NIH P01 HL112730 (RAG, JVE), NIH R01 HL132075 (RAG, JVE), Barbra Streisand Frauen-Herz-Zentrum (RAG, JVE), Dorothy und E. Phillip Lyon Stuhl in Molekulare Kardiologie (RAG), Erika Glazer Stiftungslehrstuhl im Herzgesundheit der Frau (JVE) und Tschechische Akademie der Wissenschaften Institutionelle Unterstützung RVO: 68081715 (MS).

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Polysome Profilanalyse ermöglicht die Untersuchung von Protein-Übersetzung durch die Analyse von translationalen Bundesstaat eine spezifische mRNA oder die ganze Transkriptom-6,-7. Es ist auch eine große Hilfe, wenn lokale Übersetzung werden, wie z. B. Synaptosomes8 untersucht muss. Diese Methode umfasst traditionell die Trennung von Mono - und Polyribosomes und die damit verbundenen mRNAs auf einer Saccharose-Steigung welche mit genom...

Das Herz reagiert auf die Verletzung von (I) Ischämie und Reperfusion (R) in einer dynamischen Art und Weise. Allerdings gibt es wenig Einblick in akute Veränderungen bei der Proteinsynthese während der Reaktion. Um dieses Problem anzugehen, wir nutzten die etablierte Methode der Polysome Profilierung1 zur Identifizierung von Änderungen im Protein-Überfluss, die Umverteilung von Ribosomen und Translationale regulatorische Faktoren vom Zytosol zu Polysomes, zu reflektieren und die Anstieg der neu synthetisierten Proteine (NSP). In der Umgebung von I / R, die Zunahme der neuen Protein-Synthese erfolgt in einem Zeitrahmen, die nicht übereinstimmen mit Transkription des neuen mRNAs2; Darüber hinaus wurde Discordance zwischen mRNA Ausdruck und Protein Fülle gemeldeten3. Aus diesen Gründen haben wir die Änderungen in der dynamischen Proteomforschung zu analysieren, wie Protein Übersetzung widerspiegelt. Um dies zu tun, wir mRNA in den Polysome Fraktionen zu quantifizieren, und analysieren die Proteinzusammensetzung in den Polysome Fraktionen. Schließlich, da MicroRNAs (MiRs) Verfügbarkeit von mRNAs für die Übersetzung zu regulieren und Effizienz von Protein Übersetzung4,5stören können, untersuchten wir die Verteilung der MiRs in den Polysome Fraktionen, mit Schwerpunkt auf der Antwort auf I / r.
Wir entschieden uns für die isolierten Langendorff Perfusion Mausmodell und geernteten Gewebe unter basalen Bedingungen der kontinuierliche Perfusion, nach 30 min global keine-Flow-Ischämie und nach 30 min von Ischämie, gefolgt von 30 min der Reperfusion verwenden. Wir dann solubilisiert Herzgewebe und Polysomes über einen Saccharose Gradienten getrennt gefolgt von Proteomic Analysen und selektive Detektion von mRNAs und MiRNAs durch PCR und Microarray, beziehungsweise. Diese Kombination von Methoden steht einen leistungsfähigen Ansatz zum Verständnis der dynamischen Proteom, ermöglicht simultane Detektion von mRNA und MiRNA, Nadel-, sowie die Umverteilung von regulatorischen Proteinen, MiRNA und mRNA zwischen nontranslating Brüchen, niedrige Effizienz Polysomes und Hochleistungs-Polysomes (siehe Abbildung 1). Einblicke in die dynamische Regulierung dieses Prozesses werden durch weitere Analysen der Phosphorylierung wichtige regulatorische Faktoren wie eIF2α oder mTOR verlängert werden. Diese einzelnen Schritte sind nun detailliert beschrieben.

