Hypotonic Buffer Research Articles

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle.

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

Possible roles for ATP release from RBCs exclude the cAMP-mediated Panx1 pathway.

Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.

Condensin II plays an essential role in reversible assembly of mitotic chromosomes in situ.

Condensins I and II are multisubunit complexes that play a central role in mitotic chromosome assembly. Although both complexes become concentrated along the axial region of each chromatid by metaphase, it remains unclear exactly how such axes might assemble and contribute to chromosome shaping. To address these questions from a physico-chemical point of view, we have established a set of two-step protocols for inducing reversible assembly of chromosome structure in situ, namely within a whole cell. In this assay, mitotic chromosomes are first expanded in a hypotonic buffer containing a Mg2+-chelating agent and then converted into different shapes in a NaCl concentration-dependent manner. Both chromatin and condensin-positive chromosome axes are converted into near-original shapes at 100 mM NaCl. This assay combined with small interfering RNA depletion demonstrates that the recovery of chromatin shapes and the reorganization of axes are highly sensitive to depletion of condensin II but less sensitive to depletion of condensin I or topoisomerase IIα. Furthermore, quantitative morphological analyses using the machine-learning algorithm wndchrm support the notion that chromosome shaping is tightly coupled to the reorganization of condensin II-based axes. We propose that condensin II makes a primary contribution to mitotic chromosome architecture and maintenance in human cells.

Abstract 38: Bioreactor Based Approach for Valve Tissue Engineering: Novel Application of Decellularized Porcine Pericardium

Background: Aldehyde-fixed pericardium is commonly used in valve implant manufacturing. Despite its wide employment, this tissue undergoes chronic rejection that limits implant performance and durability. In this work, we employed a method to engineer a leaflet-like tissue by seeding aortic valve interstitial cells (VIC) into fixative-free decellularized pericardium using a bioreactor based approach. Methods: Following treatment with hypotonic buffer (Tris-HCl) to induce cell lysis, porcine pericardium was incubated with TritonX-100, to remove adipose tissue and then treated with sodium dodecylsulfate to wash cellular debris. DNA was removed by incubation into a DNAse I solution. Pericardium permeability was measured on samples before and after decellularization (pressure from 735 Pa to 2200 Pa). A direct perfusion bioreactor was employed to seed (3days, 3ml/min) porcine VICs (6.5E+5 cell/scaffold) into decellularized pericardium patches (6mm diameters) and perform long-term culture (up to 14 days, 0.03ml/min). Cell seeding efficiency (Day 3) and cell proliferation (Day 7-14) were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) staining, histological analyses and fluorescence staining for quiescent/activated VIC markers (DAPI, vimentin, α-smooth muscle actin). Results: Permeability tests revealed a significant increase in decellularized samples (1-way ANOVA p < 0.05), thus supporting its use in a direct perfusion bioreactor system. MTT staining revealed homogeneous cell seeding distribution, supported by DAPI staining, showing efficient cellularization through the whole patch volume. Computer-based cell nuclei counting revealed a significant cell increase from day 3 to 7 and 14 ( p <0.05 1-way ANOVA). Immunofluorescence showed a marked reduction of αSMA in cells populating the inner layers. Conclusions: Our data show, for the first time, the capability to seed and culture VICs into a cell/fixative free pericardium with a direct perfusion system. Moreover, the use of pericardium treated with our decellularization procedure and recellularized under dynamic conditions supports a more physiological growth of VIC, as suggested by downregulation of αSMA in the inner pericardium layer.

Decellularization of human donor aortic and pulmonary valved conduits using low concentration sodium dodecyl sulfate.

The clinical use of decellularized cardiac valve allografts is increasing. Long‐term data will be required to determine whether they outperform conventional cryopreserved allografts. Valves decellularized using different processes may show varied long‐term outcomes. It is therefore important to understand the effects of specific decellularization technologies on the characteristics of donor heart valves. Human cryopreserved aortic and pulmonary valved conduits were decellularized using hypotonic buffer, 0.1% (w/v) sodium dodecyl sulfate and nuclease digestion. The decellularized tissues were compared to cellular cryopreserved valve tissues using histology, immunohistochemistry, quantitation of total deoxyribose nucleic acid, collagen and glycosaminoglycan content, in vitro cytotoxicity assays, uniaxial tensile testing and subcutaneous implantation in mice. The decellularized tissues showed no histological evidence of cells or cell remnants and >97% deoxyribose nucleic acid removal in all regions (arterial wall, muscle, leaflet and junction). The decellularized tissues retained collagen IV and von Willebrand factor staining with some loss of fibronectin, laminin and chondroitin sulfate staining. There was an absence of major histocompatibility complex Class I staining in decellularized pulmonary valve tissues, with only residual staining in isolated areas of decellularized aortic valve tissues. The collagen content of the tissues was not decreased following decellularization however the glycosaminoglycan content was reduced. Only moderate changes in the maximum load to failure of the tissues were recorded postdecellularization. The decellularized tissues were noncytotoxic in vitro, and were biocompatible in vivo in a mouse subcutaneous implant model. The decellularization process will now be translated into a good manufacturing practices‐compatible process for donor cryopreserved valves with a view to future clinical use. Copyright © 2016 The Authors Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with "heavy" amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.HCV-infected/"light" amino acids and uninfected control/"heavy" amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Heteromeric Slick/Slack K+ channels show graded sensitivity to cell volume changes.

