Kardiyovasküler Hastalıklarda Humoral Bağışıklık Yanıtları (- Bu çalışma Deutsche Forschungsgemeinschaft (DFG) ve Sonderforschungsbereich Transregio 19 (SFB/TR19, C2), Ekonomi ve Teknoloji proje ZIM-KF 2727801MD0 Federal Bakanlığı ve İnovasyon Yetkinlik Merkezi tarafından desteklenen ZIK-Yürüyüş, Eğitim ve Araştırma, Almanya Federal Bakanlığı BMBF FKZ 03Z2CN12). Hayvanlarla Konut ve deneyler Avrupa Laboratuar Hayvan Bilimi Derneği (FELASA) Laboratuvarı Zootekni (Gesellschaft für Versuchstierkunde, GV-SOLAS) ve Federasyon için Derneği&#39;nin önerilerine göre yapıldı.

1. Hasta Örneklerden IgG İzolasyonu <ol><li> Boş Econo Pac sütuna 2 ml hastanın EDTA plazma başına 3 ml anti-IgG Sepharose doldurarak mini immunoadsorbsiyon sütunlar hazırlayın. IgG izolasyon hastanın plazma, en az 2 ml gerektirir. </li><li> Anti-IgG Sepharose üstünde filtre yerleştirin, sütunun alt ucu açık kesilmiş ve yaklaşık olarak 1 damla / sn &#39;lik bir akış hızına ulaşmak için filtre düğmesine basın. Sütunun tükendi korunması çözümü olsun. </li><li> 3 cilt plazma hacmi başına% 0.9 NaCl ile sütun yıkayın. </li><li> Sütun üzerinde plazma pipet. Plazma tamamen anti-IgG Sepharose batırılır sonra, aynı hacimde% 0.9 NaCl ile yıkanır. </li><li> Pipet bir plazma sütun üzerinde 0.2 M glisin HCl, pH 2.8 hacim ve Sepharose içine batırmayın edelim. Sütun üzerine glisin HCI hacmi başka ekleyin. Bir 15 ml tüp içinde ortaya çıkan akış toplayın ve 0.5 M Tris-HCI, pH 8.0 ile pH 7.3 &#39;e ayarlayın. </li><li> Sütun yenilemek için, 3 plazma hacimleri glisin HCl ile yıkayın. 2 hacim% 0.9 NaCl ile bir son yıkamadan sonra, sütun sonraki plazma örneği için kullanılabilir. ° C ila 4 hafta için 8 - Alternatif olarak, kolon koruma çözeltisi ile doldurulmuş ve 4 de muhafaza edilebilir. </li><li> Kardiyomiyositlerde üzerinde kullanım için, toplanan IgG fraksiyonu deneme tamponu (EB) karşı diyaliz gerekmektedir. EB hazırlanması için, 117 mM NaCI, 2.8 mM KCI, 0.6 mM MgCl2, 1.2 mM KH 2 PO 4, 1.2 mM CaCl2, 10 mM HEPES ve bir manyetik karıştırıcı üzerinde, 20 mM glikoz 1 L damıtılmış suya eklenir ve pH ayarlamak 7.3. </li><li> 100 kDa kesilmiş olan bir molekül ağırlığına sahip olan bir selüloz ester diyaliz zarı tüp için IgG fraksiyonu doldurun. 1 L EB tüp koyun ve 4&#39;e bir manyetik karıştırıcı ile yavaş yavaş karıştırılır ° C. 8 saat sonra, taze EB 3 L diyaliz tüpü içine aktarmak ve 4&#39;de diğer bir 20 saat boyunca dialyze ° C. </li><li> Inactivat için bir su banyosu içinde 30 dakika 57 ° C&#39;de diyaliz için, ısı örnek takibene kompleman faktörleri. Total IgG konsantrasyonunu belirleyin ve 330 mikrogram IgG içeren her alikotları hazırlamak. Ölçümü (adım 