<table><tbody><tr><td>Pentobarbital</td><td>Vortech Phamaceuticals</td><td>9373</td><td>for euthansia</td></tr><tr><td>Heparin</td><td>Sagent</td><td>103424</td><td>used in langendorff preparation</td></tr><tr><td>forceps</td><td>Fine Science Tools</td><td>91110-10</td><td>used to hang the heart</td></tr><tr><td>Langendorff system</td><td>Radnoti + home made</td><td>n/a</td><td>A &#039;four heart&#039; system consisting of custom blown glass, tubing and water baths</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653-5KG</td><td>Krebs buffer and Sucrose gradient</td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td>Krebs buffer and Lysis buffer</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;42&lt;/sub&gt;</td><td>Sigma</td><td>P-5504</td><td>Krebs buffer</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M7774-500G</td><td>Krebs buffer</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G5767</td><td>Krebs buffer</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C1016-500G</td><td>Krebs buffer</td></tr><tr><td>Sucrose powder</td><td>Sigma</td><td>S0389-1KG</td><td>Sucrose gradient</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>208337</td><td>Sucrose gradient and Lysis buffer</td></tr><tr><td>Tris-base</td><td>Sigma</td><td>T1503-1KG</td><td>Sucrose gradient and Lysis buffer</td></tr><tr><td>Xylene Cyanole</td><td>Sigma</td><td>X-4126</td><td>Sucrose gradient</td></tr><tr><td>Cycloheximide</td><td>Sigma-aldrich</td><td>239763</td><td>Sucrose gradient and Lysis buffer</td></tr><tr><td>RNaseOUT</td><td>Life Technologies</td><td>C00019</td><td>RNAse inhibitor for Lysis buffer</td></tr><tr><td>Igepal CA-360 (NP40)</td><td>Sigma</td><td>I3021</td><td>Lysis buffer</td></tr><tr><td>Protease Inhibitor Cocktail tablets, EDTA free</td><td>Roche</td><td>5056489001</td><td></td></tr><tr><td>Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 mm x 89 mm</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>LE-80K</td><td>Ultracentrifugation of the gradients</td></tr><tr><td>Rotor</td><td>Beckman</td><td>SW41</td><td>Ultracentrifugation of the gradients</td></tr><tr><td>Biologic LP (pump)</td><td>Biorad</td><td>731-8300</td><td>Fractionation of the gradients</td></tr><tr><td>BioFrac</td><td>Biorad</td><td>741-0002</td><td>Fractionation of the gradients</td></tr><tr><td>Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube</td><td>Sigma</td><td>Z666513-100EA</td><td>Gradient fraction and RNA extraction</td></tr><tr><td>TRIzol Reagent</td><td>Life technologies</td><td>AM9738</td><td>RNA extraction</td></tr><tr><td>Luciferase Control RNA</td><td>Promega</td><td>L4561</td><td>RNA extraction</td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C606-4</td><td>RNA extraction</td></tr><tr><td>Glycogen, RNA grade</td><td>Thermo Fisher Scientific</td><td>R0551</td><td>RNA extraction</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>I9516</td><td>RNA extraction</td></tr><tr><td>Ethanol</td><td>Sigma</td><td>E7023-1L</td><td>RNA extraction</td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>BioRad</td><td>170-8891</td><td>Reverse transcription</td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>BioRad</td><td>175-5122</td><td>Quantative PCR</td></tr><tr><td>miRNeasy Micro Kit (50)</td><td>Qiagen</td><td>217084</td><td>Kit for total RNA isolation</td></tr><tr><td>miScript II RT Kit (50)</td><td>Qiagen</td><td>218161</td><td>Kit for miRNA reverse transcription</td></tr><tr><td>miScript Sybr Green PCR Kit (200)</td><td>Qiagen</td><td>218073</td><td>Kit for real-time PCR expression analysis of miRNAs</td></tr><tr><td>Centrifuge 5424R</td><td>Eppendorf</td><td></td><td>For centrifugation of 1.5 mL or 2.0 mL tubes at different temperatures. Max speed - 21130 x g</td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td></td><td>For real-time PCR plate centrifugation at different temperatures. Max speed - 2039 x g</td></tr><tr><td>My Cycler Thermal Cycler</td><td>Bio-Rad</td><td></td><td>For reverse transcription</td></tr><tr><td>CFX96 