Slick and Slack high-conductance K+ channels are found in the CNS, kidneys, pancreas, among other organs, where they play an important role in cell excitability as well as in ion transport processes. They are both activated by Na+ and Cl- but show a differential regulation by cell volume changes. Slick has been shown to be regulated by cell volume changes, whereas Slack is insensitive. α-subunits of these channels form homomeric as well as heteromeric channels. It is the aim of this work to explore whether the subunit composition of the Slick/Slack heteromeric channel affects the response to osmotic challenges. In order to provide with the adequate water permeability to the cell membrane of Xenopus laevis oocytes, mRNA of aquaporin 1 was co-expressed with homomeric or heteromeric Slick and Slack α-subunits. Oocytes were superfused with hypotonic or hypertonic buffers and changes in currents were measured by two-electrode voltage clamp. This work presents the first heteromeric K+ channel with a characteristic graded sensitivity to small and fast changes in cell volume. Our results show that the cell volume sensitivity of Slick/Slack heteromeric channels is dependent on the number of volume sensitive Slick α-subunits in the tetrameric channels, giving rise to graded cell volume sensitivity. Regulation of the subunit composition of a channel may constitute a novel mechanism to determine volume sensitivity of cells.

Decellularization and Characterization of Porcine Superflexor Tendon: A Potential Anterior Cruciate Ligament Replacement.

The porcine superflexor tendon (SFT) was identified as having appropriate structure and properties for development of a decellularized device for use in anterior cruciate ligament reconstruction. SFTs were decellularized using a combination of freeze–thaw and washes in hypotonic buffer and 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus proteinase inhibitors, followed by nuclease treatment and sterilization using peracetic acid. The decellularized biological scaffold was devoid of cells and cell remnants and contained only 13 ng/mg (dry weight) residual total DNA. Immunohistochemistry showed retention of collagen type I and III and tenascin-C. Quantitative analysis of sulfated sugar and hydroxyproline content revealed a loss of glycosaminoglycans compared with native tissue, but no loss of collagen. The decellularized SFT was biocompatible in vitro and in vivo following implantation in a mouse subcutaneous model for 12 weeks. Uniaxial tensile testing to failure indicated that the gross material properties of decellularized SFTs were not significantly different to native tissue. Decellularized SFTs had an ultimate tensile strength of 61.8 ± 10.3 MPa (±95% confidence limits), a failure strain of 0.29 ± 0.04, and a Young's modulus of the collagen phase of 294.1 ± 61.9 MPa. Analysis of the presence of the α-Gal (galactose-α-1,3-galactose) epitope by immunohistochemistry, lectin binding, and antibody absorption assay indicated that the epitope was reduced, but still present post decellularization. This is discussed in light of the potential role of noncellular α-Gal in the acceleration of wound healing and tissue regeneration in the presence of antibodies to α-Gal.

Development and characterisation of a low-concentration sodium dodecyl sulphate decellularised porcine dermis

The aim of this study was to adapt a proprietary decellularisation process for human dermis for use with porcine skin. Porcine skin was subject to: sodium chloride (1 M) to detach the epidermis, trypsin paste to remove hair follicles, peracetic acid (0.1% v/v) disinfection, washed in hypotonic buffer and 0.1% (w/v) sodium dodecyl sulphate in the presence of proteinase inhibitors followed by nuclease treatment. Cellular porcine skin, decellularised porcine and human dermis were compared using histology, immunohistochemistry, GSL-1 lectin (alpha-gal epitope) staining, biochemical assays, uniaxial tensile and in vitro cytotoxicity tests. There was no microscopic evidence of cells in decellularised porcine dermis. DNA content was reduced by 98.2% compared to cellular porcine skin. There were no significant differences in the biomechanical parameters studied or evidence of cytotoxicity. The decellularised porcine dermis retained residual alpha-gal epitope. Basement membrane collagen IV immunostaining was lost following decellularisation; however, laminin staining was retained.