4.3) için kardiyomiyositlerde için örnek eklerken, EB ile 1 ml alikotları kadar doldurun. Seyreltilmemiş alikotları daha fazla kullanıma kadar -20 ° C&#39;de muhafaza edilebilir. </li></ol> 2. Sıçan kardiyomiyositlerde izolasyonu <ol><li> Ca2 +-serbest izolasyon tampon (IB) hazırlanması için, 110 mM NaCI, 2.6 mM KCI, 1.2 mM MgSO4, 1.2 mM KH 2 PO 4, 20 mM HEPES ve bir manyetik kullanılarak 1 L damıtılmış su ile 11 mM glükoz eklemek karıştırıcı. Sterilizasyon için 0.2 mikron şişe üst filtre ile oda sıcaklığında ve filtre, 7.4 pH ayarlayın. </li><li> Termostatlı bir Langendorff perfüzyon sisteminin üst rezervuar içinde IB 80 ml doldurun. 37 ° C arasında bir sıcaklık muhafaza tampon ve% 95 O2 /% 5 30 dakika için CO2 ile tampon havalandırmak için termostat ayar ayarlayın. </li><li> Yaklaşık 10.000 tartılır - collag 12.000 Uenase tip II, 175 mg yağ asidi serbest BSA ve 100 mM CaCl2 stok solüsyonu 22.5 ul içeren 20 ml&#39;lik IB içinde çözülür. Üst rezervuar içinde tecrit tampon pH kontrolü ve gerekirse ayarlanır. </li><li> 375 mg / kg vücut ağırlığı tiyopental ile 200 gr - 175 bir Wistar sıçan Anesteziyoloji. Tiyopental çözümü 2500 IU Heparin ekleyin. Sağlam bir aort arkı ile dikkatlice torakotomi, tüketim kalp sonra ve buz gibi soğuk% 0.9 NaCl içine aktarın. Kan kalp ve çevresindeki doku ve taze% 0.9 NaCl transfer temizleyin. Aorta Açığa, 2-3/sec bir damla hızını elde etmek ve kanül için kalp eklemek Langendorff sistemin akışını düşürücü açın. </li><li> Kadar kalan kan dışarı yıkamak 1 damla / sn debi ile 3 dakikaya kadar kalp serpmek. Langendorff sisteminde IB dolaşmaya başlayın. Üst rezervuar 20 ml tampon kaldır gibi 80 ml ulaşmak için gerekli çok tampon kollajenaz çözüm ve dolum doldurun. C sirküle<html> Başka bir 27 dakika için çözelti ollagenase. Akış hızı Akış redüktör kullanarak 1 damla / sn aralıklarla kontrol ve muhafaza edilmelidir. </li><li> Kulakçıklar ve aortadan temiz kanül gelen kalp, ayırın ve küçük parçalar halinde doğrayın. Kollajenaz çözüm alt rezervuar içine çalıştırmak için izin ver, 40 ml, maksimum hacmi azaltmak ve daha 10 için kıyılmış ventriküller sindirmek -% 95 O 2 /% 5 CO 2 ile sürekli havalandırma altında 15 dk. Daha sonra, bir 50 ml tüp içinde bir 200 mikron elek gazlı bez ile hücre süspansiyonu filtre. </li><li> 200 uM CaCl2 içeren 10 ml IB içinde oda sıcaklığı ve tekrar süspansiyon hücreler 2 dakika için 43 x g&#39;da hücre süspansiyonu santrifüj: 3 adım hücrelerin Ca 2 + içerik yükler. Tekrar santrifüjlenir ve 500 uM CaCl2, 10 ml IB hücreleri tekrar süspansiyon. Son bir santrifüj 50.000 hücre / ml &#39;lik bir yoğunluğa steril filtre edilmiş EB kardiyomiyositlerde (1.8 bakınız) tekrar