ. However, with tamoxifen injection, a few positive cells were observed in kidney, eye, lung, heart and brain. All other organs, 28 in total, were negative with tamoxifen injection. However, with age, a muscle phenotype was apparent in the Sost-ERT2 Cre mice. Therefore, although this mouse model may be useful for targeting gene deletion or expression to mature osteocytes, the muscle phenotype may restrict the use of this model to specific applications and should be considered when interpreting data.Animal model: Transgenic mice offer platform to study mature osteocytesA new transgenic mouse allows DNA modifications to be targeted to mature bone cells known as osteocytes, enabling researchers to better study the function of this cell population, although the model has some weaknesses that may restrict its use. Lynda Bonewald from Indiana University, Indianapolis, USA, and colleagues engineered mice to express introduced genetic material only in cells in which a gene called Sost is normally active — namely, osteocytes — and only when experimenters introduce a drug called tamoxifen. In general, transgene activity was contained to the osteocytes, but a full characterization of the mice showed some issues. For one, spontaneously transgene expression occurred in older mice in the absence of tamoxifen, although this was limited to the mature bone cells. With tamoxifen, the researchers observed some transgene expression outside of the bone. Most problematically, there were some broad muscle defects.
Skeletal muscle and bone interact at the level of mechanical loading through the application of force by muscles to the skeleton. Recently considerable focus has been placed on signaling ...factors/molecules produced by these two tissues that may act to modulate the function of the other tissue. We sought to determine if muscle and muscle-derived factors were essential to the osteocyte response to loading. Botox® induced muscle paralysis was used to investigate the role of muscle contraction during in vivo tibia compression loading. 5–6 month-old female TOPGAL mice had their right hindlimb muscles surrounding the tibia injected with either BOTOX® or saline. At four days post injections when muscle paralysis peaked, the right tibia was subjected to a single session of in vivo compression loading at ∼2600 με. At 24 h post-load we observed a 2.5-fold increase in β-catenin signaling in osteocytes in the tibias of the saline injected mice, whereas loading of tibias from Botox® injected mice failed to active β-catenin signaling in osteocytes. This suggests that active muscle contraction produces a factor(s) that is necessary for or conditions the osteocyte's ability to respond to load. To further investigate the role of muscle derived factors, MLO-Y4 osteocyte-like cells and a luciferase based β-catenin reporter (TOPflash-MLO-Y4) cell line we developed were treated with conditioned media (CM) from C2C12 myoblasts (MB) and myotubes (MT) and ex vivo contracted Extensor Digitorum Longus (EDL) and Soleus (Sol) muscles under static or loading conditions using fluid flow shear stress (FFSS). 10 % C2C12 myotube CM, but not myoblast or NIH3T3 fibroblast cells CM, induced a rapid activation of the Akt signaling pathway, peaking at 15 min and returning to baseline by 1–2 h under static conditions. FFSS applied to MLO-Y4 cells for 2 h in the presence of 10 % MT-CM resulted in a 6–8 fold increase in pAkt compared to a 3–4 fold increase under control or when exposed to 10 % MB-CM. A similar response was observed in the presence of 10 % EDL-CM, but not in the presence of 10 % Sol-CM. TOPflash-MLO-Y4 cells were treated with 10 ng/ml Wnt3a in the presence or absence of MT-CM. While MT-CM resulted in a 2-fold activation and Wnt3a produced a 10-fold activation, the combination of MT-CM + Wnt3a resulted in a 25-fold activation of β-catenin signaling, implying a synergistic effect of factors in MT-CM with Wnt3a. These data provide clear evidence that specific muscles and myotubes produce factors that alter important signaling pathways involved in the response of osteocytes to mechanical load. These data strongly suggest that beyond mechanical loading there is a molecular coupling of muscle and bone.
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•Muscle paraylysis prevents load-induced osteocyte β-catenin signaling.•Contracted EDL muscle produces factor(s) that induce β-catenin signalning.•C2C12 myotubes factor(s) synergize with Wnt3a to induce β-catenin signaling.•A luciferase based MLO-Y4 β-catenin reporter cell line was developed.
The primary mechanism that contributes to decreasing skeletal muscle strength and size with healthy aging is not presently known. This study examined the contribution of the ubiquitin-proteasome ...pathway and apoptosis to skeletal muscle wasting in older adults (n = 21; mean age = 72.76 +/- 8.31 years) and young controls (n = 21; mean age = 21.48 +/- 2.93 years). Subjects underwent a percutaneous muscle biopsy of the vastus lateralis to determine: (1) ubiquitin ligase gene expression (MAFbx and MuRF1); (2) frequency of apoptosis; and (3) individual fiber type and cross-sectional area. In addition, a whole muscle strength test was also performed. A one-way ANOVA revealed significant increases in the number of positive TUNEL cells in older adults (87%; p < 0.05), although no significant increase in caspase-3/7 activity was detected. Additionally, ubiquitin ligase gene expression, individual muscle fiber type and CSA were not different between old and young subjects. Muscle strength was also significantly lower in old compared to young subjects (p < 0.05). In conclusion, this study indicates a preferential role for apoptosis contributing to decreases in muscle function with age.
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