Development of nanosponges from erythrocyte ghosts for removal of streptolysin-O from mammalian blood.

To produce mammalian biomimetic nanosponges from mammalian erythrocyte ghosts. Biomimetic nanosponges were studied in vitro as treatment platforms against exotoxin-related sepsis. Ovine blood was treated with hypotonic buffer to create erythrocyte ghosts and then subjected to sonication to produce erythrocyte vesicles of nonuniform size. Vesicles were then serially extruded through 400-nm and 100-nm polycarbonate membranes. Nanosponges were prepared by fusing poly(d,l-lactic-co-glycolic acid) cores with ovine erythrocyte vesicles. Ovine erythrocytes were the most susceptible to streptolysin-O lysis, making it a model to study sepsis treatment. Ovine nanosponges adsorbed streptolysin-O at 37 and 40°C. These results identify ovine nanosponges as novel therapeutic model to test adsorption of cholesterol binding toxins such as streptolysin-O.

Mechanical activation of angiotensin II type 1 receptors causes actin remodelling and myogenic responsiveness in skeletal muscle arterioles.

Candesartan, an inverse agonist of the type 1 angiotensin II receptor (AT1 R), causes a concentration-dependent inhibition of pressure-dependent myogenic tone consistent with previous reports of mechanosensitivity of this G protein-coupled receptor. Mechanoactivation of the AT1 R occurs independently of local angiotensin II production and the type 2 angiotensin receptor. Mechanoactivation of the AT1 R stimulates actin polymerization by a protein kinase C-dependent mechanism, but independently of a change in intracellular Ca2+ . Using atomic force microscopy, changes in single vascular smooth muscle cell cortical actin are observed to remodel following mechanoactivation of the AT1 R. The Gq/11 protein-coupled angiotensin II type 1 receptor (AT1 R) has been shown to be activated by mechanical stimuli. In the vascular system, evidence supports the AT1 R being a mechanosensor that contributes to arteriolar myogenic constriction. The aim of this study was to determine if AT1 R mechanoactivation affects myogenic constriction in skeletal muscle arterioles and to determine underlying cellular mechanisms. Using pressure myography to study rat isolated first-order cremaster muscle arterioles the AT1 R inhibitor candesartan (10-7 -10-5 m) showed partial but concentration-dependent inhibition of myogenic reactivity. Inhibition was demonstrated by a rightward shift in the pressure-diameter relationship over the intraluminal pressure range, 30-110mmHg. Pressure-induced changes in global vascular smooth muscle intracellular Ca2+ (using Fura-2) were similar in the absence or presence of candesartan, indicating that AT1 R-mediated myogenic constriction relies on Ca2+ -independent downstream signalling. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) reversed the inhibitory effect of candesartan, while this rescue effect was prevented by the protein kinase C (PKC) inhibitor GF 109203X. Both candesartan and PKC inhibition caused increased G-actin levels, as determined by Western blotting of vessel lysates, supporting involvement of cytoskeletal remodelling. At the single vascular smooth muscle cell level, atomic force microscopy showed that cell swelling (stretch) with hypotonic buffer also caused thickening of cortical actin fibres and this was blocked by candesartan. Collectively, the present studies support growing evidence for novel modes of activation of the AT1 R in arterioles and suggest that mechanically activated AT1 R generates diacylglycerol, which in turn activates PKC which induces the actin cytoskeleton reorganization that is required for pressure-induced vasoconstriction.

Decellularisation and histological characterisation of porcine peripheral nerves.

ABSTRACTPeripheral nerve injuries affect a large proportion of the global population, often causing significant morbidity and loss of function. Current treatment strategies include the use of implantable nerve guide conduits (NGC's) to direct regenerating axons between the proximal and distal ends of the nerve gap. However, NGC's are limited in their effectiveness at promoting regeneration Current NGCs are not suitable as substrates for supporting either neuronal or Schwann cell growth, as they lack an architecture similar to that of the native extracellular matrix (ECM) of the nerve. The aim of this study was to create an acellular porcine peripheral nerve using a novel decellularisation protocol, in order to eliminate the immunogenic cellular components of the tissue, while preserving the three‐dimensional histoarchitecture and ECM components. Porcine peripheral nerve (sciatic branches were decellularised using a low concentration (0.1%; w/v) sodium dodecyl sulphate in conjunction with hypotonic buffers and protease inhibitors, and then sterilised using 0.1% (v/v) peracetic acid. Quantitative and qualitative analysis revealed a ≥95% (w/w) reduction in DNA content as well as preservation of the nerve fascicles and connective tissue. Acellular nerves were shown to have retained key ECM components such as collagen, laminin and fibronectin. Slow strain rate to failure testing demonstrated the biomechanical properties of acellular nerves to be comparable to fresh controls. In conclusion, we report the production of a biocompatible, biomechanically functional acellular scaffold, which may have use in peripheral nerve repair. Biotechnol. Bioeng. 2016;113: 2041–2053. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