Real-Time System/C1000 Touch Thermal Cycler</td><td>Bio-Rad</td><td></td><td>For real-time PCR analysis</td></tr><tr><td>miRNeasy Serum/Plasma Spike-in Control</td><td>Qiagen</td><td>219610</td><td>For quality control of RNA isolation</td></tr><tr><td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear</td><td>Bio-Rad</td><td>HSP9641</td><td>For real-time PCR analysis</td></tr><tr><td>Microseal &#039;B&#039; PCR Plate Sealing Film, adhesive, optical</td><td>Bio-Rad</td><td>MSB1001</td><td>For real-time PCR plate sealing</td></tr><tr><td>Research plus Single-Channel Pipette, Gray; 0.5-10 &amp;micro;L</td><td>Eppendorf</td><td>UX-24505-02</td><td>For pipetting</td></tr><tr><td>PIPETMAN Classic Pipets, P20</td><td>Gilson</td><td>F123600G</td><td>For pipetting</td></tr><tr><td>PIPETMAN Classic Pipets, P200</td><td>Gilson</td><td>F144565</td><td>For pipetting</td></tr><tr><td>Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 &amp;micro;L</td><td>Gilson</td><td>17011782</td><td>For pipetting</td></tr><tr><td>Glycogen, RNA grade</td><td>Thermo Fisher Scientific</td><td>R0551</td><td>Improves total RNA isolation efficiency</td></tr><tr><td>Posi-Click 1.7 mL Tubes, natural color</td><td>Denville</td><td>C2170</td><td>RNA isolation and storage; reagent mix</td></tr><tr><td>Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically</td><td>Denville</td><td>C18098-4 (1000910)</td><td>Reverse transcription reaction</td></tr><tr><td>Sharp 10 Precision barrier Tips</td><td>Denville</td><td>P1096-FR</td><td>For pipetting</td></tr><tr><td>Sharp 20 Precision barrier Tips</td><td>Denville</td><td>P1121</td><td>For pipetting</td></tr><tr><td>Sharp 200 Precision barrier Tips</td><td>Denville</td><td>P1122</td><td>For pipetting</td></tr><tr><td>Tips LTS 1 mL Filter</td><td>Rainin</td><td>RT-L1000F</td><td>For pipetting</td></tr><tr><td>miScript Primer Assay (200)</td><td>Qiagen</td><td>(it changes according to the miRNA)</td><td>For real-time PCR analysis</td></tr><tr><td>Gradient Master ver 5.3 Model 108</td><td>BioComp Instruments</td><td></td><td>For preparation of sucrose gradients</td></tr><tr><td>trichloroacetic acid</td><td>Sigma Aldrich</td><td>T6399</td><td></td></tr><tr><td>acetone</td><td>Sigma Aldrich</td><td>650501</td><td></td></tr><tr><td>Tris hydrochloride</td><td>Amresco</td><td>M108</td><td></td></tr><tr><td>dithiothreitol</td><td>Fisher Scientific</td><td>BP172</td><td></td></tr><tr><td>iodoacetamide</td><td>Gbiosciences</td><td>RC-150</td><td></td></tr><tr><td>sequencing grade modified trypsine, porcine</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>ammonium bicarbonate</td><td>BDH</td><td>BDH9206</td><td></td></tr><tr><td>formic acid, Optima LC/MS</td><td>Fisher Chemical</td><td>A117</td><td></td></tr><tr><td>methanol, Optima LC/MS</td><td>Fisher Chemical</td><td>A454</td><td></td></tr><tr><td>acetonitrile, Optima LC/MS</td><td>Fisher Chemical</td><td>A996</td><td></td></tr><tr><td>Protein LoBind tubes 0.5 mL</td><td>Eppendorf AG</td><td>22431064</td><td></td></tr><tr><td>Protein LoBind tubes 1.5 mL</td><td>Eppendorf AG</td><td>22431081</td><td></td></tr><tr><td>HLB &amp;micro;Elution plate 30 &amp;micro;m</td><td>Oasis</td><td>186001828BA</td><td></td></tr><tr><td>SpeedVac concentrator</td><td>Thermo Scientific</td><td>Savant SPD2010</td><td></td></tr><tr><td>sonicator</td><td>Qsonica</td><td>Oasis180</td><td></td></tr><tr><td>centrifuge</td><td>Thermo Scientific</td><td>Sorvall Legend micro 21R</td><td></td></tr><tr><td>LC trap column PepMap 100 C18</td><td>Thermo Scientific</td><td>160454</td><td></td></tr><tr><td>LC separation column PepMap RSLC C18</td><td>Thermo Scientific</td><td>164536</td><td></td></tr><tr><td>mass spectrometer</td><td>Thermo Scientific</td><td>Orbitrap Elite ion trap mass spectrometer</td><td></td></tr><tr><td>MSConvert software</td><td>ProteoWizard Toolkit</td><td></td><td></td></tr><tr><td>Sorcerer-SEQUEST software</td><td>Sage-N Research, Inc.</td><td></td><td></td></tr></tbody></table>

dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion

Studien in dynamischen Änderungen im Protein Übersetzung erfordern spezialisierte Methoden. Hier untersuchten wir Veränderungen in neu synthetisierten Proteine als Reaktion auf Ischämie und Reperfusion mit isoliert PERFUNDIERTEN Maus Herzen gepaart mit Polysome Profilierung. Um die dynamischen Änderungen im Protein Übersetzung besser zu verstehen, wir zeichnen die mRNAs, die wurden mit cytosolische Ribosomen (Polyribosomes oder Polysomes) geladen und auch erholt mitochondriale Polysomes und verglichen mRNA und Protein-Verteilung in der Hocheffizienz Fraktionen (zahlreiche Ribosomen an mRNA befestigt), niedrig-Effizienz (weniger Ribosomen befestigt) darunter auch mitochondriale Polysomes und die Fraktionen nicht übersetzen. MiRNAs können auch mit mRNAs, die übersetzt werden werden, wodurch die Effizienz der Übersetzung zuordnen, untersuchten wir die Verteilung der MiRNAs in die Brüche. Die Verteilung der mRNAs, MiRNAs und Proteine wurde unter basalen PERFUNDIERTEN Bedingungen am Ende des 30 min des globalen keine-Flow-Ischämie und nach 30 min der Reperfusion untersucht. Hier präsentieren wir Ihnen die Methoden verwendet, um diese Analyse zu erreichen – unter anderem wurde der Ansatz zur Optimierung der Proteingewinnung aus Saccharose Neigung, als dies nicht vor beschrieben — und einige repräsentative Ergebnisse liefern.

mRNA-Analyse mRNA-Ergebnisse können als Gewinnausschüttung einer bestimmten mRNA in jedem Bruchteil (Abbildung 3A); Kombinieren Sie für die Quantifizierung polyribosomal Übersetzung Brüche und vergleichen Sie nicht übersetzen Bruchteil (Abb. 3 b), präsentiert ein Verhältnis von mRNA Fülle nontranslating Fraktionen zu übersetzen. Weitere Informationen gewinnt man durch die Untersuchung der hocheffiziente Poly...

Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Hier präsentieren wir ein Protokoll zur Polysome Profilierung auf das Herz isoliert PERFUNDIERTEN Maus durchführen. Wir beschreiben Methoden für Herz Perfusion, Polysome Profilierung und Analyse der Polysome Fraktionen in Bezug auf mRNAs, MiRNAs und Polysome Proteom.

Dynamische Proteomic und MiRNA Analyse des Polysomes von isolierten Maus Herz nach Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

dynamic-proteomic-mirna-analysis-polysomes-from-isolated-mouse-heart

Research

JoVE Journal

Biochemistry

8.5K Aufrufe.  Cedars-Sinai Medical Center. Hier präsentieren wir ein Protokoll zur Polysome Profilierung auf das Herz isoliert PERFUNDIERTEN Maus durchführen. Wir beschreiben Methoden für Herz Perfusion, Polysome Profilierung und Analyse der Polysome Fraktionen in Bezug auf mRNAs, MiRNAs und Polysome Proteom.

Serie: Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Beurteilung der Herzfunktion und Energetik in isolierten Maus-Herzen mit 31 P-NMR-Spektroskopie

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

Forschung

Medizin

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

Gewebespezifische MiRNA Expression Profiling in Maus Herz Abschnitte In Situ Hybridisierung

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Genetik

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

Quantitative Analyse von Chromatin Proteome in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Single Cell Transcriptional Profiling der erwachsenen Maus Kardiomyozyten

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Biologie

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolation und physiologische Analyse von Maus Kardiomyozyten

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

Analyse der Übersetzung im sich entwickelnden Mausgehirn mit Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

Neurowissenschaften

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Proteinisolierung aus den Entwicklungs embryonalen Mäuseherzklappen Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Herstellung einer Nicht-Kardiomyozyten-Zellsuspension für die Einzelzell-RNA-Sequenzierung aus einem erwachsenen Mausherzen nach Myokardinfarkt

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

Gleichzeitige Isolierung und Kultur von Atrial Myozyten, ventrikulären Myozyten und Nicht-Myozyten aus einem erwachsenen Mausherz

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

Dynamische Proteomic und MiRNA Analyse des Polysomes von isolierten Maus Herz nach Langendorff Perfusion

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Beurteilung der Herzfunktion und Energetik in isolierten Maus-Herzen mit³¹ P-NMR-Spektroskopie