Detection of Nav1.5 conformational change in mammalian cells using the non-canonical amino acid ANAP

Nav1.5 inactivation is necessary for healthy conduction of the cardiac action potential. Genetic mutations of Nav1.5 perturb inactivation and cause potentially fatal arrhythmias associated with long QT syndrome type 3. The exact structural dynamics of the inactivation complex is unknown. To sense inactivation gate conformational change in live mammalian cells, we incorporated the solvatochromic fluorescent non-canonical amino acid ANAP into single sites in the Nav1.5 inactivation gate. ANAP was incorporated in full-length and C-terminally truncated Nav1.5 channels using mammalian cell synthetase-tRNA technology. ANAP-incorporated channels were expressed in mammalian cells and they exhibited pathophysiological function. A spectral imaging potassium-depolarization assay was designed to detect ANAP emission shifts associated with Nav1.5 conformational change. Site-specific intracellular ANAP incorporation affords live-cell imaging and detection of Nav1.5 inactivation gate conformational change in mammalian cells

Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype

Author ManuscriptThe application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to β-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR, and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); NSF grant 16478 (G.V.-N.); the University of Minho MD/PhD program (D.T.); a Japan Society for the Promotion of Science fellowship (K.M.); and the Columbia University Stem Cell Initiative (L.S., M.Y.

Conversion to complete resection with mFOLFOX6 with bevacizumab or cetuximab based on K‐RAS status for unresectable colorectal liver metastasis (BECK study): Long‐term results of survival

[Background/Purpose]To investigate the long‐term outcome and entire treatment course of patients with technically unresectable CRLM who underwent conversion hepatectomy and to examine factors associated with conversion to hepatectomy. [Methods]Recurrence and survival data with long‐term follow‐up were analyzed in the cohort of a multi‐institutional phase II trial for technically unresectable colorectal liver metastases (the BECK study). [Results]A total of 22/12 patients with K‐RAS wild‐type/mutant tumors were treated with mFOLFOX6 + cetuximab/bevacizumab. The conversion R0/1 hepatectomy rate was significantly higher in left‐sided primary tumors than in right‐sided tumors (75.0% vs 30.0%, P = .022). The median follow‐up was 72.6 months. The 5‐year overall survival (OS) rate in the entire cohort was 48.1%. In patients who underwent R0/1 hepatectomy (n = 21), the 5‐year RFS rate and OS rate were 19.1% and 66.3%, respectively. At the final follow‐up, seven patients had no evidence of disease, five were alive with disease, and 20 had died from their original cancer. All 16 patients who achieved 5‐year survival underwent conversion hepatectomy, and 11 of them underwent further resection for other recurrences (median: 2, range: 1‐4). [Conclusions]Conversion hepatectomy achieved a similar long‐term survival to the results of previous studies in initially resectable patients, although many of them experienced several post‐hepatectomy recurrences. Left‐sided primary was found to be the predictor for conversion hepatectomy

Advanced maturation of human cardiac tissue grown from pluripotent stem cells

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1-6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.The authors acknowledge funding support from the National Institutes of Health of the USA (NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); Columbia University MD/PhD program (S.P.M., T.C.); University of Minho MD/PhD program (D.T.); Japan Society for the Promotion of Science fellowship (K.M.); and Columbia University Stem Cell Initiative (D.S., L.S., M.Y.). We thank S. Duncan and B. Conklin for providing human iPSCs, M.B. Bouchard for assistance with image and video analysis, and L. Cohen-Gould for transmission electron microscopy services.info:eu-repo/semantics/publishedVersio

Apelin Enhances Directed Cardiac Differentiation of Mouse and Human Embryonic Stem Cells

Apelin is a peptide ligand for an orphan G-protein coupled receptor (APJ receptor) and serves as a critical gradient for migration of mesodermal cells fated to contribute to the myocardial lineage. The present study was designed to establish a robust cardiac differentiation protocol, specifically, to evaluate the effect of apelin on directed differentiation of mouse and human embryonic stem cells (mESCs and hESCs) into cardiac lineage. Different concentrations of apelin (50, 100, 500 nM) were evaluated to determine its differentiation potential. The optimized dose of apelin was then combined with mesodermal differentiation factors, including BMP-4, activin-A, and bFGF, in a developmentally specific temporal sequence to examine the synergistic effects on cardiac differentiation. Cellular, molecular, and physiologic characteristics of the apelin-induced contractile embryoid bodies (EBs) were analyzed. It was found that 100 nM apelin resulted in highest percentage of contractile EB for mESCs while 500 nM had the highest effects on hESCs. Functionally, the contractile frequency of mESCs-derived EBs (mEBs) responded appropriately to increasing concentration of isoprenaline and diltiazem. Positive phenotype of cardiac specific markers was confirmed in the apelin-treated groups. The protocol, consisting of apelin and mesodermal differentiation factors, induced contractility in significantly higher percentage of hESC-derived EBs (hEBs), up-regulated cardiac-specific genes and cell surface markers, and increased the contractile force. In conclusion, we have demonstrated that the treatment of apelin enhanced cardiac differentiation of mouse and human ESCs and exhibited synergistic effects with mesodermal differentiation factors

サイボウナイ ニ チョゾウサレル カルシウム イオン ノ チョウセツ ニ ヒツヨウナ TRIC チャネル ノ カイセキ

京都大学0048新制・課程博士博士(薬学)甲第12973号薬博第612号新制||薬||218(附属図書館)UT51-2007-H246京都大学大学院薬学研究科生命薬科学専攻(主査)教授 竹島 浩, 教授 中山 和久, 教授 伊藤 信行学位規則第4条第1項該当Doctor of Pharmaceutical SciencesKyoto UniversityDA

Two regions of the tail are necessary for the isoform-specific functions of nonmuscle myosin IIB.

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB(-/-) fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament

Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Myosin II molecules assemble into filaments through their C-terminal rod region, and are responsible for several cellular motile activities. Three isoforms of nonmuscle myosin II (IIA, IIB and IIC) are expressed in mammalian cells. However, little is known regarding the isoform composition in filaments. To obtain new insight into the assembly properties of myosin II isoforms, especially regarding the isoform composition in filaments, we performed a combination analysis of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS), which enables us to acquire information on both the interaction and the size of each molecule simultaneously. Using C-terminal rod fragments of IIA and IIB (ARF296 and BRF305) labelled with different fluorescent probes, we demonstrated that hetero-assemblies were formed from a mixture of ARF296 and BRF305, and that dynamic exchange of rod fragments occurred between pre-formed homo-assemblies of each isoform in an isoform-independent manner. We also showed that Mts1 (S100A4) specifically stripped ARF296 away from the hetero-assemblies, and consequently, homo-assemblies of BRF305 were formed. These results suggest that IIA and IIB can form hetero-filaments in an isoform-independent manner, and that a factor like Mts1 can remove one isoform from the hetero-filament, resulting in a formation of homo-filaments consisting of another isoform