süspansiyon sonra. </li></ol>kardiyomiyositlerin &quot;&gt; 3 E_TITLE. Fura-2 Boyama <ol><li> Coat 10 mikrogram başına iyi laminin ve tamamen kurutulur beklemek bir 4 iyi odacıklıdır coverglass. </li><li> Her iyi bir kesim uçlu bir mikropipet kullanarak içine kardiyomiyosit süspansiyonu 1 ml doldurun. Hücreler, oda sıcaklığında 1 saat boyunca uymak için izin verin. </li><li> 10, 1 mg / ml &#39;lik ul Fura-2 5 ml EB stok çözelti AM. Seyreltilir Karanlıkta boyama çözüm tutun. </li><li> Coverglass ve her kuyuya boyama çözeltisi pipetle 0.5 ml tampon ve bekâr hücreleri çıkarınız. Sarsıntı orta altında 10 dakika için hücreler inkübe edin. Daha sonra, boyama çözüm take off 1 ml EB ile bir kez hücreleri yıkayın ve nihayet her oyuğuna 0.5 ml EB doldurun. Boyama işlemi sırasında doğrudan ışıktan sakının ve her zaman karanlıkta boyanan hücreleri tutun. </li></ol> 4. Hücre kısaltılması ve Ca 2 + Geçişlerine kaydedilmesi <ol><li> Ters floresan m odacıklı coverglass yerleştirinicroscope bir miyosit kalsiyum ve kontraktilite kayıt sistemine bağlı. De ilk akını ve akış tüpü ile özel yapılmış elektrot yerleştirin. 1 ml / dak EB ve (20 V, 1 Hz, 5 ms süre), elektrik uyarımı ile başlamak Superfuse kardiyomiyositlerde. Hücrelerin yaklaşık 2 dakika stimülasyon uyum sağlayın. </li><li> Net çizgili ve sürekli kasılma ile sağlam bir çubuk şekilli kardiyomiyosit seçin. Görüş alanının merkezinde hücre yerleştirin ve kamera kanal açılır. Hücre çerçeveleme adaptörü dönen ve mikroskop sahne hareket ettirerek videoyu alanı içinde yatay hücre yönlendirilmesi. </li><li> Başlangıç ​​hücre kısalma ve Ca 2 + geçici elde etmek için 15 kasılmalar - kenarı video alanı kontrol elemanları (Şekil 2) tespit ayarlayın, floresan kanal ve kayıt 10 açın. </li><li> Floresans leke ağartma önlemek için mikroskop ışık kanalı kapatın. Örnek EB aracılığıyla akışını değiştirmeHastanın IgG 1 ml ile 3-yollu vana ve superfuse kardiyomiyositlerde ile bypass. Hava iyi ulaşmadan hemen önce pompayı durdur. </li><li> Işık kanalı açın ve başka bir 10 kayıt - Akut hücre yanıtını elde etmek için 15 kasılmalar. Kayıt dondurularak ve tekrar ışık kanalı kapatın. 2&#39;den sonra kayıt ve 5 dk tekrarlayın. </li><li> Kuyunun elektrot çıkarın. EB ile iyice temizleyin ve tüpler odacıklı coverglass bir sonraki sıra ile devam edin. </li><li> Sırasıyla Hücre kısalma ve Ca 2 + &quot;Edge-Length/Length&quot; kullanarak IonWizard yazılımı ile geçici ve &quot;fura 2-Sayısal / Oranı çıkarılır&quot; penceresinde, analiz edin. Başlangıç ​​hücre kısalma ve Ca 2 + Akut için temel (bl% pik h) atıfta pik yüksekliği geçici, 2 dakika ve 5 dk yanıtı yüzde değişim hesaplayın. </li></ol>