A novel PDE1A coupled to M2AChR at plasma membranes from bovine tracheal smooth muscle

Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M2AChR pharmacological profile. Therefore, a novel Ca2+/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58 kDa) being the 58 kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [3H]cGMP and [3H]cAMP exhibiting a higher affinity as Km (μM) for cGMP than cAMP but being close values with Vmax cAMP/cGMP ratio of 1.5. The co-factor Mg2+ showed similar K(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC50 of 4.9 and 4.6 μM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M2AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed.

Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration

3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals.

Isolation of mitochondria from tissue culture cells.

The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from ∼10⁹ cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation.

Preparation of Drosophila Kc cell nuclear extracts for in vitro splicing.

This protocol describes how to prepare nuclear extracts from cultured Drosophila cells. It uses the Kc cell line, which grows without fetal bovine serum and so is suitable for large-scale suspension cultures. The suspension cells are harvested by centrifugation, washed to remove media components, and resuspended in a low-salt (hypotonic) buffer that makes the cells swell in size and easy to homogenize. Usually, nuclear extract preparations are performed on 4-32 L of cells at ∼ 3.5 × 10(6) cells/mL.

Development and characterization of acellular porcine pulmonary valve scaffolds for tissue engineering.

Currently available replacement heart valves all have limitations. This study aimed to produce and characterize an acellular, biocompatible porcine pulmonary root conduit for reconstruction of the right ventricular outflow tract e.g., during Ross procedure. A process for the decellularization of porcine pulmonary roots was developed incorporating trypsin treatment of the adventitial surface of the scraped pulmonary artery and sequential treatment with hypotonic Tris buffer (HTB; 10 mM Tris pH 8.0, 0.1% (w/v) EDTA, and 10 KIU aprotinin), 0.1% (w/v) sodium dodecyl sulfate in HTB, two cycles of DNase and RNase, and sterilization with 0.1% (v/v) peracetic acid. Histology confirmed an absence of cells and retention of the gross histoarchitecture. Immunohistochemistry further confirmed cell removal and partial retention of the extracellular matrix, but a loss of collagen type IV. DNA levels were reduced by more than 96% throughout all regions of the acellular tissue and no functional genes were detected using polymerase chain reaction. Total collagen levels were retained but there was a significant loss of glycosaminoglycans following decellularization. The biomechanical, hydrodynamic, and leaflet kinematics properties were minimally affected by the process. Both immunohistochemical labeling and antibody absorption assay confirmed a lack of α-gal epitopes in the acellular porcine pulmonary roots and in vitro biocompatibility studies indicated that acellular leaflets and pulmonary arteries were not cytotoxic. Overall the acellular porcine pulmonary roots have excellent potential for development of a tissue substitute for right ventricular outflow tract reconstruction e.g., during the Ross procedure.

Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells†

Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure. Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell attachment, apoptosis induction (Caspase 3/7 activity) and metabolic activity (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium conversion) were followed. Transcriptional activation of genes involved in pluripotency; early and late myocardial development; and endothelial, ectodermal or endodermal commitment were monitored by quantitative real-time polymerase chain reaction (rtPCR). Protein expression of selected markers was confirmed by immunohistology. cECM supported the proliferation of ESCs and iPSCs, and Caspase 3/7 activity was significantly lower compared with standard culture. Cardiac lineage commitment was favoured when ESCs or iPSCs were grown on cECM, as evidenced by the significantly increased mRNA expression of cardiac alpha myosin heavy polypeptide 6 (Myh6), cardiac troponin T2 (Tnnt2) and NK2 homeobox 5 (Nkx2.5) as well as positive immunohistology for cardiac troponin T and heavy-chain cardiac myosin protein. In contrast, Matrigel or Geltrex did not induce cardiac-specific markers. MSCs showed no evidence of cardiomyocyte differentiation. Human cardiac ECM seems to direct differentiation of pluripotent stem cells towards a cardiomyocyte phenotype. This phenomenon supports the use of cardiac ECM preparations for guided stem cell differentiation and myocardial repair, and may ultimately increase the therapeutic efficacy of cell therapy in heart failure patients.

A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1

Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPγS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi1 subunit.