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Çıkar çatışması ilan etti.

Sunulan yöntem belirsiz kökenli DCM hastalarda fonksiyonel olarak etkili kardiyak otoantikor saptamak için uygun bir yol sunar. Diğer yöntemler ile karşılaştırıldığında, cAMP seviyelerini 6 üzerindeki etkileri ile β1-adrenoseptör karşı işlevsel aktif antikorların saptanması, örneğin, bizim yöntem, belirli bir epitopa bağımsızdır. Tabii ki literatürde açıklanan diğer epitop bağımsız analizi yani yama klemp tekniği veya izole Purkinje lifleri 14 k...

<table border="1"><tbody><tr><td> Reaktif / ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Immunosorba koruma çözümü </td><td> Fresenius Medical Care </td><td> 903609211 </td><td></td></tr><tr><td> Kollajenaz tip 2 </td><td> Hücre Sistemi </td><td> LS004176 </td><td></td></tr><tr><td> BSA, serbest yağ asidi </td><td> Sigma-Aldrich </td><td> A6003 </td><td></td></tr><tr><td> Laminin </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Fura-02:00 </td><td> Sigma-Aldrich </td><td> F0888 </td><td> DMSO içinde 1 mg / ml </td></tr><tr><td> Econo Pac Kromatografi Sütunları </td><td> BioRad </td><td> 732-1010 </td><td></td></tr><tr><td> Spectra / Por Biotech Selüloz Ester Diyaliz MembranE </td><td> Spectrumlabs </td><td> 131417 </td><td></td></tr><tr><td> 4 sıra Chambered coverglass </td><td> Nunc </td><td> 155383 </td><td></td></tr><tr><td> Miyosit Kalsiyum ve Kasılabilme Kayıt Sistemi </td><td> IonOptix </td><td></td><td></td></tr><tr><td> IonWizard 6.0 analiz yazılımı </td><td> IonOptix </td><td></td><td></td></tr></tbody></table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

Dilate kardiyomiyopati (DKMP) genç yetişkinler 1 kalp yetmezliği için ana nedenlerinden biridir. Toksik maddelere genetik eğilim ve fuar hastaların yaklaşık üçte birinde bu hastalığın nedenleri bilinmesine rağmen, DCM kökeni büyük ölçüde belirsizdir. Bu hastaların önemli bir kısmında, kardiyak epitoplara otoantikorların tespit edilmiş ve hastalığın 2,3 başlangıcı ve ilerlemesinde önemli bir rol oynadığından şüphe edilir. Kardiyak otoantikorların önemi immunoadsorbsiyon 3-5 ile otoantikorların ortadan kaldırıldıktan sonra DCM hastada hemodinamik iyileşme altı çizilir. Spesifik antijenlerin çeşitli zaten 2,3 tespit edilmiştir ve bu hedeflere karşı antikorlar immunoassay ile tespit edilebilir. Bununla birlikte, bu ölçümlerin uyarıcı (ve bu nedenle de fonksiyonel olarak etkin) ile bloke edici antikorlar arasında ayırt edemez. Eviden vardır artmaktadırBu ayrım önemlidir 6,7 olduğunu ce. Aynı zamanda, cardiotropic antikorların bir dizi hedeflerin yine tanımlanamayan ve bu nedenle bağışıklık deneyleri ile tespit edilemez olduğu kabul edilebilir. Bu nedenle, kendi antijeninin bağımsız işlevsel olarak aktif cardiotropic antikor tespiti için bir metod kurulmamıştır. Yöntem için arka plan genellikle memeli 8,9 arasında kardiyak proteinlerin işlevsel bölgeleri için gözlenen yüksek bir homoloji olduğu. Bu insan antijenlerine karşı, kardiyak antikorlar erişkin sıçan kardiyomiyositler üzerine DCM hastaların IgG test veriyor insan olmayan hedef hücreleri, ile çapraz reaksiyona girer göstermektedir. Önerilen yöntem, 3 adımlardan oluşur: ilk olarak, IgG immunoadsorbsiyon sütunlar (PlasmaSelect, Teterow, Almanya) dan elde edilen Sepharose birleştirilmiş anti-IgG antikorları kullanılarak hasta plazmadan izole edilir. İkinci olarak, erişkin kardiyomiyositlerde ap kullanılarak bir Langendorff perfüzyon cihaz içinde kolajenaz perfüzyon yoluyla izole edilirotokol önceki eserlerinden 10,11 güncellenmiştir. Elde edilen kardiyomiyositlerde laminin kaplanmış odacıklı coverglasses eklenmiş ve Fura-2, kolaylıkla (Ca 2 +) içindekiler 12 hücre içi kalsiyum gözlemlemek için hücre içine getirilebilir bir kalsiyum-seçici floresan boya ile boyanır. Son aşamada, hücre katı ve Ca 2 hasta IgG etki + alan uyarılan kardiyomiyositlerde geçici bir standart ters floresan bağlı olan bir ticari miyosit kalsiyum ve kasılmasını kontrol sisteminin (IonOptix, Milton, MA, ABD) kullanılarak online olarak izlenir mikroskop.

Alanda hücresel inotropiyi ölçümü için iki örnek aşağıda verilmektedir, bir miyosit Ca 2 + ve kasılma kayıt sistemi kullanılarak erişkin kardiyomiyositlerde (Şekil 2) ile alınabilir. Şekil 3, bir kontrol ölçümü arasında bir izlenimini vermektedir, Şekil 4 içinde iken cardiodepressive antikorların etkisi bir hastanın DCM gösterilmiştir. Her iki örnek, başlangıç ​​hücre kısalma ve Ca 2 +</s...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Sunulan yöntem izole sıçan kardiyomiyositlerde hücresel kısalma ve hücre içi kalsiyum geçici üzerinde izole hasta immunglobulin etkisini analiz ederek, bağımsız spesifik antijen, dilate kardiyomiyopatili hastaların plazmasında fonksiyonel etkin cardiotropic otoantikor saptamak için bir yol sunar.

İzole kardiyomiyositlerin Hücresel Fonksiyon üzerine Antikor Etkilerinin Ölçümü

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

measurement-antibody-effects-on-cellular-function-isolated

Research

JoVE Journal

Immunology and Infection

11.8K Görüntüleme.  University Medicine Greifswald. Sunulan yöntem izole sıçan kardiyomiyositlerde hücresel kısalma ve hücre içi kalsiyum geçici üzerinde izole hasta immunglobulin etkisini analiz ederek, bağımsız spesifik antijen, dilate kardiyomiyopatili hastaların plazmasında fonksiyonel etkin cardiotropic otoantikor saptamak için bir yol sunar.

Video: Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Fonksiyonel Kardiyak Bağışıklık Hücreleri İzolasyon

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

İmmünoloji ve Enfeksiyon

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

İnsan kaynaklı Pluripotent Kök Hücre Türevli Kardiyomiyositlerde Tek Hücreli Optik Eylem Potansiyeli Ölçümü

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Biyomühendislik

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.
The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.
The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

Preklinik Kardiyak Risk Değerlendirmesi için İnsan iPSC Kaynaklı Kardiyomiyositlerin Yapısal ve Kasılma Değişikliklerini Değerlendirmek için Hibrit Hücre Analiz Sistemi

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

Bir fare modeli Metabolik sendromu ile ilgili kalp yetmezliği ile korunmuş ejeksiyon fraksiyonu atriyal Cardiomyocytes izolasyonu

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Tıp

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Olgun İnsan Kaynaklı Pluripotent Kök Hücre Kaynaklı Kardiyomiyosit Monokatmanları Kullanılarak Yüksek Verimli Kardiyotoksisite Taraması

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

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.

Fare kardiyomiyositlerde İzolasyon ve Fizyolojik Analizi

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

Biyoloji

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. However, the literature reports contrasting data on the prevalence of myocardial B-cells. B-cells have been reported to be both among the most prevalent immune cells in the rodent heart or to be present, but at a markedly lower prevalence than myeloid cells, or to be quite rare. Similarly, several groups have described that the number of myocardial B-cells increases after acute ischemic myocardial injury, but one group reported no changes in the number of B-cells of the injured myocardium. Implementation of a shared, reproducible method to assess the prevalence of myocardial B-cells is critical to harmonize observations from different research groups and thus promote the advancement of the study of B-cell myocardial interactions. Based on our experience, the seemingly contrasting observations reported in the literature likely stem from the fact that murine myocardial B-cells are mostly intravascular and connected to the microvascular endothelium. Therefore, the number of B-cells recovered from a murine heart is exquisitely sensitive to the perfusion conditions used to clean the organ and to the method of digestion used. Here we report an optimized protocol that accounts for these two critical variables in a specific way. This protocol empowers reproducible, flow cytometry-based analysis of the number of murine myocardial B-cells and allows researchers to distinguish extravascular vs. intravascular myocardial B-cells.

Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry.

Akış Sitometrisine Dayalı Miyokard B Hücrelerinin Nicelleştirilmesi ve Analizi

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

Derili Kardiyomiyositler Kullanarak Kardiyak Fonksiyonun İn Vitro Değerlendirilmesi

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.

This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.

İzole Yetişkin İnsan Primer Kardiyomiyositlerinde Kalp Kontraktilitesinin Ölçülmesi

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This ...

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.